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  • 专利说明
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    • 专利摘要:
    This invention relates to the field of biotechnology and genetic engineering. The aim of the invention is the simultaneous selection of clones carrying recombinant DNA molecules with a gene called x-e-and 0-interferon. Simultaneous selection of clones is ensured by the presence of homology between (from 5 to 7) functional IFN-y genes. The homology site is 13 nucleotides long with the following formula 5 CCTTCTGGAACTG-3. In addition, RNA from Namalva cells was isolated 9 hours after virus induction, synthesis to DNA was reduced to (L) RNA with a molecule size of 6 and 18 S, and DNA was obtained in size 600 bp. ie s
    公开号:SU1346048A3
    申请号:SU833598429
    申请日:1983-05-27
    公开日:1987-10-15
    发明作者:Дворкин-Растл Эва；Дворкин Марк-Брус；Адольф Гюнтер；Мейндл Петер；Бодо Герхард；Светлый Петер
    申请人:Др.Карл Томэ Гмбх (Фирма)；
    IPC主号:
    专利说明:

    This invention relates to the field of biotechnology and genetic engineering.
    The aim of the invention is the simultaneous selection of clones carrying recombinant DNA molecules with the gene encoding c .; - i-interferon.
    Simultaneous selection of clones is ensured by the presence of homology between (from 5 to 7) functional IFW-e-and IFN-fl-genes. Moreover, the homology region is 13 nucleotides long with the following formula 5 CCTTCTGGAA-CTG3. In addition, RNA from Namalwa cells was isolated 9 hours after virus induction, synthesis to the DNA of the axis was performed on (A) RNA with a molecule size of 6 and 18 S, and DNA was obtained in size 600 p.
    Example 1, Suitable cells are selected for the production of poly (A) RNA which contain human IFN-ciH IFN- | 3-mRNA.
    Various human cells induce Sendvirus; for the production of interferon by known methods. After 24-48 hours, the content of IFN-ci is determined and the type-specific antiserum is neutralized.
    It is found that the cells nalva produce more than 50% of IFE-et and before. 50% IFN- | i after induction by sendivevirus,
    The neutralization test is carried out as follows.
    Approximately 10 units of interferon are incubated at 37 ° C for 60–80 minutes:
    1) antiserum against IFN-c i; final dilution
    2) antiserum against human IFN-ft; final dilution l: 300j
    3) a mixture of the first and second antisera.
    After incubation, the residual activity of interferon is determined by the method of determining sterile-1 spots,
    EXAMPLE 2 Poly (A) RNA is obtained which contains human IFN-ct-. : mRNA from nalvwa cells induced by Sendai virus.
    Dissolve the cells and induce it with Sendai virus. mRNA is isolated 9 hours after induction with the Sendai virus, since after this interval the amount of interferon-specific mRNA reaches a maximum,
    Cells are separated by centrifugation for 20 minutes at 1000 g, washed once with NP-40 buffer (0.14 mol
    Iac1, 1.5 mmol MgCl, 10 mmole Tris-HCl, pH 7.4) and resuspended in NP 40 (Shell) buffer with O, -5% non-ionic detergent (Shell), and 2 mg / mol bentonite. After 5 min in ice
    In the core cell bath, centrifuged, pelletized, as described above, extracted the RNA containing cytoplasmic fraction by adding 2% sodium dodecyl sulfate, 5 mmol of ethylenedinitrilotetraacetic acid and
    50 mmol Tris-HC, pH 9, then three times with a phenol-chloroform mixture and once with chloroform and immediately after that, RNA is precipitated with alcohol,
    POLY (A) RNA is purified by oligo (dT) cellulose chromatography by centrifuging in a 5-20% sucrose gradient in Yummol sodium acetate buffer, pH 5.5,
    I mmol of ethylenedinitrilotetraacetic acid (20 hours at a speed of 25,000 rpm with a Spinco SW 27 rotor) is divided by the size of the molecule and the molecules are collected in the order of 6 S (sedimentation unit) and 18 S (for simplicity, the aforementioned poly 12S- PHK).
    The content of interferon-specific mRNA (iFN-ciH IFN, mRNA) is determined by microinjection of 12S-PHK into herononus oocytes, the activity of interferon in the oocytes is measured. At the same time, 1 uch of injected injection is obtained (12S-PHK average titer of interferon about 1000 IE is an international unit of interferon, considered
    interferon standard 69/19.
    At the same time, about 80% of the activity is neutralized with antiserum against the oi-type interferon, while the total activity is neutralized by only
    a mixture consisting of anti-o -1 -and anti- | 6-interferon antisera,
    Example 3. FULL (A) RNA, which has a molecule size of 5-18 S
    () 5 is used as a template for producing single-stranded cDNA, with 40 | 1 | r / ivm poly (A) RNA is incubated in 50 mmol Tris-HCl, pH 8.3, 10 mmol MgClj, 100 mol KCl, 1 mmol
    (fflTPS, 0.4 mmol dithiothreitol,
    4 mmol sodium pyrophosphate, 20 / cg / ml oligo (deoxy / thymidine 12-18 (P1g-biochemistry), 25 (U g / ml actinomycin D with 100 units / ml AMU reverse transcript3134
    PS within 45 minutes at 44-45 ° C. Immediately after this, the PHk of the RNA-DNA hybrid is removed by one-hour incubation in 0.3 mol NaOH at 50 ° C, after which the single-stranded cDNA is neutralized and precipitated with ethanol.
    A second strand of DNA complementary to single-stranded DNA is synthesized under the following conditions: 30 g / ml of single-stranded cDNA is incubated in 0.12 mol of potassium phosphate buffer, pH 659, 10 mmol MgCl2, 10 mmol dithio-treitol, 1 mmol dNTPS with 300 units / ml of poly1.1razy 1 DNA (Bolhringer Mann-heim), 6 h 15 ° С. Then it is extracted with fe: -hol and precipitated with ethanol.
    The double-stranded cDNA mixture thus obtained at both ends of the string is modified, the protruding ends of the individual strands are removed. For this, the DNA is incubated in 0.3 mol of NaCl, 30 mmol of sodium acetate j pH 4.5, 1 mmol of ZnCl with 1250 units / ml of S 1 nuclease (Miles Laboratories) for 30 minutes at which is then extracted with phenol and precipitated with ethanol. The double-stranded cDNA thus obtained is divided into a 5% 20% sucrose gradient in 10 mmol sodium acetate buffer at pH 5.5, in 1 mmol ethylene-nitrile tetraacetic acid and those fractions that have at least 600 o.p. are isolated. cDNA (0.5pg) is extended by approximately 15 nucleo
    by applying oligodeoxy-cytidine at the 3 -end of both as a result of incubation with 140 mmol of potassium carbonate, pH 6.9, 30 mmol of tris-HC1, pH 6.9, 2 mmol of CoC, 0.1 mmol of Ditiotreitol, 0, 1 mg / ml BSA, 5 | X mol dCTPs using 500 units. terminal transferase for 4 min at 37 ° C. Aliquot 2 1 g g pBR322 is linearized with restriction endocuclease Pst 1, the cDNA mixture is extended by overlapping oligodeoxyguanidine at the end of the 3 molecules and modifying, protruding the ends of the oligodesoxyguanine. Stable dsDNK is obtained by the main pairing of oligodeoxyguanidine ends with free
    the ends of oligodeoxycytidine cDNA molecules. To do this, both components of this reaction are incubated in 0.1 mmol-NaCl, 1 mmol of ethylenedinitrilotetraacetic acid, 20 mmol of Tris-HCl, pH 8, for 10 minutes at 65 ° C, then for 2.5 hours at 45 ° C and then in overnight at 37 C.

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    With this method, the cleavage site of the Pst 1 restriction endonuclease is regenerated and, subsequently, this can be used to cut a cDNA insert from a vector hybrid.
    The hybrid plasmids obtained from this method from pBR322 and the nalva cDNA are used to transform Escherichia coli HB 101. This method produces 30,000 clones of transformed E ccli cells, which are separated on microtitroplates.
    Example 3. The tridecanucleotide is labeled in 50 mmol Tris-HC, pH 7.5, 10 mmol MgCl, 5 mmol dithiothreitol, O 5 5 nmol spermidine, 1 rii m P / p-adenosine triphosphate with 60 units ./ml T4 polynucleotide kinase (Bethesda Research La, boratories) for 30 min at 37 ° C until specific activity is about 500 Ci / mmol at the 5th end of the molecule.
    This labeled Tridecanucleotide is used as a primer (Dsime) for the synthesis of single-stranded cDNA, with POLY {A) DNA from Sendai virus-induced cells being used as a template. The reaction is carried out with a 5–10-fold molar excess of primer relative to RNA in 50 mmol Tris-HC1, pH 8.3, 60 mmol KCl, 5 mmol dithiothreitol, 50 pg / ml actinomycin I, 4 mmol MgCl,. 4 mmol sodium pyrophosphate, 0.5 mmol dNTPS with 100 units / mp of AMU reverse transcriptase for 90 min at 37 ° C. After removing the RNA, a hybrid RNA-DNA incubator for 1 h in 0.3 mol NaOH at 50 ° C, followed by neutralization with 0.3 mol acetic acid and cDNA is used as a hybridization probe. The cDNA thus obtained carries a radioactive label of lip in molecules that have been synthesized from an interferonspecific tridecane nucleotide as a primer and, therefore, has a high specificity for identifying the interferon DNA sequence in the hybridization.
    PRI me R 4. The cells of individual cloned transformants were transferred to a nitrocellulose filter (22x15 cm, pore size 0.45, Millipore odes Schlliches), grown to a colony size of 2 mm in diameter and prepared for hybridization. The hybridization of the nucleic acid labeled at the ends of the cDNA is carried out in a mixture containing 50% formamide, 4xSET (lxSET 0.15 mol NaCl, 5 mmol ethylene dinitrilotetraacetic acid, 50 mmol Tris-HC, pH 8), 1 Denhardt solution (0 , 02% BSA, 0.02% Ficoll, 0.02% polyvinylpyrrolidone), 0.5 mg / ml denatured salmon sperm DNA, 0.1% sodium pyrophosphate and 0.1% sodium dodecyl sulfate, for 48 h at 37 ° C. The filter is washed in a solution consisting of 50% formamide and 0.2xSSC (1x SSC 0.15 mol NaCl, 0.015 mol sodium nitrate) at C for 6-8 hours and then sprayed with IxSSC solution. Filters are vyststvayut at 20-2 5 ° C and at -70 ° C exhibit with x-ray film (Kodak XB). In total, approximately 13,000 transformed bacterial colonies are obtained from screening and 190 clones are obtained. Accordingly, about 1% of all clone bank clones contain the specific sequence dCCTTCT-GGMCTG.
    Example 5. Tridecanucleotide-positive clones (microtitroplates PI and P2) and randomly selected clones of a microtiter plate (E52) are transferred to a nitrocellulose filter and diluted, prepared for hybridization of bacteria and hybridized with various P-labeled cDNA inserts. Hybridization was carried out. Adding 50 ju g / ml poly (and lOfur / ml poly (l): poly (C) to the hybridization solution, washed the filter after hybridization in 50% formamide and 1x SSC. It turned out that the clone P1H1 hybridizes more than 90% of all tridecanucleotide-positive clones of plate P1 and P2, as well as some clones of plate B; 52 Identity of many of these cloned sequences is confirmed by restriction analysis. Clone P2H10 hybridizes not only to itself, but also to the clone of 1C12 position , IF12, E52E1. Clones P1A6 and P2B10 were analyzed using the same method.
    EXAMPLE 6, salts (A) RNA is separated from induced and uninduced cells by nalv, denatured by incubation for 1 hour at 1 mol glyoxal, 50% dimethyl sulfoxide, 10 mmol sodium phosphate buffer, pH 7, divided by
    A 1.4% floating agar is transferred to a nitrocellulose filter and hybridized with P-labeled plasmd DNA using nicktranslation. Plasmid DNA, which is derived from an induced gene, in this series of experiments hybridizes only RNA from induced cells, but not RNA from uninduced cells. The plasmids used for hybridization of DNA are PCH1 (A), P1.FI2 (B, 1), P2H10 (C) P1A6 (E), P2B10 (g). Of the plasmids tested, only P1F12, P2H10, and P1A6 only hybridized with RNA from the infected cells, while P1H1 and P2B10 hybridized also with the RNA from uninduced cells. The size of the RNA molecule that hybridizes with P1F12 and P2H10, 11-12 S, the RNA value that hybridizes with P1AN, 12-14 B (these molecule sizes are known for TFN-ci or IFN- / i-HPHK.- The P1F12, P2H10 and P1A6 trains are examined, in addition, to uniquely establish their content in the interferon DNA sequence.
    PRI me R 7. Plasmid DNA was fixed on a nitrocellulose filter and incubated under hybridization conditions with POLY (A) RNA from induced cells by nalv. Prepared bound interferon DNA sequence, the unhybridized RNAs rinsed, flexible ridizirovannuyu RNA from DNA vtlavl dissolved and injected into oocytes Xenopus laevis oocytes If the protein is synthesized interferon, antiviral properties which can be demonstrated by counting plaques sterilnps, 10 g of linearized plasmid DNA denatured in 200 | tl 0.5 n. NaOH, neutralized 200 ju l 0.5 n. acetic acid and 20 x SET and filtered by binding on a nitrocellulose filter with a diameter of 2.5 mm. The filter is dried at room temperature, incubated for 2 hours at 80 ° C and then for 1 hour at 53 ° C or also at 37 ° C in a mixture containing 50% formamide, 20 mmol nHnepa3HH-N5N-bic- (2-ethane acidic acid) pH 6.5, 0.4 mol of RaCl, 5 mmol of ethylenedinitrilotetra-acetic acid, 10% dextransulfate, 1% sodium dodecyl sulfate, 20 | pg / ml E. coli ntPHK, 50 jur / ml poly (A) prehybridized, and in the buffer itself after adding 14-40 (Ig induced by namalva-poly (AG RNA was hybridized for 5 h. After that, the filter was washed 3x20 min at 25 ° C in a mixture containing 0.2-1 SET, 0.2% sodium dodecyls phosphate, 1% dextran sulfate, 5 (cg / ml tRNA, then 2 min at 25 ° C in a mixture containing 2 ml of ethylenedinitrilotetraacetic acid, pH 8.0, 0.2% sodium dodecyl sulfate, 1% dextran sulfate, 5 lg / ml t-RNA, then 5 min in a mixture of 2 mmol of ethylenedinitrilotetracetic acid, pH 8.0, 0.2% sodium dodecyl sulfate, 1% dextran sulfate, 5 (U g / ml t-RNA, then 5 min at 2 mM ethylenedinitrilotetraacetic acid, pH 8.0, 0.2% sodium dodecyl sulfate, 5 lu g / ml t-RNA. Hybridized RNA for
    4 min elute at 100 ° C in 1 ml of H, 20 s 10 / ig t-RNA. After chromatography on an oligo- ((1T) -cellulose column, RNA is precipitated with ethanol, introduced into
    5 ml of HjO and injected into Xenopus laevis oocytes. Oocytes after incubation for 2 days at room temperature are tested by counting sterile plaque for interferon activity.
    It was confirmed that clones P1F12 and P2H10 carry the interferon DNA insertion.
    The table shows the test results.
    Example. The cDNA inserts of clone P1F12 and P2H10 are examined using a restriction endonuclease, and the presence of restriction endonuclease sites B & JnHI, Bgl II, Eco RI Hind III, EstI and Pvxi II is determined. Restriction maps of P1F12 and P2H10 are compared with the gene known from the literature for IFN-p. All three clones have Pvu II,
    0
    PstI and Bgl Ii restriction sites. In addition, the DNA sequence of the insertion of clones P1F12 and P2H10 with the IFIJ gene was determined by the method of Maxam and Gilbert (right 3 Rls II - and Bgl II fragment) and compared with the published IFN- | 3 sequence. P1F12 sequences and
    P2H10 is identical with the IFN-γ gene.
    You dreamed3 both clones at the 5th end do not have the full IFN-p-sequence, they lack about 60 nucleotides of the region that
    5 dying for mature protein, Y
    The 3-terminus of P1F12 is complete, the P2H10 also contains the entire 3-region of -cDNA, including the poly, oA) sequence, which is derived from POLY (A) RICK.
    Q P2H1D, however, extends to en: Z-. (1400 bp), 3 poly (dA) in the same region as the oligo sequence (d. C), following the sequence of unknown origin, ana5 LIZ DNA sequence of different segments of this the additional P2H10 region does not give homology with the gene. Cross-hybridized with clone P2H10, clones P1C12 and E52E1 hybridize with the additional region P2H10, and not with the part of IFTJ-j Example. Clone P1A6 is hybridized with viral-induced namalo-RNA in the region of 12-14 S. It contains an insert of approximately 900 bp kdak, which, as shown by restriction analysis, does not have a restriction site for BamHI, Bgl II , Eco RI, Hind III, PstI and Pvu II.
    The evidence that clone P1A6 contains the IFK- | 9-sequence is obtained by analyzing the DNA sequence. The sequence of the cDNA insert P1A6 is determined by comparison with the IFN-oi-sequence. It turned out that clone P1A6 contains 49 base pairs a shorter version of LelFNC. The presence of a poly (A) sequence at the 3-terminus of P1A6 indicates that clone P2A6 was derived from a shorter mRNA than that published by LeFN C. Clone C. The encoding region of LeIFN C is contained in plasmid DNA.
    5 And p. 90. Clone P1A6 - the only 1P 1-L-type of clone is identified from the collection of 190 tridecan-adhesives and positive clones. 1800 clones of the original are hybrid
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    cDNA bacca with cDNV insert of clone P1A6s | The restriction assay is carried out and the DNA sequence of the clCNA of this clone is spatially determined. Clone IF7, Clone IF /, contains both LeIFN A and restriction sites for endonuclea Bgl II and C,
    Sequence T) J “parts of the IFN - (, i.-tHrfa clone (IF7) DNA are analyzed.
    For positions 120--327, parts were found that were barely blown out afterwards: Gees
    0., CCTCK ;; - CACAGATGAGGAOAATCTCTCTTTTCr CCTGCTT -.AAGGACACACG TGAT: 0 GGATTTCCC. AGGAGGA -.- TTTGGGAACCAGTTCCMAAG aST (W1 CCA TCCCTGTCGTCGAT (; AOATGATCCAGCAGA TCTTCAA.TCTCTTAGAGCACAAAaGAC TGATGv GGTGGTTGGGATGAGACGC JCCTA (;), (2014))
    For example, with the IF sequence: 4 .fi - A THna clone, lime literature, the new sequence is IFfi DNA; A-type hli; :: nd (the nucleotides list No. 120- 327 is scattered differently; in position 137 and 170, respectively: the nucleus is ((; t g a s-a) with the nucleotide G-
    P p and mers 10. Pisats of the cult of bacteria ,, trax forks of PlAi; CL IF7 5 iesmshduyug. -the content of biologically a: k; ccnog) vgerfers a, for this 1GO ml cult p p i b; lt; they crush L-Bro:, h - cps-.i-ds opt chae-mss density gi ,, 8 to rub in at that price of riful-IroBO-K ;. for 10 min. pk 7000 about. zh. prokat
    50 mmol Tris - HGl ,. RL 6. 30 i-lsl KaGl and suspender in i.5 mol tsgs same sbuffer. After 1 W: cube, qi; E
    1MG / M31 lkzoci; and in 1h: 30 mi bacteria: a gikr g :: - th amor nod in ice and with | hayav j joslg. For example, by centrifuging for 1 h at 40,000 rpm, cell fragments, Sediment filter sterile filters and sterile plaques are tested, the active tartaron 1 is obtained, and a pair of odd cells on the IF7 1 shows the presence of cells; .g1no 500 units, per ml of incherferon; Chab clone in this i ec 1-a did not show
    activity, which is explained by the belief of 1H another oriented gaz gazuki plasmid
    Example S 1, D-1I: ;; with iessy; -: interferon 3 coded IF7. s, as a promoter sequence, take the limestone from the literature on the motor of the tryptophy: the Serratia operon. marcescens ;, and Ms-Shstze G1RS - izvv






    ten



    DNA sequence from literature. The IF7-kDNA binding composition is digested with a double-stranded DNA exonuclease BAL 31S, resulting in a mixture of molecules of different lengths. Then these i-cells are ligated into the plasmid. Strain HB1 01 is transformed with the plasmid-: -Sh; some transhformants were tested for interferon production.
    0.5-1 g of TF7-3 stakes are incubated in 100 l of 20 mmol TpHc-HGl, pH 8.1
    O. mol of Na, Gl5 12 mmol MgGlg.j
    i 2 mmol of GaGI.25 1 mmol of ethylenedinitrate solution for 3 microns 30 ° G with 0; 5 units, BA1-31 rFjethesda Researcli Laborat. ) “After this, we are pumping reactively, they add 300.; l 1 mmol of ethylenedinitrilotetraacetic acid; pH 1; i., heated with 1 O-: VMH to G5 G5 extract with phenol and ether and precipitated with ethanol.
    Osgdok in I) t 70 mmol tris-KClj pH, 7 mmol MgGl, i mmol dithiothree golad 0.4 mmol adenosine tri- (phosphate, together with mmol phosphorized Hind TC-left (Bethesda Research AaO Ha;; ) and incubator-tt
    3 overnight at 14 € from O., 5-1 units.
    The finish is completed by heating for 10 minutes to 65 G,
    2 mol of H-aietate and purify the inerferon genes from unligated left
    and 3 o pp o p anol o (0,6 obm a) o The precipitate is dissolved; from 30--50 | tl 33 mmol tris-acetate; pH 7; 9. beat mmol K-a of the kit; 0 mmol Mg-aireTaTa, i00 (cg / ml serum albumen-Shna Bo vinay and digest 30-50 units of H-.nd III for 23 hours. ”Reader for 10 seconds by cutting within
    min to 65 si
    After the addition of 2 mol of NH-acetate interferon genes precipitate, give isopropa-1,0,0,0 eoem.): Sediment solution - from B 10 L 70 1 P; Tris-KS, pH 7.5, 7 Åm MgGIg S mmol ditiotreito jiE ;, Up5 mmol of adenogsintrifug sphate and with a diet of 10 units of T4 ligase ligir: from C 0, 2; Hind III - lineariso-: t; a-: iHbiM phosphatase-treated expression; the PERMUZ plasmid during the night when the PORUS is PB.R 3 2 2-pr 31E about d; -5 S which, with about e rusgt: about the sequence of the promoter of trypto-phanoperone S, marcescens and known from the literature of PPH his mono lin & redoxis with Hind III near the ORS.
    The plasmids thus obtained are used to transform E. coli HB101 and test the interferon production of individual transformants. An increase in the expression of interferon up to about 10 times the value of si is obtained.
    spontaneous expression formula of the invention
    A method for producing an Escherichia coli clone bacterium diffused with the pBE322 plasmid containing a Pst-1 insert encoding interferon ei or f, in particular, obtaining cDNA fragments containing the coding sequence for the genes of the common and interferon and in the DNA by induction of the cDNA containing the coding sequence for the genes of the common and | B cell lymphocyte synthesis cells on a poly (A) cDNA template using reverse transcriptase, prepared on a single basis, day cDNA. DNA in the presence of DNA polymerase 1, which is further treated with S-nuclease and attached to the 3-end of this DNA oligodeoxycytidine dp nucleotides using the end of
    Compiled by N. Kuzenkova Editor L. Veselovska Tehred I., Veres
    Order 5728 Circulation 500 Subscription
    VNIIPI USSR State Committee
    for inventions and discoveries 113035, Moscow, Zh-35, Raushsk nab, d. A / 5
    . Manufacturing and printing company, Uzhgorod, st. Project, 4

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    deoxyribonucleotidyltransferase, insertion of the obtained fragment into plasmid pBR322, which was pretreated with Pst-1, S-1 nuclease endonuclease, and oligodeoxyguanidine nucleotides obtained and the transformation obtained by the hybrid plasmids of Y. the desired clone, characterized in that, in order to simultaneously select clones carrying recombinant DNA molecules with the gene,
    coding for and / -interferon and mRNA from the B cells of Hamalva lymphocytes after 9 h after virus induction, cDNA synthesis is carried out on (A) RNA with a size of 6 and 18 S, get day. DNA is 600 bp, and the selection of a clone containing a complementary sequence to a tridecanucleotide labeled with radioactive phosphorus with formula 5 SSTTSTGGOAAST-3 is carried out by direct hybridization followed by identification of the clone containing the recombinant DNA molecules with the gene encoding interferon oi | B „
    Proofreader V. But ha
    权利要求:
    Claims (1)
    [1]
    Claim
    A method for producing a bacterial clone of Escherichia coli transformed with plasmid pBR322 containing a Pst-.l insert encoding interferon eb or p, comprising obtaining cDNA fragments containing the coding sequence for 6- and p-interferon genes by isolating mRNA from Sendai virus-induced cells B lymphocytes synthesis on a template poly (a) + RNA cDNA using reverse transcriptase, producing a one-based cDNA days, the DNA in the presence of DNA polymerase 1 February 5, which is then treated with nuclease S-and attached to conclude with this DNA oligodez xicitidine for nucleotides using terminal deoxyribonucleotidyl transferase, insertion of the obtained fragment into plasmid pBR322, which is pretreated with endonuclease Pst-1, S-1 nuclease, and oligodeoxig-anidine nucleotides and transformation with the obtained hybrid plasmas are attached to its 5th end. coli HB101 followed by selection of the desired clone, characterized in that, in order to simultaneously select clones carrying recombinant DNA molecules with the gene encoding the ith and β-interferon, mRNA is isolated from p-lymphocyte cells s Namalwa after 9 hours after induction of virus, cDNA synthesis was carried out on (a) + RNA molecules with the value 6 ', 18 S, prepared days, a DNA of 600 bp, and clone selection containing the complementary sequence to tridekanukleotidu labeled with radioactive phosphorus, with the formula 5 * CCTTCTGGAACTG-3 'is carried out by direct hybridization followed by identification of a clone containing recombinant DNA molecules with a gene encoding interferon οό and β>"
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    NO831903L|1983-11-29|
    DK239383D0|1983-05-27|
    JPH0678774A|1994-03-22|
    SG38692G|1992-09-04|
    JPH0774238B2|1995-08-09|
    UA8037A1|1995-12-26|
    FI831818A0|1983-05-23|
    EP0095702B1|1989-04-12|
    FI82072B|1990-09-28|
    FI82072C|1991-01-10|
    DE3220116A1|1983-12-01|
    NO168538B|1991-11-25|
    DD211359A5|1984-07-11|
    ES8403522A1|1984-03-16|
    NO168538C|1992-03-04|
    AU1504483A|1983-12-01|
    ES529360A0|1984-10-01|
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    法律状态:
    优先权:
    申请号 | 申请日 | 专利标题
    DE3220116A|DE3220116C2|1982-05-28|1982-05-28|
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