GB2208866A - Recombinant plasmid dna ppr-ifn¼-13, coding for synthesis of human fidroblast ¼-1 interferon, and strain of bacteria escherichia coli vniigenetika vl903 - Google Patents

Description

1 _t 220886o, RECOMBINAT PLASYLID DNA PHR-IFN1-13 CODING SYNTHESIS OF

PIBROBLASTIC HUMANj$ 1 - INTERFERON, METHOD OF ENGINEERING THEREOF AND STRAIN OF BACTERIA Escherichia Coli V=GENETIKA VL 903 (pHR-IFNJ1-13) PRODUCING HUMAN 5 J1-INTERFERON CONTAINING SAME

Field of the Invention

The present invention relates to the art of genetic engineering and biotechnology and, more particularly, to a novel recombinant plasmid DNA pPR-IFN,1-13 coding the synthesis of a fibroblastic human' 1-interferon, to a method for preparing thereof and to a strain of bacteria Escherichia Coli VNIIGenetika VL 903 (PPR-IFN,'l-l3), which is a producer of human flinterferon, incorporating the same.

Prior Art

Int erferons are proteins synthesized by specialized human cells in response to a viral infection or an effect of various inductors. The experimental data available at the present time poini to antiviral, antiproliferative and im- munomodulating effects of interferon upon treatment, therewith, of individual cells, tissues and the organism as a whole. As regards its versatile character and significance for the organism, the system of interferon is comparable with the system of immunity. In accordance with antigene, biological and chemical properties, as well as depending on the type of cells producing them, human interferons are di vided into three groups:o- leukocytictL - fibroblastic, r- immunic which are produced mainly by cells of leukocytes, fibroblasts and T-lymphocytes respectively.

By the present time the ranges of application of inter ferons in medicine are determined in general. These applica tions are keratitises and dermatoses, treatment of respira tory infections induced by various viruses (influenza, adeno viruses) Hepatitises B and the like.

However, the mechanism of action of interferons, their structural and functional features and the clinical poten tial have not been studied adequately enough. This is to a great extent associated with the difficulties of obtaining 1 220886r'-') preparative amounts of pure interferons by traditional methods based on the use of induced cultures of human cells as a source of this protein. Purthexmore, the interferon synthesized in this case comprises, as a rule, a mixture of its different subtypes and forms differing in their structural- and-functional characteristics. In this respect, the preparation of individual interferons by a microbiological synthesis acquires an ever- growing scale.

Known in the art is a number of methods for the pre- paration of human interferonsj/,.,,'/$ and rbased on the use of bacteria as producers, in particular various strains of Escherichia coli, Bacillus subtilis, Pseudomonas sp. containing recombinant plasmid DNAs which ensure expression of heterologic genes in a new genetic environment. In the case of a fibroblast human,, interferon (IFBfil), for ensuring expression of the gene ITV1 in cells of E.coli use is made of setting the coding portion of a mature interferon under the control of signals of transcription and translation of genes (tuf B, rec A) and operons (lac UV 5trp) of E.coli and coliphages (P,.A). A mature,l-interferon formed as a result of the microbiological synthesis (with a molecular mass of about 19,000 Dalton) is characterized by the absence of the carbohydrate component and by the availability of a formyl-methionine N-terminal re- sidue instead of the methionine one. Howevex, this distinction does not result in a change in the biological activity of a protein product of the gene IFNAl as compared to the natural glycoprotein produced in cells of fibroblasts. The,81-interferon synthesized by this microbiological me- thod can be used both for studies of molecular mechanisms of interaction with cell receptors and in medical practice.

Known in the art are recombinant plasmid DNAs coding the synthesis of a fibroblastic hiananfil-interferon s, ch as PC1857 and pPL c 245HPIP25 and the strain E.coli SG4044 containing these plasmids (Remaut E., Staussenes P., Fiers G., 1983, Nucl. Acida Res. 11, 4677-4688).

In the recombinant plasmid DNA pPL c245HFIF25 the transcription of the gene IFNl is controlled by the regu- 4 t 1 2208860' latory unit PLOL of the baCteriDphB68 and initiation of translation of the protein product of the gene is ensured at the 8CCDUMt of engineering of a hybrid region of binding of ribDSDMeS on the basis of the SD- sequence of the gene of replicase of the phage MS2. The neximum yield of huxan 'fil- interferDn ensured by culturing of the aboveidentified strain is 47o Df. the total protein of plasmidContaining cells Of ReCDlie The abDve-mentiDned strain is characterized by that the Controlled expression of the gene IFN,l ensured with the help of the P L PZDMDtor necessitates an additional plasmid pC1857 with a positioned thereon teinperature-sensitive gene regulating cIts857, of the pbage. This re- sults in a potential instability of the recombinant plasmid under conditions of the strain culturing on 8 largescale production due to a rec.A-dependent recombination between the plasmids pC1857 and pPL c245HFIF25 at hDMOlOgieel areas. FurtberMDre, the use of 8 relatively Short SD-sequence Of the gene of replocase of the pbage MS2 for organization of a hybrid region of binding of riboSDMeS 0 does not make it possible to fully realize the potential power of the translation apparatus of B.coli due to an insufficiently effective interaction witb rIbDsomes upon ini- tiatiDn of translation of fibroblastic interferon. Besides, the absence of,Pindependent transcription termin(3tors at the 3-terminal field of the gene IFNfil in the plasmid pPL c 245HFIF25 results in a substan- tial reduction of replicability and even in a Pro bable loss of the recombinant plasmid under Conditions of depression Of a highly-effective P L prD=DtDr of the ph.9 ge A ensuring initiation of transcription of the gene IFNfil.

All this does not allow to resdh a high level Of the biosynthesis of the Product of the gene IFN,61 and can re sult in a further lowering of the content offil-interfe rDn in a biomass of the producer strain upon its large scale culturing, thus CDMPIiC8ting recovery of the pure Protein and lowering the yield of the desired Product.

Disclosure of the Invention

2208860 The recombinant plasmid DNA pPR-INFJ1-13, the method of its engineering and the strain of bacteria Escherichia coli VNIIGenetika VL 903 (pPR- IFNAJ-13) containing the same are novel and hitherto unknown from the literature.

The present invention is directed to the provision of a novel recombinant plasmid DNA coding the synthesis of fibroblastic humanfi 1-interferon, a method of its engineering and a novel highly-productive strain producing human 3.0 /1-interferon containing the same which would ensure preparation of,l- interferon in a high yield.

This object is accomplished by that the recombinant plasmid DNA pPR-M1-13 coding the synthesis of human fibroblastic,fil-interferon according to the present invention has the size of 3,900 b.p. and consists of the following units: BamHI-BgIII fragment with the size of 3,4000 b.p. of the vector plasmid pPR124B produced on the basis of pPR40 and pML24-EcorI- Sau3A fragment of the plasmid PIIY-trp7 with t.h.e size of 510 b.p. and has the following features:

- incorporates a region responsible for initiation of replication and its regulation - ColEI-replicon, a gene of the repressor cIts 857 of the phage,, a gene of resistance to ampicillin Apr, a tandem of,,Pindependent terminators of transcription of the phage fd, a regulatory field PROR and a SD-sequence of the gene cro coding the sequence of a mature fibroblastic human interferon IFNJ1 with its own me.thionine codon; - ligation of the regulatory field of the gene cro, coding portion of_fil-interferon and terminators of transcription of the phage fd is effected so that before the gene IFNV ,B1 a hybrid region of combining ribosomes is formed which has the following nucleotide sequence: 51-...TAAGGAGGTTGCATG...-3', wherein TAAGGAGGT - SD-sequence of the gene cro of the phage A 9 ATG - methionine -codon of fibroblastic interferon and, directly after the terminating cod6n of the gene IFNfil a tandem of,)c-independent terminators of transcription of the phage fd is located; 2208866 - has unique regions of recognition of restrictases cla I whose coordinate is the beginning of count (0), Pvu II (about 1,110), Bgl II (about 1,480), AccI (about 1,870), Pvu I (about 3,360); deposited on 12. 01.87 at the Collec- tion 'of Cultures of Microorganisms of the All-Union Research Institute of Antibiotics and registered under No. 1825.

The recombinant plasmid DNA pPR-IFNIl-13 according to the-present invention ensures expression of the gene IFNIfil under the control of the regulatory field P.O. of the bac- teriophage.A. The gene cIts857 regulator of initiation of transcription from preceding promotors of the bacteriphage,A and the gene IFN1-13 which is under control of the PR promotor are positioned in the same recombinant plasmid pPR-IFNfl-13. This circumstance makes it possible to avoid a potential instability during culturing of the strain with the recombinant molecule of DNA, since in the structure ac cording to the present invention no recA-dependent recom bination originates due to the absence of an additional plasmid in the producer-strain.

The present invention also relates to a method of en gineering the recombinant plasmid DNA pPR-MjAl-l3 compris ing cleavage of the vector plasmid pPR124B, built on the basis of the plasmids pPR40 and pML24, by means of the res triction endonucleases EcoRI and Bgl II and ligation of the greater of the formed fragments of the vector molecule with the EcoRISau3A fragment of the plasmid pIN:f-trp7 containing the coding portion of a maturefi 1-interferon; in the thus-produced recombinant plasmid pPRIFTyl-123 the;re is effected nearing of the SD-sequence of the gene cro and of the first (methionine) codon of,,Bl-interferon$ wherefor the plasmid DNA is hydrolyzed by means of the restrictase BamHI, a portion of nucleotides is removed by means of the endonuclease activity of the DNApolymerase I E.coli revealed under conditions of an incomplete set of nucleosidetriphosphates in the reaction mixture, the DNA is fermentatively cleaved by means of the restrictase EcoRI, treated with the SI-endonuclease to remove single-chain regions of the DNA, then the two-chain terminals are restored by means of a Klenov fragment of the DNApolymerase 2208860' I E.coli and the formed linear DNA molecules are cyclized by means of the DNA-ligase of the phage T4; the resulting preparation is used to transform cells of E.coliC600 with s?_:,lection of the transformants on a medium with ampicillin upon culturing of the cells at the temperature of 28 OC9 followed by selection of the clones having a reduced growth rate at the temperature of 42 OC, whereafter from the thusselected clones the recombinant plasmid pPR-IFN$1-13 is isolated.

The use of the method for engineering of the recombi- nant plasmid pPR-INF1-13, wherein the longest (out of the known for the matrix RNA E.coli and coliphages) SD-sequence of the gene cro of the bacteriophage j\ is connected with the coding portion of mature human, 1- interferon, results in organization of a hybrid region of combining ribosomes of the gene IFN fil with the optimal primary and steric structure of the 51-terminal area of the matrix RNA. In contrast to the known method, the method of engineering according to the present invention also ensures the pre- sence of tandem off-independent terminators of transcription in the 31- non-translated area of the gene IFIVI on the plasmid PPR-IPN1-13, thus preventing interference between replication of the plasmid DNA and a highly-effective transcripti:on of the gene of fibroblast interferon under the conditions of depression of the PR promotor.

Apart from the above-said, the present invention also relates to a strain of bacteria Escherichia coli VNIIGenetika VL 903 (pPR-IFN1-13) producing human. 1-interferon which incorporates the recombinant plasmid DNA pPRIFNJ1-13 according to the present invention and obtained by the method of genetic engineering through introduction of the plasmid DNA pPR-IFN1-13 into bacteria Escherichia coli deposited on 12-01-87 at the Collection of Cultures of Microorganisms of the All-Union Research Institute of Antibiotics and registered under No. 1825.

The strain according to the present invention makes it possible to ensure a stable accumulation of humen.81interferon under conditions of a largescale culturing and to att4din a yield of the desired product of above 2X109 U/1 2208866 which corresponds to 5-10% of the total amount of the cell protein.

Best Mode for Carrying Out the Invention

The method for engineering the recombinent plasmid pFR-IFN1-13 according to the present invention is carried out in several stages.

In the first stage the vector plasmid pPR124 is engineered.

To this end, deletion by means of the endonuclease Bal3l is effected in the DNA of the plasmid pPR40 (Molekulyarnaya Biologiya, 1987, vo. 21 No. 5, Moscow, P. 13091321) in the region of recognition of the restrictase Bgl II.

In this manner, the plasmid pPRIOO is obtained, Where- in the region of recognition BamHI is located directly after the area of combining of ribosomes of the phage A so that in the region of the initiating AUG codon a sequence is formed:

AUGGATCC BamHI Afterwards, using conventional procedures of genetic engineering, the smallest PstI-BamHI fTagment of the plas mid pPRIOO is ligated with the greatest PstI-BamHI fragment of the plasmid pML24 with the formation of the plasmid pPR124, whereinto a region of recognition of the restrue- tase Bgl II is introduced by means of an oligonucleotide linker. The Dlasmid pPR124B is thus obtained. The an intermediate plasmid pPR-IFNfl- 123 is engineer- ed.

The thus-produced vector plasmid pPR124B is cleaved by means of restriction endonucleases EcoRI and Bg1 II and li gation of the greater fragment out of the formed fragments of a vector molecule with the EcoRI-Sau3A fragment incor porating the coding portion of maturefi 1-interferon from the plasmid pINfi-trP7 is effected to give the intermediate recombinant plasmid pPR-IFV1-123.

Then the third stage is conducted - engineering of the plasmid pPR-IFNfi-13. To this end, in the intermediate plas- mid pPR-IFNfl-123 the nearing of the SD-sequence of the gene cro and of the first codon of,,81-interferon, wherefor the plasmid DNA Is hydrolyzed by mean of the restrIctase BamHI,, a portion of nucleotides is removed by means of the endonuelease activity of the DNA-polymerase I E.coli, the DNA is cleaved by means of the restrictase EcoRI, treated with the SI- endonuclease, followed by a complete res toration of the two-chain terminals by means of a Klenov fragment of the DNA-polymerase I E.coli and cyclization of the resulting linear DNA molecules is ensured by means of the DNA-ligase of the phage T4. The thus-obtained preparation is used to tram form the cells of E.coli C600, the transformants are selected on a medium with ampicillin upon culturing of the cells at the temperature of 28 OC, follow- ed by selection for a lowered rate of growth at the temperatured of 42 OC. From the thus-selected clones the plasmid DNA pPR-IFN1-13 is isolated.

The strain of bacteria Escherichia coli VNIIGenetika VL 903 (pPR-IFNkl13) according to the present invention is produced by transformation of the recipient strain with the recombinant plasmid pPR-IFNJ1-13, followed by selection of the recombinant clones on a medium with ampicillin at the temperature of 28 OC and determination of the activity of fibroblastic,81-interferon in extracts of the transfor- mant cells after depression of the PR-promotor ensured by culturing of the strain for 1-2 hours at the temperature of 42 OC. As the recipient the strains E.Coli C600 and other derivatives of E.coli K12 can be used.

The strain E.coli VNIIGenetika VL 903 (pPR-IFN1-13) is characterized by the following features.

Morpholigical features. Cells straight, bacilliform (1.2-1.6)x(2.0-6.) um, low-mobile, capable of producing thread-like forms, gram-negative, non- sporiferous.

Cultural features. Cblls grow well on dense and li- quid ordin y synthetic, semi-synthetic and complex media. When grown on an agarized Hottinger's broth or L-broth they form mucus-coated, round, slightly matted colonies. When grown in liquid media such as L-broth or M9 with casamine acids they form a uniform suspension.

k 220886c") Physiologo-biochemical features. Cells are capable of growing at a temperature within the range of from 5 to 40 "C (optimum 35 OC) at a pH of from 6.5 to 7.5. As the source of carbon aminoacids and carbohydrates (for example, saccharose) are used. The source of nitrogen can be represented by mineral salts in their ammonium form, as well as organic compounds in the form of peptone, tryptone, yeast extract and aminbacids.

Resistance to antibiotics. The strain is resistant to ampicillin in a concentration of up to 100 mg/l upon growing in liquid and agarized nutrient media.

Stability of the plasmid.,Upon storage of cells on an agarized medium (for a period of up to 1 month) with a series of successive reinoculations (for at least 6 months) and during a deep culturing in a liquid medium with an antibiotic no loss or restructuring of the plasmid occurs.

The thus-prepared strain E.coli VNIIGenetika VL 903 (pPR-IFN1-13) is a highly-efficient producer of fibroblastic humanl-interferon and can be used for a commercial- scale production of,,81-interferon.

The present invention is further illustrated by examples of particular embodiments of the method for engineering of the plasmid, the method for producing the strain, its culturing with reference to the accompanying Figures, where- in:

Fig. 1 physico-genetic chart of the recombinant plas mid pPR-IFN Fig. 2 diagram of the preparation of the plasmid pPR -IFNAl-13 according to the present invention.

Example 1

The recombinant plasmid pPR-IFNJI-13 (Fig 1) is produc ed in several stages. Stage I - engineering of the vector plasmid pPR124B.

To this end, the plasmid pPRII'@ is engineered in the following manner. 3pg of the plasmid pPR40 are cleaved by means of the restrictase Bgl II in 60,jul of a buffer for restriction-I containing 10 mM of tris-HC1, pH 8.0, 6 mM of MgCl 2, 6 mM of 2-mercaptoethanol, 150 mM of NaCl. The DNA is repricipitated with 2 volumes of ethanol. The precipitate 220886o' is dissolved in 20,ALl of H 2 0. The treatment of the DNA with the endonuclease Bal 31 is effected for 3 minutes at the temperature of 30 OC in 30J11 of a buffer containing 3 mM NAC1, 60 mM of CaCl 2, 60 mM of MgC121 100 mM of tris-]Cl, pH 8-Ot 5 mM of ethylenediaminetetracetic acid. The DNA is precipitated with 2 volumes of ethanol. The precipitate is dissolved in 10 ul of H2 0. The treatment with the restrictas6 BamHI is effected in a 3Vul sample containing the above-mentioned buffer for restriction-I. The completion of the single-chain 3'-terminal regions of the DNA is effected by treatment with a Klenov fragment of the DNA-polymerase I E.coli in a 20,,Ul sample containing 10 mM of tris-HC1., pH 8.0, 10 mM of MgCl 2 9 2jUg of the DNA preparation, by 30JUM of each of desoxyribonucleoside triphosphates, 5 units of the enzyme. The DNA from the reaction mixture is repricipitated with ethanol and dissolved in 100jil of H 20. The ligation of the linearized plasmid DNA is effected at the temperature of 16 OC for 12 hours by mean of the DNA-ligase of the phage T4 in a sample containing a buffer for ligation (60 mM of tris-HC1, pH 7.6, 10 mM of MgCl2 10 MM of 2- mercaptoethanol, 0.4 MM of adenosinetriphosphate) and l.ug of the DNA. The resulting mixture is used for transformation of cells of E.coli C600. The efficiency of tram former is 5xlO 6 colonies per ljag of the native plasmid pPR40. The clones resistant to ampicillin (100jug/ml), the plasmid DNA is recovered therefrom by a modified method suggested by Birnboim and Doli and then employed for a restriction analysis. As a result, the plasmid pPRIOO is obtained which contains a unique region of cleavage BamHI. Thereafter., the plasmid pPR124 is engineered. To this end, 2 /Ug of the DNA of the plasmid pML24 are jointly cleaved by means of the restrictases BamHI and PstI in 40,AA of a buffer for restriction-I. The ligation is effected by means of the DNA-ligase of the phage T4 at the temperature of 0 OC with the grafments obtained in hydrolysis of the DNA of the plasmid pPRIOO with the restrictases BamHI and PstI. The resulting DNA preparation is used for transformation of cells E.coli C600 with selection of tran - k -112208866 formants on a medium with ampicilling followed by selection of Cm r clones on a medium with 300 ug/ml of chloramphenicol upon culturing of the cells at the remperature of 42 OC. From the thus-selected clones the plasmid DNA is isolated and studied by means of a restriction analysis. As a result, the plasmid pPR124..is obtained which contains a unique region by means of the restrictase BamHI. Thereafter 3JLIg Of the DNA of the plasmid pPR124 are cleaved by means of the restrictase XbaI in 70-ul of a buffer for restriction-I containing 10 mM of tris-HC1,, pH 7.91, 6 mM of MgC12, 6 mM of 2- mercaptoethanol, 150 mM of NaCl. The DNA is reprecipitated with 2 volumes of ethanol. The precipitate is dissolved in 15 dil of H 2 0. The completion of thesingle-chain 31terminal regions of the DNA is effected by treatment with a Klenov's fragment of the DNA-polymerase I of E.coli in a 20,il sample containing 10 mM of tris-HC1, pH 8.0, 10 mM of MgC12, 2, pg of the DNA preparation, by 30'W of desoxyribonucleisidetriphosphates, 5 units of the enzyme. The DNA from the reaction mixture is precipitated with ethanol and dissolved in 100',l of H 2 0. The ligation of the linearized plasmid DNA with BglII linkers is effected at the temperature of 16 OC for 12 hours by means of the DNA-ligase of the phage T4 in a sample containing a buffer for ligation (60 mM of t:is-HC1, pH 7.6, 10 mM of MgC1 21 10 mM of 2- mercaptoethanol, 0.4 mM of adenosinetriphosphate, 2,/ag of the plasmid DNA and 0.5 ug of the linkers. After the ligation the DNA from the reaction mixture is precipitated with ethanol, dissolved and subjected to an enzymatic hydrolysis by means of the restrictase BglII in a 40"il sample containning a buffer for restriction-2 (6 mM of tris-HC1,, pH 7.61 6mM of MgCl 2 6 mM 2-mereaptoethanol, 50 mM of NaCl). The DNA is again precipitated with ethanol, dissolved and cyclization of the DNA molecules is ensured; to this end, 1 jug of the plasmid DNA preparation in 240,Aa of a buffer for ligation are treated with the DNA-ligase of T4. The resulting mixture is used to tran former cells of E.coli C600. The transformation efficiency is up to 5X10 6 colonies per 1',ug of the native plasmid pPR124. The clones resistant to ampicillin are chosen (100,pg/ml), the plasmid DNA is isolated therefrom following a modified procedure suggested by Birnboim and Doli and this DNA is used for a restriction analysis. As a result, the plasmid pPR124B is obtained, in contrast to the starting vector molecule pPR124, has a unique of restriction Bgl II instead of the previously availabrle sequence of recognition XbaI (Fig. 2).

Stage 2 - engineering of the intermediate plasmid pPRIFN 1-123- Into the vector pPR124B the coding sequence of fibroblastic human interferon is integrated from the plasmid pIN-trp 7. To this end, 10,lag of tha DNA pIN!fi-trp 7 are treated jointly with the restrictases EcoRI and Sau 3A in a 100,pl sample containing a buffer for restriction-2. The resulting preparation is applied onto a gel of a 1.1% low-melting agarose and subjected to electrophoresis in a tris-acetate buffer system. The gel strip containing the DNA fragment 510 b.p. long is cut out and the DNA is eluted from the gel. The thus-produced DNA fragment (about lju.g) is integrated into the plasmid pPR124B. To this end, lyg of the DNA pPRI24 is cleaved by means of the restrictases EcoRI and BglII in a 20yl sample in a buffer for restriction-2. The resulting preparation is subjected to a phenolic deproteinization, the DNA is precipitated with ethanol and dissolved in 10_zl of H 20 0.5jug of the clea- ved plasmid pPR124B is combined with 1 ug of the isolated fragment of the plasmid pIN 8-trP 7 and treated with the DNA-ligase of T4 in a 30 dal sample in a buffer for ligation. The thus-produced DNA preparation is used for tran formation of cells of E.coli C600 with selection of transfor- mants on a medium with amPicilling followed by selection of ems-clones on a medium with 200jug/ml. of chloramphenicol upon culturing of the cells at the temperature of 42 OC. Prom the thus-selected clones the plasmid DNA is isolated and studied by way of a restriction analysis. In the resul35 ting plasmid the EcoRI-Bg1II fragment coding the C-terminal portion of chloramphenicolacetyltran ferase in the vector pPR124 is replaced with the coding sequence of mature humanBl-interferon. This plasmid is denoted as pPR-IPN 1123 and it is used at the next stage of the engineering 1 220886o process.

Stage 3 - engineering of the plasmid pPR-IFN 1-13- ug of the DNA of the plasmid pPR-IFNl-I23 are cleaved by means of the restrictase BamHI in 150_pl of a buffer for restriction-I, the DNA is subjected to a phenolic deprote inization and precipitated with ethanol. The precipitate is-dissolved in 40,)Al of I H20-I For the limited degradation of the DNA by m6ans of 3 --115 exonuclease activity of the DNA-polymerase-I. E.coli, the resulting DNA preparation is subjected to incubation in a 150 P sample containing 50 mM of tris-HC1 (pH 8.0)0 l0jA of ehtylenediaminetetracetic acid,, 5 MM of MaC'21 100 _IuM of desoxyadenosinetriphosphate (and then desoxyguanosinetriphosphate) and 20 units of the DNA-polymerase. The reaction is stopped, the DNA is preci- pi.tated and the precipitate is again dissolved in 40')21 of H 2 0. 10', pZ of the resulting preparation are treated with the restrictase EcoRI in 50jul of a buffer for restriction-2, followed by a phenolic deproteinization and precipitation of the DNA with ethanol; the precipitate is dissolved in 30 ul of H 0. 2 The removal of the single-thread terminal regions of the resulting DNA preparation is effected using the SIendonuclease in a 100."il sample containing 30 MM of CH 3 COONa (PH 4-4)9 4.5 mM of ZnSO 4' 250 mM NaCl. 10yg of the DNA, 200 units of the SI-endonuclease. The reaction is conducted at the temperature of 20 OC, whereafter the mixture is subjected to a phenolic treatment and precipitation of the nucleic acid with ethanol. The precipitate is dissolved in 20 Jul of H 2 0. 4yg of the thus-obtained DNA preparation are treated with a Klenov fragment of the DNA-polymerase I E.coli under the above-described conditions to complete probable existing single-chain molecule terminals to the two-chain ones. The reaction is stopped by a phenolic depoteiniziation, the nucleic acids are pre- cipitated with ethanol and dissolved in 20JUl of H20' The ligation of the linear DNA molecules with the twochain terminals is conducted at the temperature of 16 OC in a 50 ul sample containing 60 mM of tris-HC1 (pH 7.6), "208866 MM of MgCl 29 10 mM of dithiothreitol, 4 MM of adenosinetriphosphate, 8% by mass of polyethylene glycol-6000, 4 ug of the DNA, 100 units of the DNA-ligase of T4. On completion of the reaction the sample is diluted by 4 times and precipitation of nucleic acids is effected by means of ethanol. 2 ug of the DNA dissolved in water are treated with the restrictase Bgl II in 30 ul of a buffer for restriction-2. the DNA is precipitated from the reactioTi mixture with ethanol, dissolved in water and treated with the DNA-ligase of T4 in 200,ul of a buffer for ligation. The resulting DNA preparation is used for transformation of cells of E.coli C600 as it has been described hereinbefore, followed by selection of the transformants on a medium with ampicillin upon culturing of the cells for one day at the temperature of 28 OC. Out of the thus-prepared transformants the ones having a reduced ability for growth at the temperature of 42 OC are singled. From these clones the plasmid DNA is isolated as described hereinabove and subjected to a restriction analysis. To identify the primary structure of the hybrid region of combining r

bosomes before the gene IFN/l in the thus-produced plasmid pPR-IFN,'1-13, 30 ug of the corresponding DNA are treated with the restrictase Hind II in 100 ul of a buffer for restriction-2, the preparation is applied onto a gradient (4-12%) polyacrylamide gel and subjected to electrophoresis in a tris-acetate buffer system. The DNA fragment about 190 b.p. long is eluted from the gel using the Maxam Gilbert method; it is then extended, by means of the DNA-ligase of T4, with BamHI linkers in a manner similar to the above-described procedure of the addition of Bgl II- linkers to the linearized plasmid pPR124. The DNA is precipitated from the reaction mixture by means of ethanol and the precipitate is dissolved in 20,ul of H 2 0, cleaved by means of the restrictase BamHI in 30.ul of a buffer for restriction-1, the DNA is subjected to a phenolic deproteinization, again precipitated with ethanol and dissolved in H 2 0.

The molecular cloning of the DNA fragment in the replicative form of the DNA of the bacteriophage M13MPIO 2 2 0 111, 8 6 ES formed upon cleavage with BamHI, selection of recombinant phages on an indicator medium containing 5,-bromo-4-chloro-3-indolyl- -D-galactoside, isolation of the single-chain phage DNA and determination of the primary structure of the 5 cloned fragment by the method of a limited matrix copying (method suggested by F.Senger) are carried out following standard procedures. The nucleotide sequence of the hybrid region of combining ribosomes found by the Senger method in the plasmid pPR- IFN 1-13 is the following: 5 1 -...

TAAGGAGGTTGCATG...-3 I, wherein TAAGGAGGT - SD-sequence of the cro of the phageAand ATG - methionine codon of fl-interferon.

Example 2

The strain E.coli VNIIGenetika VL 903 (pPR-IYV1-13) - producer of fibroblastic human 1-interferon is produced in the following manner.

The plasmid pPR,IFh$1-13 coding the synthesis of fibroblast1c human interferon is introduced, by transformation in a manner similar to that described in Example 1 hereinabove, into cells of the strain E.coli VNIIGenetika VL 903 (deposited at the All-Union Collection of Industrial Microorganisms of the All-Union Research Institute of Genetics and Selection of Industrial Microorganisms and registered under No. BKRM B-3546). The efficiency of transformation of cells of E.coli VNIIGenetika VL 903 is about 10 6 clones per)ug of the native DNA of the plasmid pPRIFN P-13. The transformants are selected on a medium containing ampicillin (100,pg/ml) after culturing of the cells for one day at the temperature of 28 0 C. From the selected clones the plasmid DNA is recovered and its identity with the DNA preparation pPR-IFL:1-13 is proven by means of a restriction analysis. The strain E.coli VNIIGenetika VL 903 (PPR-IFB 1-13) is thus obtained.

For the detexmination of productivity of the strain E.coli VNIIGenetika VL 903 (pPR-IFNJI-13) the plasmid containing cells are grown at the temperature of 28 0 C on a slant agarized standard Hottinger medium containing ug/ml of ampicillin for 14 hours. The biomass grown on 2208866 the slants is used for the preparation of the inoculation material. To this end, the cells are transferred into 750 ml Erlenmeyer flasks with 100 ml of the Hottinger medium containing 100"pg/ml of ampicillin and grown at the temperature of 28 0 C on a shaker at 240 r.p.m. for 6 hours. The optical density of the inoculation culture is 1.52.5 units.

The fermentation is carried out in a fermenter provid-ems for controlling pH, temperature, stirring ed with syst and aeration rate. For the fermentation a Hottinger medium with 100jug/ml of ampicillin and 10 g/l of glucose is used. The inoculation culture is introduced in an amount of 5-10% by mass. The culturing is conducted at a pH of 6.6-6.8 while maintaining this level by supplying ammonia water.

The first part of the fermentation is conducted at the tem- perature of 28 OC till the optical of 3.5 at 550 m, where after thermoinduction is effected by elevating temperature to 42-45 0 C for 5 minutes, then the fermentation is conti nued for 2 more hours at the same temperature.

On completion of the process, to determine the activi ty of interferon, cells from I ml of the cultural liquid are precipitated by centrifugation, the precipitate is sus pended in I ml of a 1% solution of sodium dedecylsulphate in a 0.02Mphosphate buffer with the pH of 7.2 containing 1% of 2-mercaptoethanol and heated for 2-4 minutes at the temperature of 100 0 C. Afterwards, the precipitate is separated by centrifugation and the activity of interferon contained in the supernatant fraction is determined by standard procedures or by protection of human diploid fibroblasts from the cytopathic effect of the virus of vesicular stomatitis or by the method of an immuno-enzymatic - analysis. As the standard references preparations - of_fi-interferon ("Torey". Japan) are used which are titrated against the standard leukocytic interferon MPC B 69/19 (Great Britain).

The activity of fibroblastic human interferon synthesized in cells of the strain E.coli VNIIGenetika VL 903 WR-IFN1-13) detexmined by various methods is more than 1 2 1 2208866 2xlO9 international units/litre of the bacterial culture.

For the determination of the share of the synthesized fibroblastic interferon in the total of the cell protein, the cells from 1 ml of the culture liquid are precipitated by centrifugation and treated ad described hereinbefore; the preparation is separated by way of electrophoresis in the presence of 0.1111o' of sodium dodecylsulphate in a 1% polyacrylamide gel following a standard procedure. The proteins separated in the polyacrylamide gel are dyed in a solution Kumassi R-250 "Servall (West Germany) following a standard procedure. The quantitative content of proteins in the zones is determined after scanning the gel in an automated lase densitometer. The identification of the zone corresponding to the mature fibroblast interferon synthesized in the cells (19 kD) is effected on the basis of comparison with the electrophoretic mobility of the marker proteins, as well as by means of immunoblotting of separated protein fractions onto nitrocellulose filters and identification of the zone of interferon by treating the filters in solutions containing mive monoclonal antibodies against.8- interferon and anti-mice rabbit antibodies conjugated with peroxidase from horse radish. After a corresponding dyeing procedure the zone of interferon appears as a dark brown band against a non-dyed background of the nitrocellulose filter.

The content of protein in the zone corresponding to 1-interferon is about 10% of the total protein of the plasmid-containing cells of E.coli VNIIGenetika VL 903 (PPR-IFN1-13)- Industrial Applicability

The recombinant plasmid DNA PPR-IFN1-13 according to the present invention coding the synthesis of fibroblastic human.fil-interferon is useful in the preparation of strains producing human,,El-interferon with a high activity.

The strain of bacteria Escherichia coli VNIIGenetika VL 903 (pPR-IFN1-13) according to the present invention - a producer of human_$l-interferon, is useful in the microbiological and medical industries.

1 - 18 - 220,8866

Claims (3)

C L A I M S:

1. A recombinant plasmid DNA pPR-Iffill-13 coding the synthesis of fibroblastc human_l-interferon characterized in that it has the size of 3, 900 b.p. and consists of the following units:

- BamHI-Bgl II fragment with the size of 3,400 b.p. of-the vector plasmid PPR134B produced from pPR40 and plIL23; - EcoRI-Sau3A fragment of the plasmid PIN fi-trp 7 with the size of 510 b.p. and having the following features:

- incorporates a region responsible for initiation of replication and its regulation - Col El-replicon, a gene of repressor cIts 857 of the phage_A, a gene of resistance to amicillin Ap r. a tandem of,,P-independent terminators of transcription of the phage fd, a regulatory area PROR and a SD- sequence of the gene_Acro coding the sequence of a mature fibroblastic human interferon IFNji wiih its own methionine codon; - the ligation of the regulatory area of the gene, cro,, coding part of.81-interferon and terminators of transcription fd is effected so that before the gene IFNfI a hybrid region of combining ribosomes is formed which has the following nucleotide sequence:

5,-...TAAGGAGGTTGCATG...-3 I. wherein TAAGGAGGT - SD- sequence of the gene cro of the phage_ ATG - methionine codon of interferon and directly after the terminating codon of the gene IFN l a tandem ofP-independent terminators of the transcription of the phage fd is located; - has unique regions of recognition of the restricta- ses ClaI whose coordinate is the beginning of count (0).

Pvu II (abount 1,110), Bgl II (about 1,480). AccI (about 1,870), Pvu I (about 3,360); deposited at the Collection of Cultures of Microorganisms of the All-Union Research Institute of Antibiotics and registered under No. 1825.

2. A method foe engineering a recombinant plasmid DNA pPR-IFNil-13 according to Claim 1. characterized in that the vector plasmid pPR124B engineered on the basis of the plasmids pPR40 and pML24 is cleaved by means of the res- Z _1 triction endonucleases E.coRI and BglII and ligation of the greater of the formed fragments of the vector molecule with the EcoRI-Sau3A fragment of the plasmid pIX A-trp 7 containing the coding portion of mature human 1-interfe5 ron; in the thus-produced recombinent plasmid pPR-IMIl123 nearing of the SD- sequence of the gene cro and the first (methionine) codon of 1-interferon is effected, wherefor the plasmid DNA is hydrolyzed by means of the restrictase BamHI, a portion of nucleotides is removed by means of the exonuclease activity of the DNA-polymerase I of E.coli manifesting itself under conditions of an incomplete set of nucleosidetriphosphates in the reaction mixture; the DNA is enzymatically cleaved by the restrictase EcoRI, treated with the SI-endonuclease to remove single- chain DNA regions, then the two-chain terminals are restored by means of a Klenov fragment of the DNA-polymerase I of E.coli and the resulting linear DNA molecules are cyclized by means of the DNA-ligase of the phage T4; the resulting preparation is used to transfer cells of E.coli -' transformants on a medium with am- C600 with selection o. picillin upon culturing of the cells at the temperature of 28 OC, followed by selection of clones exhibiting a reduced rate of growth at 42 OC, vinereafter from the selected clones the recombinant plamid DNA pPR-IMT,'1-13 is isolat25 ed.

3. A strain of bacteria Escherichia coli VNIIGenetika VL 903 (pPR-IFVI13) a producer of human_l-interferon containing the recombinant plasmid DNA pPR-ITVl-I3 according to Claim 1, produced by the method of genetic engineer30 ing by way of introducing the recombinent plasmid DNA pPR-INV1I3 into the bacteria Eschericia coli, deposited on 12.01.87 at the collection of cultures of microorganisms of the All-Union Research Institute of Antibiotics and registered under No. 1825.

Published 1985 a, The Patent Office, State House. 6671 Hig! Ho'borr.. London WClR 4TP Further copies inky be obtained froM The Patent Office. Sales Branch, St Mary Cray. Orpingtcn. Kent BR5 3RD- Printed by Multiplex techniques ltd, St Mary Cray, Kent. Con. 1/87.