DD286820A5 - METHOD FOR THE PRODUCTION OF THE RECOMBINANT PROTEIN HBCAG DELTA-HIVP24, WHICH CONSTITUTES A FUSION FROM THE COREPROTEINS OF HEPATITIS B VIRUS AND HUMAHEM IMMUNODEFICIENCY VIRUS 1 - Google Patents

Abstract

The invention relates to a method for the production of the recombinant protein HBcAgd-HIVp24, which is a fusion of the core proteins of hepatitis B virus and human immunodeficiency virus 1. The essence of the invention is that the fragment of the gag gene of HIV 1, which encodes 13 amino acids of the p17, 231 amino acids of p24 and 74 amino acids of p15, is inserted into the codon for the 144th amino acid of HBcAg. Termination of the translation occurs immediately behind the gag region at a stop codon of the pUC segment. The producer strain of HBcAgd-HIVp24 is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pHIV 24-5. This producer strain allows the production of the recombinant protein HBcAgd-HIVp24, which is a fusion of the core proteins of hepatitis B virus and human immunodeficiency virus 1. {Kapsid; Core antigen; Hepatitis B virus; Human immunodeficiency virus; epitope; recombinant DNA; genetic engineering; Escherichia coli; Diagnostic Virology; Vaccine}

Description

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Field of application of the invention

The invention relates to the recombinant protein HBcAgA-HIVgp24, which is a fusion of the core proteins of hepatitis B virus and human immunodeficiency virus 1.

Characteristic of the known state of the art

Recombinant structures are known which carry epitopes of the core protein ρ 24 of HIV 1 and are obtained on the basis of bacterial proteins, either by direct expression (1) or in fusion with bacterial proteins (2). Such recombinants are intended for diagnostic use only because they are not capable of self-assembly and formation of native protein structures. This results in the main disadvantage of the known recombinant structures, which is in a low immunogenicity.

A prospective carrier foreign to antigenic determinants is the coreantigen (HBcAg) of the hepatitis B virus (HBV) (3), which retains the ability to self-assemble even in the presence of extensive extraneous insertions. In addition, the inserted epitopes on the surface can be exposed on the basis of HBcAg conserved capsids. But even in the absence of self-assembly of the monomeric subunits of HBcAg to capsids, z. B. when inserting long foreign sequences (100 and more amino acids), the HBcAg and its derivatives are characterized by high immunological activity, because the HBcAg component is able to stimulate the T cell immune response (4).

Recombinant structures that carry epitopes dos ρ24 from HIV 1 and are capable of forming capsids, as well as recombinants of ρ24 based on eukaryotic proteins are not known.

Object of the invention

The object of the invention is to obtain a recombinant protein which has the immunological activity of both the HBcAg and the ρ 24 of HIV 1 and can be used both for diagnostic purposes and as a potential vaccine.

Explanation of the essence of the invention

It is an object of the invention to provide a recombinant protein which comprises a fusion of the core proteins of the bovine hepatitis B virus and the human immunodeficiency virus 1, and the creation of a producer strain for this recombinant protein.

This object is achieved according to the invention by obtaining as a result of the construction of the recombinant gene HβcAgA-HIVp24 which codes for the synthesis of the corresponding protein and the expression plasmid pHIV 24-5 o in the pathogen strain E. coli (pHIV 24-5), which ensures the production of the recombinant protein HBcAgA-HIVp24. The recombinant plasmid pHIV 24-5. its construction scheme is shown in Fig. 1, consists of the following elements: - DNA of the plasmid pHBc 5 (3), a variant of the plasmid pHBc 3, which contains the gene of HBcAg with an optimized initiation area for translation (5), and a deleted HBcAg gene that lacks the sequence for the 39 C-terminal amino acids of HBcAg and that has a unique restriction site for the Eco Rl restriction enzyme in the 144th amino acid codon; Länge6700Bp

Fragment of the HIV1 genome isolated from the plasmid pBH 10 (6) corresponding to the section between the restriction sites Pvu II _MI and Dgl H ₁₄₄₀ and comprising a region of the gag gene containing 13 amino acids of ρ 17.221 amino acids of ρ24 and 74 amino acids of ρ 15 encoded; Length 949 bp

Fragment of Piasmidi pUC 19 which corresponds to the section between the restriction sites Bam HI ₄₁₇ and Pvu H ₆₂₈ and has a stop codon in the same reading frame as the gag protein sections; Length 211 bp

The size of the plasmid is 7900bp (molecular weight 5.06MD). The DNA of the recombinant plasmid pHIV 24-5 contains the genes:

- gene HBcAgA-HIVp 24, which ensures the synthesis of the final product;

- Gene bla, which confers ampicillin resistance.

The gene HBcAgA-HIVp24 is under the control of a tandem promoter P " _p . The plasmid pHIV 24-5 is amplified after addition of chloramphenicol in the medium nlcht-conjugative. The essence of the construction of the plasmid pHIV 24-5 is that a Pvu II fragment of the plasmid plNG 3, which contains the Genome section of HIV 1 between the restriction sites Pvu ΙΙ _Μ ι and BgI ΙΙι _Μ0 and a Barn HU ^ -Pvu ll _iJe -Fragmont of Plasmid pUC 19 contains, in the restriction site Eco Rl of the vector plasmid pHBc 5 untor formation of the recombinant gene HBcAgA-HIVgp24, which is a protein of 144 amino acids HBcAg, 13 amino acids ρ 17.231 amino acids ρ24 and

74 amino acids p16 encoded.

The producer strain of HBcAgA-HIVgp24 is obtained by transformation of E. coli K802 cells with recombinant Plasmid DNApHIV24-5. Morphological features: gram-negative rod-shaped cells, Cultural features: Cells grow on common nutrient media, form colonies of medium size, Physico-biochemical characteristics: optimal cultivation temperature 37 ⁰ C, pH optimum 7.0-7.4. As a carbon sequestration

Hydrocarbons, used as nitrogen source both mineral salts and organic compounds in the form of pepion, tryptone and amino acids.

Antibiotic resistance: resistant to ampicillin due to the presence of the plasmid. The

Presence of plasmid DNA in the cell strain is confirmed by control of ampicillin resistance, as well as recovery and analysis of plasmid DNA.

The cell strain is in the collection of the Central Museum of Industrial Microorganisms of the USSR under the number WKPMW4970deponieit.

exemplary The invention will be illustrated in more detail below using an exemplary embodiment. The accompanying FIG. 1 shows the construction scheme of the recombinant plasmid DNA pHIV 24-5 according to the invention. The realization of the method according to the invention takes place in 2 process steps:

1. Construction of recombinant plasmid DNA pHIV 24-5

2pg plasmid pHBc 5, obtained by standard methods, is cleaved with 5 units of the restriction enzyme Eco Rl in 25 μΙ of

Solution A (10OmM Tris-HCl, pH 7.5; 1OmM MgCl _2; 5OmM NaCl) for one hour at 37 ⁰ C. The restrictase inactivating 15 minutes

1OmM MgCl _2;; 1OmM at 65 ^C C. The filling of the protruding ends (5OmM Tris-HCl, pH 7.5 is carried out in the solution B ΙΟΟμΙ

DTT; 25 mM NaCl; 100 μΜ dATP, dCTP, dGTP, dTTP and 5 units DNA polymerase from E. coli / Klenow fragment) in 1 hour at 12 ° C. After purification in the agarose gel, by means of a known method, the DNA is dissolved in 20μΙ H ₂ O.

20 μg of the plasmid pIVG 3, which carries the fragment Sac I ₂₂₄ -BgI Hie ") of the HiV 1 genome, with 50 units of

Restrictionase Pvu II in ΙΟΟμΙ of solution C (1OmM Tris-HCl, pH 7.5, 5OmM NaCl, 1OmM MgCl ₂ ) in the course of 2 hours at 37 ° C.

cleaved and the fragment mixture applied to a 1.5% agarose gel. The 1159 bp Pvu II fragment (molecular weight 0.74 mM), which encodes the complete ρ24 with flanking sequences, is extracted from

Agarose gel in light melting agarose according to known method (DNA2).

0.25 μg of DNA 1 and 0.1 μg of pgDNA2 are combined by means of Ti-DNA ligase in 10 μl of solution D (50 mM Tris-HCl, pH 7.5; 10 mM

MgCl ₂ ; 1OmM DTT; 50μΜ ATP and 10 units of T ₄ DNA ligase) within 12 hours at 4 ⁰ C. To the reaction mixture was 100μΙ cells E. coli RR1, which had been vorhor with 10OmM CaCl ₂ bohelt

(Final concentration 5x10 'inches / ml) added to transform the recombinant plasmid DNAs.

The selection of the clones is carried out by means of the restriction analysis of the plasmid DNA, which had been isolated by a rapid procedure. The plasmid with the expected restriction cleavage pattern receives the name pHIV 24-5 (FIG. 1).

2. Transformation of the Inventive Plasmid Into a Prodocyzone Strain

Don Produzenttnstamm obtained by transformation of E. coli K802 with dom recombinant plasmid pHIV 24-5. The cells were grown in M9 medium supplemented with 10g / l Kasaminsäuron, 2g / l glucose and 0.02 g / l ampicillin grown to optical density of OD einor _e «o 4-5. The cells are collected by centrifugation and lysed in four times the volume of a buffer containing 0.05M Tris-HCl, pH 8.0, 0, 15M NaCl, 0.1% Triton X 100, 005MEDTA, and 2 mg / ml lysozyme. Nach30minütiger incubation at 4 ^C C, the cells are ausgeseit a three-times freezing / thawing. Subsequently Desoxyribonukleaso and MgCl ₂ are added to final concentrations of 70μg iu / ml and 1OmM and incubated again for 30 minutes at 4 ^C C. The Lysnt is centrifuged (1000Ox g; 4 ^C C) and the pellet in a buffer of G2.5 mM Tris-HCl, pH 7.8; 2% SPS and 2% ß-mercaptoethanol resuspended

 The characterization of the recombinant protein according to the invention is carried out as follows:

Determination of the Immunological Activity of the Recombinant Protein HBcAgA-HIV ρ24 by Immunoblotting For immunoblotting the cells are resuspended (6 mg) in 100 μl Laemmli sample buffer containing 2% SOS and 2% β-mercapto-ethanol and lysed by heating in a boiling water bath for 2 ml. The resulting lysates (2-4 mg / ml protein) are applied to a gradient polyacrylamide gel (12-23%) (size 150mm χ 150mm x 0.75mm). The electrophoresis is carried out for 15 minutes at a current of 7 mA.

After electrophoresis, the proteins are transferred to nitrocellulose HAWP. The filters are incubated with murine monoclonal anti-p24 antibody 4/1/1 at 1: 500 dilution in TBBS buffer (5mM Tris-HCl, pH 7.8, 15 mM NaCl, 0.1% Triton X100) with 1% BSA for 15 hours Incubated at 20 ° C. After washing three times in TBS-buffer by incubating the filters for 2 hours at 2O ⁰ C with the peroxidase conjugate the antibodies against mouse immunoglobulin (1: 500 dilution). In parallel, the filters are incubated with the monoclonal antibody 14 EII (anti-HBc) at a dilution of 1: 5015 hours at 2O ⁰ C, washed as described above, and incubated with the peroxidase conjugate of the protein A of S. aureus 2 Hours at 2O ⁰ C. After incubation with the peroxidase conjugates, the filters are washed in both batches with TBS buffer (3-5 times) and developed with diaminobenzidine.

The cell lysates of the producer strain thereby show the occurrence of specific staining zones which correspond to a protein with a molecular weight of 49 kD (or a length of 462 amino acids), which is in accordance with the construction scheme in FIG. The control rate of E. coli K802 cells (pHBc 3) does not show such zones.

Claims (3)

1. A method for producing the recombinant protein HBcAgA-HIVp2, which is a fusion of the core proteins of hepatitis B virus and human immunodeficiency virus 1, characterized in that in the 144th amino acid of HBcAg a gag portion is incorporated, the 13 Amino acids ρ 17.221 amino acids p24 and 74 amino acids ρ 15. 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pHIV 24-5 with a size of 7900bp (molecular weight 5.06 MD), consisting of the DNA of the plasmid pHBc 5 with a length of 6700 bp, which encodes the gene bla, which mediates the ampicillin resistance, and the HBc gene, which encodes only the 144N-terminal amino acids of the HBcAg and under the control of a tandem promoter P _trp , stands, contains, the fragment of the HIV 1 genome isolated from pBH 10 which corresponds to the sequence between the restriction sites Pvu H ₆₉₁ and Bgl II _1M o with a length of 949 bp, and the fragment of the plasmid pUC 19 which corresponds to the sequence between the restriction sites Bam HI ₄₁₇ and Pvu II ₆ 28 with a length of 211 bp, allows the synthesis of the recombinant protein HBcAgA-HIVgp 24. 3. The method of claim 1 and 2, df - "urch in that one obtains the producer strain of HBcAgA-HIVp24 by transformation of E. coli K802 cells with recombinant plasmid DNA pHIV 24-5.