CS274041B1 - Genically recombined bacterial cells escherichia coli - Google Patents

Abstract

The invention concerns gene recombined bacterial cells of Escherichia coli expressing protein p60 of antigen specificity of the shell glycoprotein of beef cattle leukaemia virus. The cells are stored in the collection of bacteria cells of the Experimental Oncology Institute of SAV in Bratislava under the designation UEO pEK17-5. The p60 protein produced by these cells is suitable for use in diagnostics of animals infected with beef cattle leukaemia virus by setting of the concentration of circulating antibodies against BLV antigens by methods of radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Potentially p60 protein produced by the bacterial cells pEK17-5 can be used for vaccination of animals against beef cattle leukaemia virus.

Description

SUMMARY OF THE INVENTION The present invention relates to a gene recombined bacterial cell of Escherichla coli producing a protein encoded by an expression cloned gene plasmid for the synthesis of envelope cells (BIT) of bovine leukemia virus (BLV).

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Bovine leukosis is an infectious disease, horizontally spreading, whose pududum is IILV. Virus-induced lymruprolllututively uchoronio Jo accompanied by zmonuini in the blood of infected animals, which was used in diagnostics. Recent early radioimmunoassay (R1A) and immunoczymatic (ELISA) tests are indirect and based on the presence of BLV antibodies in sera of infected animals / Devare, S.G., Chander,

5., Samagh, B.S., Stephenson, 3R .: Evalution of Radioimmunoprecipitation for Detection of Bovine Leukemia Virus Infection in Domestic Cattle. J. Immunol. 119 (199), 277; Ton1toner, C., Zajac, V., Bán, J .: A Simple and Inexpensive Method for Detection of BLV Infected Cattle Baued on Mudlfiod ELISA Prlnciplo. Zbl. Vet. Med. B. 29, (1902), 503.

In these assays, the antigens are Structural BLV proteins. The virus producers are predominantly embryonic sheep kidney burrows (FLK) infected with BLV / Van Rer Maaten, M.J., Miller, J.M .: Replication of Dovine Leukemia Virus in Monolayer Coli Cultures. Bibllo. Kacmatol. 43, (1976), 360. The virus produced by FLK cells into growth medium is concentrated from the medium and purified by high-speed centrifugation (Beckman centrifuge). The reduction of FLK virus by cells has a certain limit and decreases with long-term cultures. BLV CULTIVATION AND CELL PRODUCTS It is very demanding in terms of excellence, centrifuges, sterility and durable and expensive growth media enriched with nourishing calf sera. The calf serum proteins from the growth medium are very difficult to separate from the virus, which makes diagnostic evaluation more difficult because they are homologous proteins present, which are caused by pseudopositivity.

These deficiencies are eliminated by the gene recombined bacterial cells of Eschrichia culi ÚLU pEK17-5 producing p60 proteins of the antigen specificity of the envelope glycoprotein of bovine leukemia virus. The cells are stored in a collection of bacterial cells of the Institute of Experimental Oncology of the Slovak Academy of Sciences in Bratislava, ul. MS. Army 21, under the designation ÚEO pEK17-5.

UEO cells pEKL7-5 were obtained by transforming Escherichla cells with a strain of TK1046 / Wcinstock, GM, Rhys, C., Bermon, ML, Hampar, B., Jackson, D., Silhavy, TJ, Weisemann, J., Zweig, M. ; Open reading frame expression vectors: A general method for antigen production in Escherichia coli using protein fusions to O-galactosidation. Why. Natl. Acad. Sci. USA 80, (1983), 4432, an expression vector recombinant pORF1 in which a gene for the synthesis of envelope glycoproteins BLV-gp51 and gp30 is cloned. The bacterial cells thus prepared synthesize, in addition to their proteins, a novel non-glycosylated protein having a molecular weight of 50,000 to 60,000 daltons (hereinafter p60), which is the product of the introduced gene. The immunological properties of the new protein were tested by immunoelectrophoretic method (Western blotting) with sera from BLV infected cows. The p60 protein-producing ULU cells pCK17-i are immunologically reactive with sera from BLV infected animals. The protein does not react with the sera of non-infectious animals. No protein by mol. weight of 60,000 bacterial cells transformed with an expression plasmid without a gene that encodes BLV envelope glycoproteins does not react with sera from BLV infected animals. UEO cells pEK17-5 can be propagated in standard bacterial cell media - I.B, M9 / Maniatis, T., Fritch, E.F., Sambrook, 3 .; Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, 1982. Cells can be stored frozen at a temperature between -20 ° C and -20 ° C, respectively. in agar. Thawing yields viable cells that serve as a source of p60 protein.

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The literature on bovine leukemia has not been published to date on the preparation of antigenic structures via recombinant bacterial cells.

An example of the plasmid pIIYJD905-20, which contains a practically complete BLV gonome, with the restriction enzyme BglII (Biorad), yields a 1224 bp DNA fragment that represents U4 '. a BLV gene encoding envelope glycoproteins. This fragment is incubated with nuclease S1 and S4, respectively. Bal 31, and cloned into the pORF1 vector opened with the BamHI restriction enzyme (Biorad) and also nuclease S 4. E. coli strain MH3000 was transformed with this recombinant plasmid. The recombinant plasmids were separated from blue colonies grown on LB agar containing X-gol (5-bromo-4-chloro-3-indolyl-QD-galactoside) and red kiilones (niirii!) Containing Me Conkoy ngnro. Recombinant plasmid DNA was hybridized to a DNA fragment encoding the ³² P-labeled BLV envelope glycoproteins (Amersham). Restriction enzymes PvuII, PstI, Smal (Boehringer) were used to determine the orientation of the insert.

DNA from plasmids with the correct insert orientation was transfected into E. coli strain TK1046, and the isolated clones were monitored for bacterial protein expression by increasing the culture temperature from 30 ° C to 37 ° C. Bacterial cells were further disrupted by ultra-2-1-3-1 sound and lysed in 1.10 mol.l Tris pH 7.0, 2.10 mol.l phenylmethylsulfonyl fluoride (PMSF) and 2 mg lysozyme and electrophoretically separated in 10% polyacrylamide gel / Leammli. , UK: Cleavege of structural proteins during assembly of T4 bacteriophage head. Nature 227. (1970), 680]. Divided proteins were analyzed by Western blotting / Towbin, E., Staehelin, T., Gordon, J .: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procodure and soine applications. Why. Natl. Acad. Sci. USA 76, (1979), 4350] on nitrocellulose (Amersham). The tested bovine sera and monoclonal antibodies against different epitopes of gp51 were riaΰυιύύ 1: 2UU in puírl / 5 dried milk of either bow, 5.1Ο ^-2 mul.i ^{-1 of} Iris pil II.0, II. I 0 ² mol l ^'1 NaCl, 2.10 ^"3 mol l' ₀ ¹ CaCl, 0.2% Triton X-100 /. After 24 hours incubation at 4 ° C, -2 -1, protein-bound nitrocellulose was washed in TEN buffer / 1.10 mol.l Tris pH

7.B, 1.10 ¹ mol.l ^-1 NaCl, 1.10 ' ³ mol.l. ¹ EDTA / with 0.05h Tween 20 and incubated with ¹²⁵ Labeled Protein A (Pharmacia) for 2 hours. After nitrocellulose was washed in TEN buffer with 0.05 Tween 20, dried and bound radioactivity was monitored on film (Kodak). Explanation of the trivial names used: Tris = tris / hydroxymethylaminomethane; NaCl - sodium chloride; CaCl 2 = calcium chloride; Triton X-100 = octylphenolpolyethylene glycol ester;

EDTA = disodium salt of ethylenediaminotetraacetic acid.

The amount of bacterially produced p60 protein under optimal conditions is 1 to 4% of total bacterial protein, which is sufficient to utilize the protein in diagnostics. It is highly sensitive and specific in response to antibodies to glycoprotein gp51.

The obtained bacterial cells of UEO pEK17-5 have industrial use at very low production costs (fermentation culture) as a source of p60 protein, which can be used in the diagnosis of BLV infected animals, in radioimmunoassay (RIA) or immunoenzymatic assay (ELISA) can replace viral proteins and served to quantitatively and qualitatively determine circulating antibodies against the virus. Potentially, the use of these cells, respectively, is possible. isolated p60 protein for animal vaccination.

Claims (1)

Gene recombined bacterial cells of Escherichia coli ÚEO pEK17-5 producing the p60 protein of the antigen specificity of the envelope glycoprotein of bovine leukemia virus,