Method of leukocyte interferon preparation

专利摘要:
Recombinant DNA molecules and hosts transformed with them which produce polypeptides displaying a biological or immunological activity of human interferon, the gene coding for these polypeptides and methods of making and using these molecules, hosts, genes and polypeptides. The recombinant DNA molecules are characterized by structural genes that code for a polypeptide displaying a biological or immulogical activity of human interferon. In appropriate hosts these molecules permit the production and identification of genes and polypeptides displaying a biological or immunological activity of human interferon and their use in antiviral and antitumor or anticancer agents.
公开号:SU1764515A3
申请号:SU813233703
申请日:1981-01-08
公开日:1992-09-23
发明作者:Вайссман Чарльз
申请人:Биоген Инк (Фирма)；
IPC主号:

专利说明:

This invention relates to genetic engineering, in particular, to the production of human leukocyte interferon.
A known method for producing leukocyte interferon involves the cultivation of strains of cultured animal cells, the isolation and purification of the target product.
The aim of the invention is to increase the purity of the target product.
In this method, the detection of DNA sequences. encoding CheIFN-av cells of E. coli.
Using this method, polypeptide (s) are obtained which exhibit immunological or biological activity of CheIFN-a
The present method involves the detection and isolation of DNA sequences and the construction of recombinant DNA molecules encoding a polypeptide having the immunological or biological activity of CheIFN-a.
The obtained recombinant DNA containing the DNA sequence Z-pBR322 / Pst) (HCIF4c, Z-pBR3 22 (Pst) / HclF-2h, Z-pBR322 / Pst / HclF-SN 35, Z-pBR 322 / Pst / HclF-SN42, Z-pKT287 / Pst / Hc F-2h-AH6, as well as DNA sequences that hybridize with said inserts.
The method is illustrated by the following examples.
PRI me R 1. Human leukocytes are stimulated for 5 hours at 37 ° C with the Sendai virus and extracted from polystyrene (A) RNA containing human mRNA of leukocyte interferon (H eIFN-a mRNA). Stimulated leukocytes are collected and 10 cells are suspended in 1 l of a solution containing 8 g of NaCI, 0.2 g of CS, 1.15 g of N32HP04-2H20 and 0.2 g of KH2P04, dissolvedXI
About 4
listened to
eight
1 liter of water (PBS), and added with vigorous stirring to 17 l of 20 mM Tris-HCl (pH 7.5), 1 mM Edtuck (TE-buffer), 2% sodium dodecyl sulfate (SDS) in a separatory funnel 50 l. Pronaz is added to 200 / and g / ml and the solution is stirred for 1 hour at room temperature. 10 counts / min 125 1-globin mRNA are added as a marker for poly / A / RNA isolation. 2M Tris-HCl (pH 9) in an amount equal to 1/20 of the total volume (1/20 volume) is added, and the mixture is extracted with vigorous stirring 15 liters of distilled phenol for 10 minutes. 3 L of chloroform is added and the mixture is stirred for 5 minutes. After 30 minutes of settling, the aqueous phase is removed to separate the phases, a total of 19.1 liters are combined with 60 g of SDS. Nucleic acids precipitated from the aqueous phase 1/10 vol. ZM sodium acetate (pH 5.5) and 2 volumes of ethanol.
After being stored overnight at -20 ° C, the precipitate of nucleic acids is filtered through a plastic tea sieve. This material is then mixed with 200 ml of THE (50 mM Tris-HCl (pH 7.5), 100 MMNaCI, 5 mM EDTA) containing 0.5% SDS. It is subsequently dissolved by adding another 350 ml of this solution. The precipitate is collected by centrifugation in 1 L bottles in a Sorvoll RC-3 centrifuge for 15 minutes at 5000 rpm and dissolved in 350 ml of THE containing 0.5% SDS. Both THE solutions are combined, extracted 3 times with 1 volume of phenol, 3 times 1/2 volume of ether and 3 times with 1 volume of ether. Only 775 mg of RNA is isolated from the aqueous phase.
Poly (A) RNA mixtures are isolated by adsorption on oligo () cellulose. Add 2.7 g of oligo () cellulose to 500 ml. After stirring for one hour at room temperature to adsorb poly (A) RNA to oligo (oT) cellulose, the cellulose and mRNA mixture bound to it are centrifuged, washed once with 50 ml of THE and second time with 15 ml of THE. Then, the bound poly (A) RNA is eluted with five successive washes with 2 ml of NZO. 860 / g of poly (A) RNA are obtained. The supernatant solution of RNA from the first adsorption is subjected to two subsequent cycles of adsorption. With the second and third adsorption, 600 / and g and 170/4 g of RNA are obtained.
RNA was tested for CheIFN-a activity by injection into Xenopus laevis oocytes. The PNH was dissolved in 15 ml of Tris-HCl (pH 7.5), 88 mM NaCl (TH K buffer) to obtain a concentration of about 1 mg / ml. Inject 50 ml of this solution into every 50 oocytes.
Oocytes are incubated overnight at room temperature in Bart's medium. Incubated oocytes are then washed and homogenized with a Pasteur pipette in a tube.
for 1.5 ml Eppendorf centrifuges in 0.5 ml of 52 mM Tris-glycerol buffer (pH 8.9). The mixture is centrifuged for 2 minutes in an Eppendorf centrifuge, the supernatant is drained and frozen at
-20 ° C for testing. One unit of IFN-a reduces the amount of virus on the plate by 50%. The activity of the preparation IFN-a is expressed in relation to the well-known human CheIFN-a 69/19. Extract
oocytes have an activity of 300 IU of IFN-ana / and g RNA, while the oocytes are incubated for 48 hours. For further purification of the poly (A) RNA, add 0.5 M ethylenediaminetetraacetic acid (EDTA) to
poly (A) RNA preparation A for binding to a concentration of 5 mM EDTA. The resulting solution is extracted twice with an equal volume of THE-saturated phenol and 5 times with an equal volume of ether. Then it is passed through a column with 0.1 ml heated over 90 s to 100 ° C and in a 13 ml layer with a sucrose gradient of 5-23% containing 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0 , 2 M NaCI. As a marker, 10,000 spm of 5-terminal 32 p-labeled DNA fragments, produced while simultaneously assimilating pBR 322 and endonucleases Hind III and Pst I, are added. Centrifugation is carried out in SW 40 rotor at 10 ° C and 35,000 rpm for 16 h.
Fractions (0.6 ml) are collected with an ISCO gradient collector at 1 ml / min. The fractions for CeIFN-ohmRNA are tested, their position in relation to the 32 R-DNA markers. During the subsequent centrifugation, the fractions containing CheIFN-amRNA are identified relative to the markers of the Fraction with the activity of CheIFN-a mRNA containing 80 g of poly (A) RNA. They are mixed with 2 volumes of THE containing 0.5% SDS and
0.02% polyvinyl sulfate (in the last preparations polyvinyl sulfate was excluded), using a column with 50 / and 1 oligo (oT) cellulose. After washing the column 40 / and g of the mixture, the RNA is eluted with 4 washes with 0.6 ml of distilled water. After precipitation with ethanol, the RNA is dissolved in 1 mg / ml in 0.5 mM EDTA.
The test for the activity of CheIFN-a mRNA was carried out as described above, in portions of a poly (A) RNA precipitate. It has a specific activity of 3600 IU interferon //. Consequently, the sucrose gradient was enriched with poly (A) RNA about 10 times the ratio of cHIFN-ccmRNA. Get cooking with about 40-fold enrichment.
Synthesis of DNA mixture containing Che-IFN-asDNA.
Poly (A) RNA enriched with IFN-a mRNA is used as a template (model) for producing single strand complementary DNA (cDNA). The 800 / hl reaction mixture contains 40 mM Tris-HCl (pH 7.5), 30 mM NaCI, 5 mM MgCl2, 0.5 mM DTT, 20 mg / ml oligo (ot) 12 - 18, 5 mM dGTP, dCTP and dTTP, 5 mM 32 P-dATP (HEH, specific activity 10,000 spm / nmol), 60 mg / ml poly (A) RNA and 280 units. reverse transcriptase. After incubation for 1 hour at 37 ° C, 0.5 M EDTA and 20% SDS (recrystallized) are added to 10 mM EDTA and 0.1% SDS. The mixture is extracted with 1 volume of phenol (distilled). The phenol phase is washed with 200 / l L 200 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.1% SDN, and the aqueous phases are combined. They are extracted with an equal volume of ether and chromatographed on a column of 5 ml Sephadex G-100 in THE. Collect fractions of 0.1 ml at 0.3 ml / min. Fractions showing radioactivity (as measured by Cherepkov radiation) are pooled and GW is added. Nucleic acids are precipitated with 2.5 volumes of ethanol. After being stored overnight at -20 ° C, the samples are centrifuged, the supernatant layer is discarded. The precipitate is dissolved in 180 / g l of distilled water and transferred to a siliconized Eppendorf tube. 20 / and 1 ml of SMNaOH are added and the mixture is kept at room temperature for 40 minutes. 20 ml of 5M sodium acetate, 100 ml of distilled water and 500 ml of ethanol are added. After cooling overnight at -20 ° C, the resulting precipitate is collected by centrifugation at a force equivalent to 10,000 times the force of gravity (1 OOOOhd) for 20 minutes at 0 ° C. The output of single-stranded cDNA is 10
The single strand cDNA product is in a real complex mixture of a large number of different cDNAs transcribed from the corresponding mRNA found in a mixture of poly (A) RNA. Only very few of these cDNAs are related to IFN-a, for example, HilFN-asDNA.
The sizes of various single-stranded cDNA were determined by electrophoresis of small on alkaline 2% agarose gel using 30 mM NaOH, 2 mM EDTA as electrolyte. The 32P-cDNA has a length of 600-1000 nucleotides related to the single-stranded cDNA globin, 32 P-labeled DNA fragments are used as size markers.
PRI mme R 2. Single-stranded cDNA is converted to double-stranded by treatment with DNA polymerase 1. The precipitated single-stranded cDNA is dissolved in 200 / g l of NaO heated to 100 ° C for 2 min and incubated in 500 l of 0.1 M heated denatured potassium phosphate buffer (pH 6.9) 10 mM MgCl2, 10 mM DTT, 1 mM each of dATP, dGTP and dCTP, 1 mM 3H-dTTP (HEH, specific activity 100000 spm / nmol) and 150 units / ml DNA Polymerase T. After 6.5 hours at 15 ° C, add 0.5 EDTA and 20% SDS to 10 mM EDTA and 0.1% SDS. The mixture is then extracted with 500, P1 of phenol, the phenol phase is re-extracted with 250 / hl of 20 mM Tris-HC (pH 7.5), 5 mM EDTA (TE buffer). The two aqueous phases are combined and chromatographed on a column with 5 ml of Sephadex G-100 under the same conditions. Add 3 M sodium acetate to 0.3 Mi 2.5 volumes of ethanol, mix to precipitate the DNA. Just collect.
DNA is treated with nuclease S1. The precipitated DNA is dissolved in 250 ml of buffer (0.2 M NaCl, 50 mM sodium acetate (pH 4.5), 10 mM zinc sulfate and heated at 37 ° C for 30 minutes. 1.5 Si of the enzyme is added (11 units (/ 4p), the mixture is incubated at 37 ° C for 30 minutes, the SDS and EDTA are added to 0.1% SDS and 5 mM EDTA and the mixture is extracted with 250 ml of phenol. The phenol phase is washed with 100 ulTE of buffer. The aqueous phases are combined and chromatographed on a Sephadex G-100 column in THE, fractions of 0.1 ml are collected at a rate of 0.3 ml / min and Cherepkov radiation is determined for each fraction. After precipitation from ethanol and sodium acetate fleecing 8 / g of the DNA double helix.
Example 3, A plasmid (in particular, pBR 322) is treated with the Pst I endonuclease and dGMP tails are added with a terminal transferase. dGMP is added to the 5 end of the plasmid to regenerate the Pst I site and are ligated with a cDNA fragment carrying complementary tails. The double-stranded gDNA is extended by the addition of dCMP tails to the 3 terminal. The plasmid and cDNA are then treated with a ligase, which ensures that the cDNA is introduced into a suitable plasmid site and the hybrid DNA is obtained,
EXAMPLE 4 Plasmid pBR322 (20 g) is hydrolyzed with 21 units of Pst I endonuclease in 150 ml of 10 mM Tris-HCl (pH 7.5), 6 mM MgCl, 50 mM NaCl, 6 mM MgCl, 50 mM NaCI, 6 mM 2-mercaptoethanol, 200 mg / sp bovine serum albumin (BSA), After 2 h
at 37 ° C, the mixture is extracted with 1 volume of phenol-chloroform mixture (1: 1) and 1 volume of ether and precipitated with ethanol.
DGMP homopolymer tailings were added using terminal deoxynucleazide transferase (TdT) in 328 ml of reaction volume containing 100 mM sodium cacodylate (pH 7.2), 10 mM NaH2P04, 5 mM MgCIa, 1 mM dGTP, 50 mg / ml BSA and 3 - 6 units. TdT (purified as above) per 1 mg of DNA. Incubate at 37 ° C for 20 min. Add EDTA to 10 mM, extract the mixture as above and dialyze for two days against THE buffer.
The double-stranded DNA was extended with dCMP residues according to a standard procedure. Incubate 150 ml of the double-stranded cDNA described above, a 8, W1 100 mM sodium cacodylate (pH 7.2), 2.5 mM CoCl2, BSA, 0.2 mM dCTR containing 3 - 6 units purified TdT per mg of DNA for 8 min at 27 ° C, and then frozen at -20 ° C.
One colony of E. coli XI776 is inoculated in 100 ml of tryptone medium with 100 / g / ml of diaminopimelic acid, 10 cg / ml of nalidixic acid and 10 / g / ml of tetracycline added. Culture is grown at 37 ° C to an apparent optical density of 0.6 at 650 nm (ODb5o) and cooled on ice for 30 minutes. The culture is then sedimented at 4000 rpm in the rotor, the cells are washed with 50 ml of 10 mM NaCI, separated in a centrifuge and resuspended in 20 ml of 100 mM CaCl2. The suspension is cooled on ice for 30 minutes, centrifuged and again suspended in 4 ml of 100 ml of 100 mM CaCl2 and stored on ice overnight for use, Aliquots (0.5 ml) are stored frozen at -70 ° C.
3 ng of dCMP-extended DNA is mixed with 22 ng of dCMP-extended Pst I treated with pBR322 in 50 ml of THE buffer. Incubated at four successive stages at 65, 46, 37, and 20 ° C. 20 / sp of 100 mM Tris-HCl (pH 7.5), 100 mM CaCl2, 100 mM MgCl2, and 50 L of THE buffer are added, the mixture is cooled on ice for 20 minutes.
The recombinant RNA molecules are added to 100 / ip of the treated E. coli cells and the mixture is cooled on ice for 20 minutes, heated to 20 ° C for 10 minutes, and 0.6 ml of tryptone medium is added. The mixture is placed on 2 plates with tryptone agar medium prepared as previously described. The transfection efficiency is 3.3,104 colonies on / Ig hardened pBR322 transfected DNA, native pBR322 gives 3-10 colonies per fir.
Since the plasmid pBR322 includes the tetracycline resistance gene, E. coli recipient, which has been transformed with the plasmid, will grow in a culture containing such an antibiotic, in contrast to bacteria that have not been transformed. Therefore, growth in a tetracycline culture allows selection of host organisms transformed with a recombinant DNA molecule or recycled vector.
After 48 h at 37 ° C, individual colonies are picked at the peak and suspended in 100 ml of tryptone medium (prepared as described above) in the recesses of microtiter plates. After incubation at 37 ° C overnight, 100 ml of 40% glycerol are mixed in each well. Plates are kept at -20 ° C and a set of 100,000 individual transformed E. coli X1776 clones is prepared.
EXAMPLE 4 Recombinant DNA molecules are cleaved, denatured, and hybridized to poly (A) RNA leukocytes containing IFN-omRNA. Hybrids of recombinant DNA-poly (A) PH K molecules are separated from unhybridized poly (A) RNA (stage C). Poly (A) RNA is isolated from hybrids and purified (step D). The isolated RNA is harvested for the activity of IFN-omRNA (stage E). A mixture of the obtained recombinant DNA molecules contains a recombinant DNA molecule with a nucleotide sequence capable of hybridizing with poly (A) RNA under stringent hybridization conditions, and also causes the formation of oocytes. If a group of 512 clones gives a positive reaction, the clones are rearranged into 8 batches of 64 and each batch is tested. This process is continued until a single clone is identified. However, the obtained Recombinant clones do not contain the complete sequence of IFN-a DNA
Bacterial clones are inoculated onto tripton medium agar plates. After incubation at 37 ° C, each clone grows into a colony a few meters in diameter. All colonies were washed from the plates and harvested; an inoculum was obtained, used to inoculate 1 L of tryptone medium, which, as described above, filled 2 L of an Erlenmeyer flask. The culture is shaken at 37 ° C until the appearance of an ODBBO of approximately 0.8 (visually assessed). One volume of tryptone medium and chloramphenicol is added to the culture, which is shaken again at 37 ° C for 16 hours. 20 ml of chloroform is added
and culture shaken again 10 minutes at 37 ° C to remove bacteria. The culture is decanted with chloroform, the cells are harvested by centrifugation for 15 minutes at 6000 rpm at 4 ° C. Approximately 1 to 2 g of cells are obtained from each liter of the formulation. The cells are suspended in 30 ml of 20 mM Tris-HCl (pH 7.5), centrifuged for 20 min at 5000 rpm and 4 ° C, and resuspended in 30 ml of 50 mM Tris-HCl (pH 7.5). 0.25 volumes of lysozyme solution (10 mg / ml in 50 mM Tris-HCl (pH 7.5)) are added, and after cooling for 10 minutes at 0 ° C, 0.33 volumes are added (counting on the volume of the initial suspension 50 mM Tris-HC -cultures), 0.5 M EDUC (pH 8.0), gently mixed without shaking, After another 10 min at 1 ° C, 1/16 of the volume was added (again counting on the initial volume) 2% Triton X- 100. After 60 minutes, the sample is centrifuged for 60 minutes at 1000 rpm and 0 ° C in the rotor. The supernatant layer is transferred to a beaker with a magnetic stirrer and 3 M NaOH is added with stirring until a pH of 12.5 is reached, measured at 20 ° C using a glass electrode and a pH meter standardized by standard carbonate buffer. Beckmann pH 10 (No. 3505). After stirring for 10 minutes at 20 ° C, the pH is adjusted to 8.5. After another 3 minutes of stirring, 1/9 volume of 5 M NaCi and 1 volume of phenol (distilled and equilibrated with 9.5 M NaCI) are added and mixed vigorously for another 5 minutes. Separate the phases by centrifugation at 10,000 rpm at 0 ° C for 10 minutes. The supernatant layer containing form 1 DNA (circular double-stranded DNA) is carefully removed from the intermediate phase (which contains single-stranded DNA) and extracted 3 times with chloroform. (Phenol must be completely removed at this stage). Fraction 1 DNA contains recombinant DNA molecules (pBR 322-cDNA insert), originally used to transform these cells of the recipient organism, which form part of 512 clones chosen for the test.
Pancreatic RNase A (5 mg / ml), preheated for 10 minutes at 85 ° C) was added to Form 1 DNA to a concentration of 20 / g / ml and the mixture was incubated for 60 minutes at 37 ° C. 1/5 volume of 5 M NaCl is added and the mixture is treated with 30% polyethylene glycol 6000 to a final concentration of 7.5% PEG. After 2-16 hours at -10 ° C, the precipitate is collected in the rotor for 20 minutes at 8000 rpm and 0 ° C, dissolved in 0.075 M NaCl, 0.0075 M sodium citrate to an absorbance of 20 at 260 nm and brought to before
0.5% SDS. The solution is incubated for 30 min at 37 ° С with 0.5 mg / ml pronase (20 mg / ml, 2 h at 37 ° С) and extracted 3 times with 1 volume of distilled phenol and 2 times with 1 volume of chloroform. Centrifuge the sample (up to 2 ml of DNA solution per 1 mg / ml) with a sucrose gradient from 5 to 23% in 50 mM Tris-HCl (pH 7.5), 1 mM EDTA for 15 hours at 21,000 rpm and 15 ° WITH. Collect fractions and control OD260. The fractions containing the DNA are collected and the DNA is precipitated with sodium acetate and ethanol. Collect by centrifugation 20 - 100 / g of DNA mixture.
2Qjur DNA is treated in 10 mM
Tris-HCl (pH 7.5), 6 mM MgCl2, 50 mM NaCI, 6 mM 2-mercaptoethanol, 200 / g / ml BSA or gelatin, and 20 units of Hind III. The Hind III endonuclease cleaved the plasmid pBR 322. After 2 hours at 37 ° C, aliquots (1%) were subjected to electrophoresis on a 1% agarose gel in 50 mM tris-acetate (pH 7.8), 2 mM EDTA for 1 h at 50 mA to ensure that the restriction is complete. If the processing is not finished add another
Hind III and continue incubation for another 2 hours. When the DNA is completely cleaved, Pronaz, EDTA and LTO are added in the amount of 0.5 mg / ml, 10 mM and 0.5%, respectively. After 30 min at 37 ° C, the solution is extracted with 30 ml of phenol / chloroform (1.1). Washed with 50 / lp r-ra 20 mM Tris-HCl (pH 7.5), 1 mM EDTA, the combined phases are extracted 3 times with ether, filtered through a column of 0.1 ml, treated with EDTA, and
1/10 volume of sodium acetate and 2.5 volumes of ethanol are precipitated. After storage overnight at -20 ° C, DNA is harvested by centrifugation.
Prepare two hybridization mixtures.
Mixture I contains 4 / hl 10-fold concentrated buffer (4M NaCI, 0.1 PIPS (pH 6.4, 1, 4-piperazine diethanesulfonic acid), 50 mM EDTA, 0.5 / lp (about 5 ng 125 L-globin mRNA ( 5000 spm) and 6 ml induced
leukocyte poly (A) RNA (2, ug /// l). Mixture II contains IQjU-processed Hind III DNA and 0.1 ftr Pst-Z-pBR322 (H3) 4.13 DNA (pBR322 derivative, which contains the / 3-globin sequence at the Hind III site). Both mixtures are dried in gaseous nitrogen vapor. 40 ml of 80% formamide is added to the rest of mixture II and the solution is denatured for 10 minutes at 100 ° C and cooled quickly on ice. Denatured Solution
used to dissolve mixture I, and the resulting solution is incubated at 56 ° C for 4 hours.
After dilution to 1 ml with hoadlike 0.9 M NaCl, 0.09 M sodium citrate and 100% formamide to 4% (by volume), the solution is filtered at 0.5 ml / min through a Millipore filter (pore size 0.45 mm ), the filter is first tested for its ability to retain DNA / RNA hybrids, because not all filters obtained from the manufacturer are equally effective,
Immerse the above filter with poly (A) RNA hybrids in 1 ml of 0.15 M NaCI, 0.015 M sodium citrate, 0.5% SDS for 10 min at 37 ° C, washed with 50 mM Tris-HCl (pH 7, 5), 10 mM MgCl2, 2 mM CaCl2 and placed in 0.6 ml of fresh buffer. 5 / g l of DNase treated with iodoacetate (5 mg / ml) is added. The filter is incubated at 37 ° C for 10 minutes. Remove the filter and extract the solution with 1 volume of phenol and 1 volume of ether and pass through a column of 0.1 ml. Add RNA carrier solution (purified yeast RNA) and precipitate RNA with sodium acetate and ethanol. The precipitate is collected by centrifugation at 50 ° C, dissolved in 100 ml of 1 mM EDTA, heated for 90 s at 100 ° C. THE and SDS are added to 2 THE and 0.5% SDS. RNA is adsorbed on a column with 100 jur oligo dT / cellulose, eluted with four washes with 0.3 ml of distilled water and precipitated with sodium acetate and ethanol. After 16 hours at -20 ° C, the precipitated RNA is separated by centrifugation and dissolved in 2 ml of TNC buffer.
The poly (A) RNA solution obtained previously was injected into 40 oocytes (approximately 5 nl into the oocyte). Oocytes are incubated at 23 ° C for 24 to 48 hours, homogenized, and centrifuged (or the harvested medium is incubated) and tested as described previously for IFN-cg.
Most of the subsequent experiments were carried out with a recombinant DNA molecule from a single clone with DNA bound to a DVM or DRT paper. Paper DRT gives a smaller background.
Sheets of Whatman paper 540 (20 g) are stirred for 16 hours at 20 ° C with a mixture of 70 ml of 0.5 M NaOH, 2 mg / l of NaBH and 30 ml of 1,4-buty-di-di-glyndyl ether. Then the paper is transferred into a solution of 10 ml of 2-aminothiophenol in 40 ml of acetone and stirred for 10 hours. Washed with acetone, 0.1 H.HCI, H20, 0.1 H.HCI, HzO, dried. Paper APT diazotiruet to paper DRT.
DNA (up to 15 / IG) is bound to 50 mm2 of diazotized AVM (MDA) or diazotized APT (DPT) paper,
The hybrid DNA plasmid is hydrolyzed with Pstl, treated with 500 / Ig of pronase per ml of 0.5% SDS and 10 mM EDTA for 30 minutes at 37 ° C, extracted with phenol and ether, passed through a column with 0.1 ml and precipitated with ethanol. Denatured by heating DNA is incubated (up to 5 / g with small amounts of added P-DNA as a label) at 0 ° C overnight with 1 cm2
of paper DSM or DGT in 200, 25 mM potassium phosphate buffer (pH 6.5), the filters are washed 3 times for 5 minutes at room temperature with 50 mM potassium phosphate buffer (pH 6.5), 1% glycine and 3 times 99% to
recrystallized formamide. Next, they are incubated in 99% formamide for 2 minutes at 68 ° C, then washed 3 times in 50 mM potassium phosphate buffer (pH 6.5) at 20 ° C and washed twice in 0.4 M NaOH
at 37 ° C for 10 min. About 40 to 60% of the radioactivity remains on the filters. Filters are incubated for 3 hours at 38 ° C in pre-hybridized medium A, filled with 1% glycine, using 330 / hl
filter. Medium A contains 50% formamide, 5xSCK, 0.4% polyvinylpyrrolidone, 0.04% ficoll, 0.1% SDS, 25 / igpoli (A) (P and Z) and 100 / рож yeast RNA / VDN, extracted 6 times with phenol and precipitated with ethanol.
The filters are washed twice in medium A and then hybridized for 16 hours at 38 ° C (poly (A) RNA, as indicated, usually 5-8 mg) in medium A in paraffin oil. RNA is added as follows; one wet
The DNA filter is blotted and put into a sterile Petri dish, 20-40 / un of the RNA solution is pipetted onto this filter, and the second DNA filter (or duplicate or control) is placed on top and the sandwich is covered with sterile paraffin oil. After hybridization, the filters are washed successively in medium A (2 times) in a solution containing 1xSCK, 0.2% SDS, 1 mM EDTA (3 times, 10 min at 20 ° C each, medium A (2
h at 38 ° C) and in 50% formamide, 5xSCK, 0.1% SDS (3 times, 10 min at 20 ° C). Hybridized RNA is eluted by heating for 1 min at 100 ° C in 200 / yl 10 mM Tris-HCl (pH 7.4), 1 mM EDTA and 0.1% SDS.
Clone Ш-4-С, which contains a recombinant DNA molecule capable of hybridizing IFN-ai RNA, is selected.
The recombinant DNA molecule in this clone is designated: Z-pBR322 (PstllHclF4c / Hif-4c), and the bacterial strain containing it: E. coli XI776 (Z-pBR322) Pst HclF04C (E. coli Hif-4c).
Example 6: Since primary clones from transformed cells sometimes contain more than one type of recombinant DNA molecules, Hif-4c is isolated from E. coli XI776 (Hif-4C) clones and purified. The Hif-4C and pBR322 oerases hydrolyze with Pstl and are identified by electrophoresis on a 1% agarose gel.
E. coli HB101 transform Hif-4C DNA. Six clones of transformed tetracycline-resistant bacteria are selected, isolation of DNAs is obtained, purified by Pstl hydrolysis, by agarose gel electrophoresis, One DNA is designated Z-pBR322 / Pst (HclF-4c (Hif-4C) and used).
Hif-4C and the insert are hybridized with IFN-a and RNA, hydrolyzed with 125 units of Pstl, extracted with phenol and chloroform, and precipitated with ethanol. An aliquot (10 jur is dissolved in a 100 in 50 mM Tris-HCl (pH 7.5), passed through a 0.1 ml column and treated with 0.6 units of bacterial alkaline phosphatose for one hour at 65 ° C. 10 multiply concentrated THE buffer (40 ml) and the solution was extracted 3 times with 1 volume of phenol and 3 raes with 1 volume of chloroform.The DNA was precipitated with 2 volumes of ethanol at -20 ° C overnight and collected by centrifugation. For further purification, a sample of 0.5 ml of TNA are adsorbed on 0.25 ml of DEAE cellulose, which is prewashed with 2 ml of 150 mM Nad, 50 mM Tris-HCl (pH 7.5), 2 mM EDTA (NO-b ufer), washed with 2 ml of NO buffer, eluted with 0.4 ml of 1.5 M NaCI, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA and precipitated with ethanol. DNA is incubated with cy32 P-ATP (specific activity about 5000 Curie / mmol) and polynucleotide kinase, and purified by chromatography on a 3 ml Sephadex G-SOsTHE column. The eluted fractions were collected and precipitated with 32 P-DN K ethanol.
Mix 90, “g of unlabeled Pstl Hif-4c split DNA with 6 105 dpm 32P-labeled Pstl Hif-4c split DNA and conduct electrophoresis after 10x20x0.7 cm, 2% horizontal agarose gel in 50 mM Tris-acetate buffer (pH (7.8) using a 2.5 cm gel. The film on the gel is exposed to X rays and the position of the 320-bp fragment is determined. The gel strip containing the radioactive band (1.3-105 dpm) is cut out, crushed, pressed through a 2 ml plastic syringe, and extracted overnight at 4 ° C with stirring 10 times the volume of the buffer NO in relation to the volume of the gel. The DNA is adsorbed onto a 0.1 ml hydroxyapatite column previously washed with 1 ml of NO buffer. The column was washed with 1 ml of 0.1 M potassium phosphate buffer (pH 7.5) and DNA was eluted with 0.2 ml of 1 M potassium phosphate buffer (pH 7.5). The eluate is diluted with a 10-fold volume of sterile distilled water and DNA is adsorbed, and eluted from DEAE, precipitated with ethanol. This DNA is called the Hif-4c fragment.
A fragment of Hif-4c (120 ng) is bound to FFS paper (0.5 x 0.5 cm). For control, 120 ng of the yS-globin cDNA fragment is treated with Hind III from the hybrid plasmid Rc / 3G-4.13 Z-pBR322 and is treated similarly. Hybridization of double filters to the poly (A) RNA (B 20 µl), washing of the filters and isolation of the RNA from the filters are carried out as described above. After introduction into the oocytes, the following values of the activity of IFN-a are determined:
)
1 μg of this RNA gives 4600 IU / ml. The supernatant oocyte after 48 h of incubation, analysis to reduce cytopathic
effect
EXAMPLE 7 The insertion of cDNA in a recombinant Hif-4c molecule is only about 320 bp or one-third of the estimated size of the IFN-amRNA. The purified Hif-4c fragment described above is used as a probe for the selection of bacterial clones containing recombinant DNA molecules,
having related hybrid inserts
DNA
The 64 bacterial clones described above, constituting subgroup A-III, were crushed into a milliporous membrane (diameter 8 cm) and placed on an agar plate (with
the addition of diaminopimellinic acid, nalidixic acid and tetracycline, as
above) and incubated for 24 hours at 37 ° C. The filter is placed on a 0.75 ml drop of a 0.5 M solution of NO. After 2-3 minutes, it is transferred onto a paper towel to remove excess liquid, this step is repeated. The filter is neutralized using a 1.5 M Tris-HCl buffer (pH 7.5) and washed with a mixture of 1.5 M NaCi-0.5 M Tris-HCl (pH 7.4) and air dried. The filter is dipped in a 0.3 M solution of NaCI, dried in air and heated under vacuum for 2 hours at 80 ° C.
The Hif-4c Pst fragment (30 ng) is labeled with the 32P isotope by translating the label using / Pp and a 32P o-CTF (specific activity, 40 Curie / mmol for each). The filter carrying A-III colonies is prehybridized in 4 x CET (CET is 0.15 M NaCI, 30 mM Tris-HCl (pH - 8), 1 mM EDTA). 0.1% (w / v) ficoll, 0.1% polyvinylpyrrolidine, 0.1% (w / v) albumin of serum (ABS) 0.5% DSP and 200 μg / ml of denatured, fragmented salmon sperm DNA within 7 hours at 68 ° C and hybridized with 2.105 cpm of the 32P-labeled Hif-4c fragment in 4 x CET, 0.02% (w / v) Ficoll, 0.02% polyvinylpyrrolidine, 0.02% (ABS, 0.5% SDS and 2300 µg / ml denatured sperm DNA elsol at 68 ° C for 16 hours). The filter is washed with a mixture of CET-0.5% SDS at room temperature, washed with 2 x CET-0.5% SDS for 5 hours at 68 ° C, replacing the solution once, and 3 mM Trism base at room temperature for 4 hours , replace the solution once. After the filter has dried, the x-ray film is exposed to this filter for 80 hours using a screen. Three colonies give a strong positive signal, namely A-III-7D A-III-2H and A-1I-4c, and two colonies give a weak signal, namely A-III-IE and IJ-3-ZO. z
Small cultures are prepared from related Hif-4c clones, DNA Form 1 is purified, digested with Pstl and analyzed by agarose gel electrophoresis. All forms of DNA 1 create a large fragment (plasmid pBR 322 functional group) and a small fragment (hybrid insert). The recombinant DNA molecules A-III-2H contain the largest insert, and approximately 900 billion h. This recombinant DNA molecule is designated Z-pBR 322 (Pst) / HCIF-2H / (Hif-2H), and its insert is a Hif-2H fragment.
The ability of Hif-2H to bind IFN-amRNA is determined by hybridization to poly (A) RNA (0.3 µg / µl).
In the subsequent experiment, an additional set of E. coli clones containing recombinant DNA molecules was prepared, and hybridization of the colonies to the labeled Hif-4c fragment was determined. In order to ensure a high yield of plasmids with long cDNA inserts, part of the P labeled leukocyte cDNA, obtained enzymatically from leukocyte poly (A) RNA, is fractionated by centrifugation through a sucrose density gradient, using a technique similar to that described for poly centrifuging ( A) RNA. Fractions containing cDNA with sedimentation rate corresponding to 600 billion h. A DNA fragment or more is enclosed and, after precipitation with ethyl alcohol, the cDNA is isolated. This cDNA is extended with dCMP residues. The dBMP-extended pBR322 was digested with Pstl and the hybrid DNA was used to transform E. coli strain HB101. Bacteria are applied to 8 mm diameter microporous filters, placed on tripton medium agar plates (containing 10 µg / ml tetracycline) and grown until small colonies appear. The filter of the replica is prepared by pressing a fresh, wet millipore filter onto the filter containing the colony, put it face up on an agar plate containing 4.4% i lysterine, and incubate it until small colonies appear. This colony-containing filter is covered with an additional million-fold filter, frozen at -55 ° C and stored. 18 filters are prepared containing a total of about 5,000 colonies. One replica of each filter was used to hybridize to a labeled P Pst Hit-4c DNA fragment. On the autoradiogram, about 185 positive colonies are identified, which are subcloned on the milliporous filters and re-identified by hybridization. The 95 clones giving the strongest hybridization response are designated from Z-pBR 322 (Pst) / HclF-SNI to SN95 and are used for further studies.
Obviously, with the ability of Hif-2 to produce a polypeptide that exhibits immunological or biological activity of CeIFN, Hif-2h, and other related DNA base sequences, can be used in this method of selecting clones containing interferon ' 2 DNA encoding sequences.
PRI me R 8. The cleaved fragment of Z pBR322 (H3) 4.13 is disunited in a Yumkl mixture of 80% (vol.) Deionized formide — 20 mM Paypes buffer (pH 6.4) for 10 min at 80 ° C, The solution is added to an Eppendorf tube in which a mixture of leukocyte poly (A) RNA (5 µg), NaCI (4 µmol) and EDTA (10 nmol) is dried. The mixture is heated for 7 hours at 48 ° C under a layer of paraffin oil, cooled and diluted with 20 µl of water. Two samples are divided into equal parts and each of them is heated for 30 s at 100 ° C. Nucleic acids are precipitated with ethanol, dissolved in 3 µl of water and analyzed for the activity of IFN-a mRNA in oocytes.
When Hif-2h hybridizes poly (A) RNA, inhibition of the translation of IFN-a mRNA into poly (A) RNA is inhibited; after denaturing the hybrid, IFN a. mRNA can be translated again. This experiment confirms that Hif-2h contains a sequence complementary to IFN-amRNA.
Plasmid DNA is obtained and treated with various restrictases, except that bovine serum albumin in the enzyme buffer is replaced with 200 µg-ml gelatin.
Leanurized DNA (20 µg) is extracted with phenol, precipitated with ethyl alcohol, dissolved in a 0.05 M solution of Tris-HCl buffer (pH 8) and passed through a small Chelex-100 column. Fragments with 5-sticky ends are dephosphated by treatment with 0.2 units of calf intestinal alkaline phosphatose on pmol ends of 5 DNA in 200 μl of a 0.02 M Tris-HC2 solution (pH 8) for 60 minutes at 37 ° C. The enzyme is deactivated by heating at 65 ° C for 60 minutes. Bacterial alkaline phosphatase is used for DNA fragments with 3-fold ends, with the exception that phosphorylated DNA is incubated at 65 ° C for 30 minutes by adsorption and elution with DEAE-cellulose or subjected to polyacrylamide gel electrophoresis. The fragments isolated from the polyacrylamide gel (or ehgaroz) in a 0.15 M NaCI solution, 0.05 M-Tris-HCl (pH 8), are adsorbed on a 0.1-ml oxypatite column, washed 4 times with 1 ml of 0.1 M potassium phosphate buffer (pH 7) and eluted with 0.3 ml of 1M potassium phosphate buffer (pH 7). The solution was diluted ten-fold, and the DNA adsorbed on DEAE-cellulose was isolated.
After precipitation with ethanol
DNA is labeled 5-terminally with
(12-34 microns for pmol 5 -ends
DNA), and polynucleotide kinase DNA is not
denature before the kinase reaction. Get
by
specific activities of 1-1.5 μCiR of phosphate per pmol of 5-DNA ends.
To determine the amino acid sequence, the labeled fragments are digested with a second limiting enzyme, and the products are separated by electrophoresis through a 5% polyacrylamide gel into Tris-Borate-EDTA buffer. The target fragments are extracted from the gel and purified. Various fragments for determining the sequence of amino acid residues are prepared as follows:
1) splitting Hif-2h with BSpl, isolating BSpl-BSpl-232 and BSpl-BSpl-949 by gel electrophoresis through 5% polyacrylamide in Doening buffer;
2) splitting Hif-2h with BSpl, labeling, splitting with Pstl, isolation of Bspl-Pst1-83 and 1-227;
3) Cleavage of Hif-2h using VdSh, labeling, cleavage with Psti, isolation of Bglllx-Pst 1-336 and Bgli-Pst1-570;
4) cleavage of Hif-2h using M1011, labeling, hydrolysis with Pst1 and Hindll, splitting of the pBR322 fragment, 350 billion hours, isolation of Mbo11 -Pst 1-519 and Mbo11 -Pst 1-351;
5) Hif-2h cleavage with EcoR1, labeling, cleavage with Pst1, isolation of EcoR1-Pst 1-707 and EcoRI Pstl-198;
6) cleavage of Hif-2h with Pst1, labeling, cleavage with Bd | D. isolating Pst1 -BglO-570 and Pst1 -BgHl-336;
7) splitting Hif-2h with Avail, labeling, splitting with Pst1 and Bglff, highlighting Avall-Pst 1-186 and Avall-BglI: 147.
8) Hif-2h cleavage with Pvull, labeling, cleavage with Pst1 and Bglfl, isolation of Pvu l-Pstp-486.
The products are fractionated into 0.1x25x36 cm 12% polyacrylamide gels (acrylamide / bisacrylamide 18/1) in 50 mM tris-borate, 1 mM EDTA (pH 8.3) with a duration of operations 2, 8, 18 and 26 hours at 90 in with a preliminary exposure of 6 h at 700 in. The best results are obtained when the gels are kept at room temperature for 2-3 days prior to use.
The nucleotide sequence to the DNA insert is determined.
Moreover, the heteropolymer part of the insert is sideways with 23G residues at the 5th end and 7A residues (probably related to the terminal group of poly (A) mRNA), followed by 15C residues at the ends-3. The insert is numbered from the first nucleotide after the dG-tail to the last nucleotide to the poly (A) residues.
It is determined that the ATC located in nucleotides 57-59 is actually the first ATC, an authentic mRNA.
The codon in the Hif-2h fragment, corresponding to the first amino acid from lymphoblastoid interferon, is 22 code on from the first AuG (and 14 codons from the second), which indicates that the sequence coding for interferon may be preceded by a detecting signal peptide consisting of 23 (or, less likely, 15) amino acids. The longer of the putative signal sequences contain a continuous series of 11 hydrophobic amino acids (and shorter ones of 6 hydrophobic amino acids).
The nucleotide sequence corresponding to the matured IFN-a polypeptide appears to include 498 nucleotides encoding 166 amino acids. The basic composition of the coding sequence corresponds to 50% GC.
A DNA chain having the same sequence as m-RNA is designated as the plus chain, and its complement as the minus chain. Hif-2h DNAs are cleaved with the Bglli restriction enzyme, the end group is labeled with P-phosphate and the DNA is digested with Pst1, resulting in a longer fragment 545 bp, / 570 bp. and a shorter 340 bp radioactive fragment. These fragments are denatured and converted into poly (A) RNA from induced leukocytes in 80% formamide, 0.4 M NaCI, i.e. under conditions in which secondary association of DNA-DNA does not occur (see above). The nucleic acids are cleaved by the nucleotide SI, which destroys all single-stranded nucleic acids, especially non-hybridized 32 P-DNA, and the products are separated on a polyarylamide gel.
The same experiment was carried out as described in the previous section, but using poly (A) RNA from uninduced human leukocytes obtained by the same method as the Sendai leukocyte induced by the virus. Compared to the results of the previous section, poly (A) RNA from uninduced cells contains 1/20 of the amount of hybridization capable of Hif-2h m-RNA, containing in poly (A) RNA from induced cells.
The pstl site in the pBR322 is in a / 3-lactamase (penicillinase) gel. Consequently, if the coding DNA segment (for example, c-DNA, including the entire gene or part of a gene) is located in a position with the appropriate orientation, then the result is sticky (fused)
protein. The protein consists of the amino terminal portion of the / J-lactamase and the subsequent amino acid sequence that encodes the target protein. To ensure that the Hif-4c is inserted in the correct reading frame for the expression of the / 3-lactamazone gene, a set of pBR322 derivatives, namely pKT279, pKT280 and pKT287, is used. Each of these derivatives has Pst1, which is localized in such a way that the heterogeneous DNA embedded in this site will be in the correct reading frame. A Pstl-withdrawn insert from Hif-2h is also prepared as described for the Hif-4c fragment, the Hif-2hPst fragment (10 ng) is mixed with the Pstl-digested pBR322, pKT279, pCT280 or pCT287 in each case 10 ng in 20 microliters of 10 mM Tris-HC (pH 7.5), 6 mM MgCte, 100 mM NaCI, 6 mM / -mercaptoethanol, 200 micrograms / ml gelatin and 0.1 mM ATP and incubated with 0.1 unit of T4 DNA ligase for 16h at 10 ° C. The resulting recombinant DNA molecules are designated as Z-pBR322 (Pst) (HclF-2h Z-pKT279 (Pst) (HclF-2hZ-pKT280 (Pst) HclF-2h and Z-pKT287 (Pst) (HclF-2h), E. Coli HB101 transformed with each of these recombinant DNA molecules by and transformed colonies were selected on agar plates containing tetracycline Such tetracycline resistant clones of transformed bacteria contain the recycled vector, bacterial colonies of each set were grown on Millipore filters, and colonies hybridized 23P-labeled Hif-4c fragment were identified and selected as described above The strains are described as follows:
E. coli HB101 (Z-pBR322 / Pst (HclF-2h-AN1) to (-ANZ);
E. coli HB101 (Z-pBR322 (Pst) / HclF-2h-AN1) to (-ANZ);
E. coli HB101 (Z-pKT279 (Pst) / HclF-2h-AN1) to (-AN8);
E. coli HB101 (Z-pKT280 (Pst) / HclF-2h-AN1) to (-AN8);
E. coli HB101 (Z-pKT287) (Pst) / HclF-2h-AN1) to (-AN8).
Extracts of some of the above strains, as well as some of the strains of Z-PBR 322 / Pst / (HclF-SN1 95), are tested for IFN activity - “Bacteria are grown on tripton seed to the stationary phase, collected, nicknamed 1/20 of the volume (relative to culture volume) 50 mM Tris-HCl (pH 8), 30 mM · S and frozen. After thawing, the cells are suspended again in the following volume of the above buffer and
lysozyme up to 1 mg / ml. After 60 minutes and at 0 ° C, the suspensions are frozen (ethanol-dry ice in a bath) and thawed (at 37 ° C) 5 times and centrifuged for 10 minutes at 12000 rpm in a centrifuge. In some cases, a portion of the supernatant is further centrifuged by a centrifuge at an AORO and the supernatants are separated. Such supernatants are tested for IFN-o activity: by detecting a decrease in the cytopathic effect. Colonies giving a positive reaction in Example 1 are redefined along with 49
The resulting proteins have immunological or biological activity of IFN.
The Hif-SW35 plasmid is treated with the Pst1, the LAC-Alu fragment is inserted into the plasmid. The structure of the modified plasmid Z-pBR322 (Pst) (HclF-SN35-AH16) is determined, as well as the amino acid sequence with the terminal amine for the protein produced in E. coli. The nucleotide design corresponds to the sequence in which the LAC-Alu fragment is attached to the first amino acid from INF-a 1 (SN 35), the amino acid sequence of the amino terminal portion of the protein obtained in E. coli confirms that a sticky protein is obtained that has six amino acids embedded in the sequence of IFH-o: l (SN 35). However, a strain transformed with a modified plasmid produces approximately 100 times more of a polypeptide possessing the biological and immunological properties of human leukocyte interferon as compared to a host organism transformed with unmodified Z-pBR322 (Pst) (HclF-SN35).
The hybrid plasmid was cleaved with BSpI. After thermal inactivation (65 ° C, 30 min), the enzyme mixture is adjusted to a concentration of 50 mM Tris-HCl (pH 8) and heated (37 ° C, 30 min). After extraction with phenol and ether, the largest fragment 1 cD K is isolated at a low temperature on gelatinized agarose (0.8%) and Hind III binders are added. Then
0
clones from cepnMZ-pBR322 (HclF-SN-1 to SN-95.
Some of the most active manufacturers listed are checked more thoroughly. Cultures are grown to the late logarithmic phase (the observed ODBBO is approximately 0.9) and the cells are lysed in 1/50 of the culture volume. The following activities are detected using Z-pBR322 (Pst) (HclFSN32) as a negative control.
five
0
five
0
five
0
five
the modified fragment is added to the Hind III cleaved plasmid HS-pBR322 / EcoRI (a acuv 5-150 / LAC-150) by separating out gel particles from the fragment containing particles (each approximately 20 microliters) at 65 ° C, cooling to 37 ° C and by adding 20 units / ml of T4 DNA ligase. After 16 h at 15 ° C, a binding occurs in the solidified gel. One tenth of a volume of 100 mM Tris-HCl (pH 7.5), 100 mM CaCh, 100 mM MgCl2 was added to the sample, heated for 5 minutes at 65 ° C and cooled to 37 ° C. The samples are then used to transform the treated Ca. E. coliHB101, incubated at 0 ° C for 20 minutes, heated at 42 ° C for 1 minute and for 10 minutes at 20 ° C. After the addition of 1 M tripton medium, the samples are incubated for 60 min at 37 ° C and placed on agar plates containing ampicillin. Then, the plasmid DNA and the hybrid plasmid containing IFN-1 with its LAC-fragment attached at the S-terminus are separated from these cultures, identified by restrictive analysis. The plasmid was then digested with EcoRi and digested with BaL-31 exonuclease (0.06 U / ml, 2–4 min at 30 ° C) to remove the EcoRI protruding end of the LAC fragment and shorten the D-galactisidase coding segment.
The plasmid was digested with Bd | P, the largest fragment was separated on an agarose gel (0.8%). This fragment is then combined with a fragment of BSpl-Bgl {from Z-pBR322 / Pst / / HclF-SN35 and the resulting hybrid
plasmid. used to transform E. coli HB101. Transformed colonies are subjected to screening tests for IFN activity and one clone is selected that has high IFN activity. This clone is designated E.coli HB101 (C8-IFN-a 1), and its hybrid plasmid C8-IFN-a1.
Analysis of the C8-IFN-a1 DNA sequence shows that the coding sequence following the initiating triplet is determined by the first seven amino acids / 3-gadlacosidase; the Pro residue is formed by fusing amino acids from 16 to 23 in the signal sequence of the IFN-c1 sequence of the IFN- tzl (sn 35). The E.coli mini-cell strains (DS 410) transformed with the C8-IFN-a1 hybrid plasmid produce approximately 50 million units of IFN per liter, or approximately 2500 times more polypeptide, which has immunological or biological activity of CheFNP compared to the transformed Therefore, IFN-ct extracts are sensitive to trypsin and, therefore, to rotein.
Hif-35 S100 Extract
(50 units)
ABOUT
0.1
one
ten
50
thirty
20
20
2
ABOUT
Hif-35 extract (1 ml) and E. coli HB101 extract S100 (Z-pBR322 (Pst) (HclF-SN32) (Hif-32 extracts) are chromatographed on a 32 ml Sephadex G-100 column at 4 ° C in 50 millimolar K -phosphate buffer (pH 7.4). The flow rate is 2 ml / h and fractions of 1.0 ml are collected. Absorbance at 280 nanometers, 405 nanometers (cytochrome c) and IFN-a activity is determined.
0
Z-pBR322 (Pst) (Hif SN 35). The amino acid sequence of the polypeptide obtained with the plasmid C8-IFN-a1 confirms that the product is a sticky protein having seven amino acids from / 3-galactosidase, one acid, resulting from the fusion of amino acid 16-23 from the IFN signal sequence -a1-entering VIFN-a1.
EXAMPLE 9: 50 microliters of samples of authentic CheIFN-c (specific activity 1.2x106U (mg, 50U), extracts of S100, described above E. coli HB101 (Z-5 pKT287 (Pst) / HclF-2h ANS ( H t-287-6 extracts) (200i / ml, 10U) and E. coli HB101 (Z-pBR322 (Pst) / HclF-SN 35) (Hif-35 extracts) (100011 / ml, 50U) are incubated with a different amount of trypsin, as shown below, for 30 minutes at 37 ° C, Since extracts of S100 have a high content of protein, and CheIFN-anet, the mixture of CheIFN-a and control extract S100Hif-32 is tested in parallel:
0
IFN-a and the activity of Hif-35 extracts are eluted before cytochrome with a cd value of 0.45. Consequently, the apparent molecular weight of the substance is between 20,000 and 30,000, in fractions of the Hif-32 control extract.
no activity was detected.
CheIFN-a (specific activity 1.2x106 lu / mg) and Hif-35-Hif-287-6 S100 extracts are incubated with various diluted solutions of sheep antiserum against CheIFN-a (specific activity 450000 units / ml) in 100 microliters of modified Eagles medium (MCE) with 10% calf serum for 30 min
at 37 ° C and 45 microlites are analyzed for IFN activity by reduction analysis of the cytopathic effect.
Antibodies directed against Che-IFN-a specifically inhibit IFN-activity of polypeptides obtained in E. coli by transformation with some recombinant DNA molecules containing the HclF-2h DNA sequence. The reduced effect of the antibody on IFN-a, obtained in E. coli, may reflect the structural differences between the latter and the natural CheIFN-cr, for example,
These results show that Hif-35 and Hif-287-B extracts exert a protective effect on human cells and on mouse cells, showing a weak effect (10%), as is typical for human interferon.
Extracts of E. coli HB101 (Z-pBR322 (Pst) / Hif-SN 3-AH 6 are compared with authentic IFN for its effect on certain cell functions. IFN-α obtained from E. coli exhibit the following properties of natural IFN-α a: (1) it increases the killing activity of human lymphocytes, (2) it enhances the cytotoxicity of the antiel-dependent intermediate cell, (3) it inhibits the inhibition of antigen and mitogen-induced migration of leukocytes, and (4) it shows an increase in IFN-sensitivity of cells. These properties are induced actively0
the absence of a hydrocarbon fragment, the presence of a signal sequence, or fusion with a part of the / 3-lactamase sequence.
CCL23 human cells or L929 mouse cells are treated with E. coli extracts from Che-IFN-a (specific activity 1.2 x U6 units / mg) or mouse IF, infected with a virus and IFN-a activity is determined by analyzing the cytopathic effect.
stv IFN-orls synthesized E. coli against human tumors and cancer.
PRI me R 10. IFN-a without amino-terminal sequences is obtained in E. coli
and confirm that he is active in accordance with the activity of IFN.
In order to obtain the corresponding recombinant DNA molecule, the plasmid Hif-2h is hydrolyzed with EcoRI and BamHI and
the fragment containing the IFN-da coding sequence is separated on an agrosous gel and merged with the sequence coding for non-IFN-a — EcoRI / BamHI fragment of the Hif-SN plasmid
35, in which there is no Pst1 site adjacent to the Z1 end of the hybrid insert compared to Hif-2h. The resulting plasmid is treated with the Pstl / Bgll restriction enzyme of the amino terminal fragment of the IFN-a1 coding sequence. A number of IFN-a1 fragments obtained by hydrolysis of the plasmid Hif-2h with Pvu II, treatment of Ba1 exonuclease, addition of Pst 1 linkers, followed by treatment with Bgl II are inserted in its place. The plasmids thus obtained contain sequences encoding IFN-a I, in which different parts of their amino terminal sequences are missing. Plasmid 2H-M8 is digested with Pstl and its nucleotide sequence is determined. Plasmid 2H-M8 contains several nucleotides between the Pst site and the first codon of IFN-a I. Plasmid 2H-M8, hydrolyzed with Pstl, is treated with polynucleotide kinase / dATP, SI exonuclease and Sal 1. These fragments are placed under the control of the LAC by attaching them to the LAC fragment , obtained from the plasmid Lac by treating EcoRI with endonuclease S1 and Sail. The coding sequences 1/1 of the FN- “1 series of plasmids thus obtained lack the different amino-terminus fragments, which are connected in a counterclockwise direction by AUG with a fragment containing the LAC promoter.
In E. coli cells (DS 410), both of these plasmids express polypeptides that exhibit IFN activity.
Clones that show poor hybridization to the Hif-4c fragment are checked and the E. aoli clone HB101 (Z-pBR322 (Pst) / HclF-11-206) is identified.
The hybrid plasmid Z-pBR322 (Pst) HclF-11-206 (HCIF-11-206) from the clone and its insert Hif-11-206 poorly hybridized to the Hif-4c and fragment. Transformation of E. coli cells with the Hif-II-206 plasmid provides polypeptides exhibiting the biological or immunological activity of CheIFN-a.
The nucleotide sequence of the Hif-11-20 fragment (Pstl) of a plasmid fragment of DNA of 790 bp, isolated from a culture of HclF-Y) is determined using well-known methods.
Hydrolyzed DNA (usually 10 micrograms) is labeled in 5. The labeled fragments are separated by a second restriction enzyme, the products are separated by electrophoresis through a 5% polyacrylamide gel in Tris-borate-EDTA buffer, extracted from the gel and purified. Receive the following various fragments:
25 and 26 - cleavage of Hif-11-206 with Pvu II, labeling, cleavage with Bg III, isolation of the Pstl-Bglfl fragment (257 bp) (25) and Pstl Bglll fragment (279 bp) (26).
21, 22 and 23 - Hif-11-206 cleavage with Pvull, tagging, splitting with Bglll, selection of Pvull -Bglll fragment, (88 BP) (21) Pvull -Bglll fragment (176 BP) (22) and III fragment (214 BP) (23).
11,12,13 and 14 - cleavage of the Hif-11-206 using the HVIII, tagging, cleavage with the help of Pstl, isolation of the Bglll-Pstl fragment (279 bp) (14) and the comma blend of the Bglll-Pstl fragment and the Bglll -Bglll fragment. Splitting the mixture with Pvu II and isolating the Bglll-Pst fragment (257 bp) (13), the Bglll-Pvull fragment (176 bp) (12) and the Bglll-Pvul fragment (88 bp) (11).
27L, 27U, 41, 43, 44 and 45 - Hif-11-206 cleavage with Hinfl, labeling of initial fragments: Hinfl-Hinfl (113 bp) (27), Hinfl-Hlnfl (146 bp) (28): Hlnfl - Hinfl (159вр) (ЗОР). Hinfl-Hinl (379Bp) (31P) n Hinfl-Hinfl (1522 BP) (32P). Cleavage of 28P with Mvo11 and isolation of the Hinfl fragment of Mvo11 (112 bp) (41). Splitting of the ZOR using Mvo11 and the selection of the fragment Hinfl -MBoll (125 BP) (43). Cleavage of 31P with Pstl and isolation of a fragment of Hinfl-Pstl, (151 bp) (44). 32P cleavage with Pstl and isolation of a fragment of Hinfl-Pstl (139 bp) (45). Separation of gravity 27P with the production of two tay (27u 27L).
From the comparison of the amino acid sequence of the inserts, it can be seen that the Hif-11-206 fragment encodes an interferon-like protein having one amino acid less than the Hif-2h fragment (amino acid 44 (ASp) present in Hif-2h, is not contained in Hif-11-206 ). In addition, 10% of nucleotide sequences and 17% of residues of amino acid derivatives of two fragments are different. A comparison of 35 amino-terminal amino acids with lymphoblastoid interferon shows that the Hif-11-206 insert encodes a protein that is distinguished by 5 residues. Therefore, there must be at least three different IFN gene of the leukocyte type (alpha ™ na) -Hif-2h fragment.
E. coli HB101 cells containing the hybrid plasmid are grown on a triptone with shaking until an ADOB is 1 to 2 times. The cells are harvested, weighed, resuspended in a ratio of 1/100 or 1/20 of the initial volume of the culture and lysed. S-30 supernatants were tested by CPE reduction analysis in microtiter plates. Control ji3 extracts were negative
(1 I.U./ml). Human CCL 23 cells and bovine fetal kidney (BEP) cells (FLOW) are grown in MEM-10% of fetal calf serum. The cells are infected with an appropriate diluted solution of Mengo virus after 24 hours. The amount of protein is assessed visually relative to the partially purified IFN leukocyte (PIF preparation) of a known titer. This drug was three times more active in human cells than in bull cells.
Therefore, IFN-cd is about 30 times less active on human cells than IFN-o2, They both show the same activity on bovine cells. Thus, IFN can be, in addition to their use as antiviral and antitumor or anticancer agents in humans, used to treat these diseases in cattle. For example, CheIFN-a preparations are used in the treatment of FMDV and other well-being. known viral infections of cattle. This is even more acceptable for IFN-a.
1, since its activity on bovine cells is approximately 20 times greater than the activity
on human cells.
Improved yield is achieved in the case of IFN-cP with the C8 design (indicated above), the same design is obtained for IFN-a
2, Z-pBR322 (Pst) / Hif-11-206 was digested completely with Bspl and partially with Pvull and the 867 bp fragment was isolated from 6% polyarylamide gel. This fragment is then ligated to 2590 BP Pvull fragment C8-IFN-a1. The resulting hybrid plasmid is transformed with E. coli HB101 and the clones are screened for IFN activity. One high activity clone is selected
and administered in E. coli HB (C8-IFN-O2).
The DNA sequence of the hybrid plasmid C8-IFN-a2 is treated in such a way that, like C8-IFN-a, it is named after the initiator triplet, which was first determined by the seven amino acids of yS-galactosidase, Pro residue and 16-23 IFN- a2 signal sequence. Cells transformed with this plasmid give 100–200 million units per liter of IFN and the yield of IFN-a2 was 20,000–40,000 times higher than when cells transformed in unmodified Z-pBR322 (Pst) / Hif-11-206 were used.
A comparison of the yields of C8-IFN-a1 (50x 106 units / l) and C8-IFN-02 (100–200 million units / liter) at first glance is unexpected, since IFN-a 2 is about 30 times more active, than IFNsP on human cells (shown above). However, a comparison of the amount of 2 proteins obtained by culturing cells transformed with two C8 plasmids shows that the C8-IFN-a1 protein is 5-6 times more than the C8-IFN- 2.
A plasmid containing the LACAlu fragment is obtained by treating the known Lac promoter with A1i1 and the extension of the fragment with EcoRI linkers. The resulting fragment is inserted into BR322 at the EcoRI site and a small fragment of EcoRI-EcoRI is removed from the construct. The resulting plasmid is designated 404, digested with Hindlll and Pvull to insert an IFN-2 containing fragment. This fragment of IFN-a2 is obtained by partial hydrolysis of Z-pBR322 (Pst) / HclF-11-206 (206), extension of the Sau3A fragment using a Hindlll linker and cleavage with Bsp1. After the Hindlll-Pvull fragment of plasmid 404 is stitched, the resulting plasmid is hydrolyzed with Hindlll and RcoRI, treated with S1 nuclease to approximate the LAC promoter to gene 2 and are ligated again. This construction identified as the plasmid LAC-AU ("2) has an IFN-o2 DNA sequence under the control of the LAC promoter. In addition, the IFN- "2 sequence is located immediately after the initiating AUG codon of this promoter. Consequently, at least part of the IFN obtained using these plasmids will be the apparent IFN, for example, IFN without any amino acid signal sequence. B. Miniclet yield of IFN-a2 obtained with the plasmid LAC-AU (a 2) was 5-10 million units per liter.
The genetic marker from pBR322 is linked to the AUG and IFN- 2 genome from Hif-11-206. This construct is obtained by hydrolyzing pBR322 with Mvo11, processing S1, attaching the linker as previously, and re-recloning the fragment to the EcoRI site of the pBR322 and removing the EcoRI site. The resulting plasmid ((3-Lac-AUG plasmid)) is linked to the Hindlll linker-Bspl fragment 206, processed with S1. After digestion with EcoRI and treatment with S1 and phosphatase, the expression plasmid yS-Lac-AUG (a 2) is isolated. Constructs with other genes are obtained by similar fusion using the UZ-Lac-AUG plasmid for cloning other genes or constructs using the Sau HA website in plasmid / 3-Lac-AUG (a2).
This plasmid, designated as D-Lac-AUG (a2), upon transformation of the recipient cells, allows to obtain IFN-a 2 without fusion with other protein sequences. In the cells, yields of 50-100 million units per liter are observed. It is preferable to use the strain E. coli DS410.
Example 11: A series of hybrid molecules, IFN-a 1 and IFN-2, are designed to find that these hybrid constructs differ quantitatively with different properties and activities compared to each of the related IFN-a 1 or IFN-a. 2
Plasmids I, II, III and IV in these constructs are digested with Bspi restrictases. Partial DNA cleavage is carried out with reduced amounts of the enzyme. After thermal inactivation (65 ° C, 30 min) of the restriction, the samples are adjusted to 50 mM Tris-HCl (pH 8) and, if necessary, alkaline phosphatase of the calf intestine (1 volume per µgDNA) is added. After 30 min at 37 ° C Samples extracted with phenol and simple ether. In most cases, DNA fragments were isolated on low temperature gelling agarose (0.8%). To bind, jelly pieces containing the fragment (about 20 µl each) are melted at a temperature of about 65 ° C, cooled to 37 ° C, and 20 U per µl of T4 DNA ligase is added. The resulting mixture was kept at 15 ° C for 16 hours and binding was obtained in a solidified gel. Then one tenth of a volume of 100 mMTris-HC (pH 7.5), 100 mM, 100 mM MgCIa is added, the sample is heated for 5 minutes at 65 ° C and cooled to 37 ° C. Then the samples are added to the treated Ca cells, incubated at 0 ° C for 20 minutes, heated for 1 minute at 42 ° C and 10 minutes at 20 ° C, and 1 ml of tryptone medium was added. After incubation for 60 minutes at 37 ° C, the cultures are placed on agar plates containing the appropriate antibiotics.
Hybrid molecule I, a-1 / Pvull / "-2 hybrid, was constructed by partial cleavage of the C8-IFN-a1 (C8-a1) Pvull, deforporylation, hydrolysis of Pstl, and the Pstl-Pvull fragment (1346 bp) was isolated. This fragment is associated with 2135 BP Pstl
(a) -Pvull (P2) fragment obtained as a result of cleavage of C8-IFN-a 2 (Supra) (C8-a2) Pvull and treatment with Pstl.
Hybrid molecule II a-1 (Bgl) a-2 design, split hybrid molecule
Jbglll After dephosphorylation, a large fragment of the VSD is isolated and linked to a small fragment of C8-IFN-a 2. After cloning, the hybrid plasmid containing the small Bd1 U fragment in the correct orientation is identified by restriction analysis.
Hybrid molecule A-2 / Pvull / a-1 design processing C8-IFN- "1 Pvull,
by dephosphorylation, splitting Aval with the release of 1686 BP Pvull (P2) Aval and 3233 BP Pvull (Pi) -Aval fragments. These fragments bind to a 300 bp Pvull (P1) -Pvull (P2) fragment of HclF-11-206 and a plasmid containing
A small fragment of Pvull is identified by testing transformed E. coli strains for IFN-a activity.
The a-2 / BgllJ / a -1 hybrid IV molecule is constructed by hydrolyzing C8IFN-a1 BglLr and Aval m to isolate a fragment of 1776 bp. This fragment is linked to fragment 3543 bp of BgllT-Aval hybrid molecule III.
Cell cultures (DS410) transformed with different plasmids are grown and the bacteria collected by centrifugation are washed with PBS, suspended in PBS (about 1/20 of the original volume), incubated for 60 min at 0 ° C with 1 mg / ml lysozyme , 10 mM EDTA, freeze and thaw four times, mow, skip five times through a syringe and split by centrifugation.
The activity of human, mouse, guinea pig and bull cells was as follows:
It was said that all interferons have approximately the same activity with respect to bovine cells, but IFN-a1 and two hybrid IFN1 a (I and II), which have two final amino fragments of IFN-a1, have from about 10 to 1000 times less activity against human cells, rather than IFN-a 2 and two hybrid IFN1 a (III and IV) containing terminal amino fragments of IFN-a 2. Two hybrid IFN (I and II) with terminal amino-parts IFN-a 1 have lower activity in relation to human cells than in IFN-a itself 1. One of the hybrids (III) with con IFN-a 2 has amino acid moieties having approximately the same activity with respect to human cells as IFN-a2 itself,
Example 12: A collection of hybrid phages derived from human chromosomal DNA fragments is obtained by partial hydrolysis of Haelll and Alul, by attaching EcoRI binding to the I Haron 4A branches. This gene bank is screened by an in situ method, using as probe insertion of 32P labeled IFN-a 1 cDNA cut from pBR322 / Pst / Hif 2h. Sixteen positively hybridized phage clones were isolated from 240,000 plaques by re-purifying plaques. Ten hybrid phage DNA preparations were cleaved with Hindlll, Tacl, Hhal, Baml, E.coRI and BglD, the resulting fragments were separated by agarose gel electrophoresis, transferred to Millipore membranes and hybridized with a 32P-labeled Hif-2h with a DNA insert.
The chr-3 and chr-26 clones are DNA segments that overlap for the most part of their length, since they have several EcoRI and Hindlll fragments in common. The hybridizing part of the chr-l and one of the hybridizing parts of chr-10 are the same as the Hind Ill-Hindlll and EcoRI-EcoRI fragments, which hybridize with the Hif-2h probe and have the same length (3.2 kv and 0.95 kv, respectively) . Hybridizing - the part of chr-16 is identical to the hybridizing part of chr-35, although it is oriented in the opposite direction, since each of the two clones contains a 1.4 Bglll-Bglll fragment of 1.4 and 2 EcoRI-EcoRI, 2 kv, which hybridize with Hif -2h cDNA probe. Therefore, the inserts chr-16 and chr- -35 overlap.
The genome of an individual person contains at least 10 different DNA sequences that cross-hybridize with Hif-4h. This conclusion is supported by the fact that the ratio
the Hif-2h fragments that are hydrolyzing, as defined in the klopov bank, are about 1 in 16,000.
TakkakIFN-a1 and IFNa-2sDNK have
The 1 and 2BglD sites, respectively, within their coding sequences, apparently Hif-chr 35 is a copy of one of two previously cloned interferon genes. Hif-chr 35 strongly hybridizing H1t-2psDNK fragment (containing only 3 non-coding regions) when compared with weaker hybridization with another chromosomal DNA of this probe confirms the close correspondence of Hif-chr
35 and Hif-2h.
For further analysis, Hind-chr 35 Hindlll-BamH was isolated from chr-35. This fragment (3.4 kV) contains the hybridizing part (Hif-chr 35) chr-35. This fragment
the pBR322 site is subcloned into the Pstl using the well-known dC-dG method, and E. coli HB101 is transformed with the resulting recombinant DNA molecule.
The clones of these transformants are screened by in situ hybridization of the colony with the labeled
P Hif-2h fragment and then plasmid
Z-pBR322 (Pst) / Hchru DNA (| -35HB HchrIF-35HB) was isolated from positive
clones. Hybrid Insert Orientation
The Hchr IF-35HB fragment in the plasmid with respect to the β-lactamase gene pB R322 is determined by EcoRI cleavage and the size of the fragments obtained. An insert oriented in the correct reading frame with / 3-lactamase is designated a and oppositely oriented
The cultures of these positive clones are grown to an apparent Odezo 0.8, the bacteria are harvested and lysed by the method
lysozyme-freeze-thaw. Seven out of 10 studied clones demonstrate IFN-a activity in the order of 75 to 500 units / g of cells (cytopathic effect of reduction analysis).
A DNA insert of one of these seven IFN-producing E. coli HB101 clones (Z-pBR322 (Pst) Hchr IF-35HB) is further characterized by restriction analysis and the determination of the nucleotide sequence.
A DNA plasmid (But | - | gIF-35NB) is obtained from a clone and restriction sites are determined. Hchr IP-35HB is treated with EcoRI, labeled at end 5 and hydrolyzed and Pstl to cleave an unnecessary fragment of about 1
kV, 1.04 kV Ecorl Bglfj (31 proximal) and 0.96 kv EcoRI-BglQ (5-proximal) fragments were recovered by agarose gel electrophoresis. Both fragments are partially hydrolyzed with Hinfl, Bspl, and MvoM, respectively, and the resulting products are isolated on a 1% agarose gel in Tris-acetate buffer (pH 7.8) containing 1 µg / ml ethidium chromide. After maturation, the radioactive bands are detected by autoradiography. Similarly, the BstNl and HgiAl sites are determined at 1.04 in the 3 proximal fragment.
For the determination of the Hchr nucleotide sequence, the IF-35GVa was cleaved with various restriction enzymes, the resulting products were isolated by electrophoresis through a 5% polyacrylamide gel in Tris-borate-EDTA buffer and extracted from the gel and purified.
The nucleotide sequence is determined as follows: 1 and 2 - Hchr-IF-35HB cleavage and using Bglfl, labeling, splitting EcoR and Pstl and isolating the BglFl -EcoRI fragment (940 bp) (1) and BglQx -EcoRI (360 bp) (2); 3 and 4, Hchr cleavage of IP-35NB ac using EcoRI, labeling, cleavage of Bspl and isolation of the EcoR-Bspl (680 in) fragment (3) and EcoRI-Bspl (880 bp) fragment (4); 5, 6, 7, and 8 cleavage of Hchr IF-35IV and using Pvull, labeling, splitting Bglll and EcoRI and isolating Pvull -EcoRI fragment (780 bp) (5), fragment Pvull -Bgl (215вр) (6), fragment Pvull -Bg lJ (90 BP) (7) and a fragment of Pvul-EcoR (290 BP) (8);
9 and 10 cleavage of Hchr IF-35 HB and using EcoRI, isolation of a 1% agarose gel by electrophoresis in a tris-borate EDTA buffer of the fragment with 1300 para EcoRI, further cleavage of the Hinfl and isolation of the Hinfl-Hinf fragment (459 bp) and the Hinfl fragment and 45 -Hinfl (180 bp), labeling a larger Hinfl-Hinf fragment and splitting Mbol made it possible to isolate Hinfl - Mboll (190 bp) (9). The labeling of the shorter Hinfl-Hinfl fragment and the splitting of Avail allowed us to isolate the Hinfl - Avall fragment (150 BP);
11 - Hchr cleavage IF-35NV and with the help of Mboll, labeling the splitting of Vd1P and fragment selection (465 BP) (11);
12, 13, and 14 cleavage of Hchr VF-35HB with Bspl and BglQ, isolation by electrophoresis on an agarose gel as previously described in fragment 1200 bp Bspl-Bspll (a) splitting HgiAl, labeling, splitting Mboll, and isolating HgiAI -Mbo l fragment (300 BP) (12) and fragment HgiAl-Mbolll (360 BP) (13), or (b) cleavage of the BstNl, labeling, EcoR cleavage and isolation of the BstNI -EcoRI fragment (380 BP) (14). Sequences in different
The fragments are determined according to the McGuilbert method.
Comparison of the nucleotide sequence of the Hchr IF-36HB a coding region and the Hif-2h sequence (the coding region) shows that they are identical. In particular, it is surprising that there is no indication of the presence of introns within the Hchr coding sequence of the IF-35NB fragment, i.e. between the Hinfl center in 5 non-coding regions and EcoRi centers in 3 non-coding regions.
The chr-3 and chr-26 genes, which appear to be identical according to the heteroduplex data, are distinguished by a single BD | D site, and are examined for the nucleotide sequence. Five nucleotides differ in 725 base pairs. Apparently, Hif-chr3 and Hif-chr 26 are allelic forms of the same gene, since not only genes, but at least 3.5 square kilometers of the preceding and 6.0 square kilometers of the next ones form an excellent heteroduplex of - due to the relatively small difference, which covers only 2 amino acid changes. They are designated IFN- «4a (Hif-chr3 and IFN-a4b / Hifchr 26). The nucleotide sequence and the corresponding amino acid sequence of IFN-a4b are determined according to the usual procedure.
In this case, the proteins encoded by each of the sequences differ from each other in approximately 15% of their residues. This discrepancy is typical for products of nonallelic genes that have already diverged 20 to 90 million years ago,
Example 13. Plasmid Z-pBR322 (Pst) (Hchr HIF-35HB a) is used as a source of the Hif-chr35 fragment for expression in mouse cells. This plasmid is digested with Pstl and treated with 5 exonuclease to remove 5 dG tails. Then this fragment is inserted into the 5dG-Kpnl fragment of the plasmid obtained by attaching the Bam Hi-Bam HI fragments of pBR322 and the polynomial DNA. The resulting vector is used to transform mouse ETZ cells. These transformed cells denote the ZTZ mouse (Hif chr 35 polynomial). After 20-40 hours, the activity of IFN-α in the order of 300 units / ml of IFN-a per human cells and about 300 units / ml per bovine cells is determined.
E. coli HB101 lysates are infected with ten hybrid lambda phages and analyzed for the presence of IFN. Seven of the eleven phages (all except chr-10, chr-12, chr-19 and chr-27) give lysates containing IFN with activity in the interval from 30 to
37176451538
50 u / ml In the case of chr-Yu and chr-12, hybrids and purification of the target product, which differ (to Hif-2h) Hindlll-Hindlll or EcoRI-y and so that, in order to increase
EcoRI fragments are subcloned into the Pstl site of the purity of the target product, the strain of E. coli.5 of Escherichia coli ATCC 31633 is used as an bioassay of biological proteins and express the activities of IFN-logical cultures.
or 31634, a transformed recombination formula of the invention with nantes plasmid DNA with an insert
The method of obtaining leukocyte in-Z-pBR322 (Pstl) HclF-11-206 or Zterferon, involving the cultivar-pBR322 (Pstl) / HclN SN35-AHL6c nucleotidation of biological cultures, isolation of the 10 sequence
ATGGCCTCGCCCTTTGCTTTACTGATGGTCCTGGTGGTGCTCAGCTGCAAGTCAA
GCTGCTCTCTGGGCTGTGATCTCCCTGAGACCCACAGCCTGGATAACAGGAGGAC
CTTGATGCTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCCTGTCTGATGGAC
AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGG
CTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCTTCAACCTCTTTAC
CACAAAAGATTCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCTGCACC
GAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGAGAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAAGAAATACTT
CCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAG
GTTGTCAGAGCAGAAATCATGAGATCCCTCTCTTTATCAACAAACTTGCAAGAAA
GATTAAGGAGGAAGGAA, TGTGATCTCCCTGAGACCCACAGCCTGGATAACAGG
AGGACCTTGATGCTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCC t crj CTGA
TGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAQTTCCA
GAAGGCTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCTtCAACCTC
TTTACCACAAAAGATTCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCT GCACCGAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGA GAGGGTGGGAGTUVACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAAGAAA TACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCT GGGAGGTTGTCAGAGCAGAAATCATGAGATCCCTCTCTTTATCTVACAAACTTGCA AGAAAGATTAAGGAGGAAGGAA, TTACTGGTGGCCCTCCTGGTGCTCAGCTGCA AGTCAAGCTGCTCTGTGGGCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAG GAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTG AAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAA
AGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTT
CAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTAC
ACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGG
GGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATA
CTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGG
GAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAG
AAAGTTTAAGAAGTAAGGAA, TGTGATCTGCCTCAAACCCACAGCCTGGGTAGC
AGGAGGACCTTGATGCTCCTCGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCT
TGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCA
AAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTC
TTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCT
ACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGT
GGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAA
TACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCT
GGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCA
AGAAAGTTTAAGAAGTAAGGAA, ATGGCCCTGTCCTTTTCTTTACTGATGGCCG
TGCTGGTGCTCAGCTACAAATCCATCTGTTCTCTGGGCTGTGATCTGCCTCAGAC
CCACAGCCTGGGTAATAGGAGGACCTTGATACTCCTGCAACAAATGGGAAGAATC
TCTCATTTCTCCTGCCTGAAGGACAGACATGATTTCGGATTCCCCGAGGAGGAGT
TTGATGGCCACCAGTTCCAGAAGACTCAAGCCATCTCTGTCCTCCATGAOATGAT
CCAGCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCTTGCCA / CAG
ASSSTSSTASAAAAATTTTSSLSTSLASTTTLSSASSAAUTSAATSLSSSTSOLLGG
CATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAAfCtCCACTC
CATCCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTTTATCTAACAGAGAAG
AAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGAGA7CCCTCT
CGTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGGAT, and TGTGATCT GCCTCAGACCCACAGCCTGGGTAATAGGAGGACCTTGATACTCCTGCAACAAATG
GGAAGAATCTCTCATTTCTCCTGCCTGAAGGACAGACATGATTTCGGATTCCCCG AGGAGGAGTTTGATGGCCACCAGTTCCAGAAGACTCAAGCCATCTCTGTCCTCCA TGAGATGATCCAGCAGACCTTCAATCTCTTCAGCACAGAGGACTCATCTGCTGCT TGGGAACAGAGCCTCCTAGAAAAATTTTCCACTGAACTTTACCAGCAACTGAATG ACCTGGAAGCATGTGTGATACAGGAGGTTGGGGTGGAAGAGACTCCCCTGATGAA TGTGGACTCCATCCTGGCTGTGAGGAAATACTTCCAAAGAATCACTCTTTATCTA ACAGAGAAGAAATACAGCCCTTGTGCCTGGGAGGTTGTCAGAGCAGAAATCATGA GATCCCTCTCGTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAT;
or a sequence that hybrid-hybridizes with a labeled DNA insert and dies with an insertion or said post-DNA sequence fourfold under the following conditions of ficoll buffer (May / vol), 0.02% polyhybridization; nitrivinylpyrrolyrone prehybridization, 0.02% (May, / v) cellulose filter carrying DNA-5 bovine serum albumin, 0.5% sequence or DNA insert 3 times dodecyl sulfate, 200 μg / ml four-fold buffer with 0.1% (May / Rev) of salmon sperm extracted DNA, at 68 ° C in Ficoll, 0.1% polynylpyrrolidone, 0.1% for 16 h, rinsing the filter with (b.about) bovine serum albumin-room a temperature of 0.53 SDS, washing on, 0.5% sodium dodecyl sulfate with 200 Y at 68 ° C 2x0.53 SDS with a single replacement of µg / ml of denatured fragmented alignment and 3 ml of a base with lockjaw com- salmon sperm DNA at 68 ° C for 7 hours, Montana temperature for 4 hours.

权利要求:
Claims (1)
[1]
Claim
A method for producing leukocyte interferon, comprising cultivating biological cultures, isolating and purifying the target product, characterized in that, in order to increase the purity of the target product, Escherichia coli ATCC 31633 or 31634 microorganism strain transformed with recombinant plasmid DNA with Z- insert is used as biological cultures pBR322 (Pstl) HclF-1 1-206. or ZpBR322 (Pstl) / HclN SN 35-AHL6 with a nucleotide sequence
ATGGCCTCGCCCTTTGCTTTACTGATGGTCCTGGTGGTGCTCAGCTGCAAGTCAA GCTGCTCTCTGGGCTGTGATCTCCCTGAGACCCACAGCCTGGATAACAGGAGGAC CTTGATGCTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCCTGTCTGATGGAC AGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAGTTCCAGAAGG CTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCTTCAACCTCTTTAC CACAAAAGATTCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCTGCACC GAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGAGAGGG TGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAAGAAATACTT CCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCTGGGAG gttgtcagagcagaaatcatgagatccctctctttatcaacaaacttgcaagaaa GATTAAGGAGGAAGGAA, TGTGATCTCCCTGAGACCCACAGCCTGGATAACAGG AGGACCTTGATGCTCCTGGCACAAATGAGCAGAATCTCTCCTTCCTCCTGTCTGA TGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGATGGCAACCAQTTCCA GAAGGCTCCAGCCATCTCTGTCCTCCATGAGCTGATCCAGCAGATCT'rCAACCTC
TTTACCACAAAAGATTCATCTGCTGCTTGGGATGAGGACCTCCTAGACAAATTCT GCACCGAACTCTACCAGCAGCTGAATGACTTGGAAGCCTGTGTGATGCAGGAGGA GAGGGTGGGAGAAACTCCCCTGATGAATGCGGACTCCATCTTGGCTGTGAAGAAA TACTTCCGAAGAATCACTCTCTATCTGACAGAGAAGAAATACAGCCCTTGTGCCT GGGAGGTTGTCAGAGCAGAAATCATGAGATCCCTCTCTTTATCAACAAACTTGCA AGAAAGATTAAGGAGGAAGGAA, TTACTGGTGGCCCTCCTGGTGCTCAGCTGCA AGTCAAGCTGCTCTGTGGGCTGTGATCTGCCTCAAACCCACAGCCTGGGTAGCAG GAGGACCTTGATGCTCCTGGCACAGATGAGGAGAATCTCTCTTTTCTCCTGCTTG AAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCAAA
AGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTCTT CAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCTAC ACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGTGG GGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATTCTGGCTGTGAGGAAATA CTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCTGG GAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCAAG AAAGTTTAAGAAGTAAGGAA, TGTGATCTGCCTCAAACCCACAGCCTGGGTAGC Aggaggaccttgatgctcctggcacagat'gaggagaatctctcttttctcctgct TGAAGGACAGACATGACTTTGGATTTCCCCAGGAGGAGTTTGGCAACCAGTTCCA AAAGGCTGAAACCATCCCTGTCCTCCATGAGATGATCCAGCAGATCTTCAATCTC TTCAGCACAAAGGACTCATCTGCTGCTTGGGATGAGACCCTCCTAGACAAATTCT ACACTGAACTCTACCAGCAGCTGAATGACCTGGAAGCCTGTGTGATACAGGGGGT GGGGGTGACAGAGACTCCCCTGATGAAGGAGGACTCCATT.CTGGCTGTGAGGAAA TACTTCCAAAGAATCACTCTCTATCTGAAAGAGAAGAAATACAGCCCTTGTGCCT GGGAGGTTGTCAGAGCAGAAATCATGAGATCTTTTTCTTTGTCAACAAACTTGCA AGAAAGTTTAAGAAGTAAGGAA, ATGGCCCTGTCCTTTTCTTTACTGATGGCCG TGCTGGTGCTCAGCTACAAATCCATCTGTTCTCTGGGCTGTGATCTGCCTCAGAC CCACAGCCTGGGTAATAGGAGGACCTTGATACTCCTGCAACAAATG GGAAGAATC TCTCATTTCTCCTGCCTGAAGGACAGACATGATTTCGGATTCCCCGAGGAGGAGT ttgatggccaccagttccagaagactcaagccatctctgtcctccatgaoatgat · ccagcagaccttcaatctcttcagcacagaggactcatctgctgcttcccaAcag agcctcctagaaaaattttccactgaactttaccagcaactgaatgacctggaag catgtgtgatacaggaggttggggtggaagagactcccctgatgaaTctccactc catcctggctgtgaggaaatacttccaaagaatcactctttatctaacagagaag aaatacagcccttgtgcctgggaggttgtcagagcagaaatcatgagatccctct CGTTTTCAACAAACTTGCAAAAAAGATTAAGGAGGAAGGAT, -and TGTGATCT gcctcagacccacagcctgggtaataggaggaccttgatactcctgcaacaaatg ggaagaatctctcatttctcctgcctgaaggacagacatgatttcggattccccg aggaggagtttgatggccaccagttccagaagactcaagccatctctgtcctcca tgagatgatccagcagaccttcaatctcttcagcacagaggactcatctgctgct tgggaacagagcctcctagaaaaattttccactgaactttaccagcaactgaatg acctggaagcatgtgtgatacaggaggttggggtggaagagactcccctgatgaa tgtggactccatcctggctgtgaggaaatacttccaaagaatcactctttatcta acagagaagaaatacagcccttgtgcctgggaggttgtcagagcagaaatcatga gatccctctcgttttcaacaaacttgcaaaaaagattaaggaggaaggat;
4L or a sequence that hybridizes to an insert or said sequence under the following hybridization conditions; prehybridization of a nitrocellulose filter carrying a DNA sequence 5 or DNA insert in a four-fold buffer with 0.1% (May./vol) of ficol, 0.1% polynylpyrrolidone, 0.1% (mae) of bovine serum albumin, 0.5% sodium dodecyl sulfate with 200 ¹ θ μg / ml denatured fragmented salmon sperm DNA at 68 ° С for 7 h, and hybridization with labeled DNA insert and DNA sequence in four-fold Ficoll buffer (May./vol.), 0 , 02% polyvinylpyrroliron, 0.02% (May / v) bovine serum albumin, 0.5% sodium dodecyl sulfate, 200 μg / ml denate ingly salmon sperm DNA at 68 ° C for 16 hours, rinsing the filter at room temperature 0,53 SDS, washing at 68 ° C 2x0,53 SDS-once replacement solution and 3 ml trismus base at room temperature for 4 hours.

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同族专利:

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公开号 | 公开日

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UA13380A|1997-02-28|

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IN153751B|1984-08-18|

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MD431C2|1996-07-31|

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JPH0321150B2|1991-03-22|

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JPH0321151B2|1991-03-22|

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US4530901A|1985-07-23|

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JPS62122590A|1987-06-03|

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JPS56150100A|1981-11-20|

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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EP80300079|1980-01-08|LTRP1005A| LT2517B|1980-01-08|1993-09-20|THE BUDGET FOR RECEIVING THE LEUKOCITIVE INTERFERENCE|

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LV931083A| LV5676A3|1980-01-08|1993-09-22|Satisfaction with leukocyte interferon acquisition|