DD286819A5 - METHOD OF GENERATING THE RECOMBINANT CAPSIDAL STRUCTURE HBCAG DELTA-HIVGP41 (78-129), WHICH SHOWS THE COREANTIGEN OF THE HEPATITIS B VIRUS WITH THE EPITOP HIVGP41 EXPOSED TO ITS SURFACE (78-129) - Google Patents

Abstract

The invention relates to a process for the production of the recombinant capsid structure HBcAgd-HIVgp41 (78-129), which represents the coreantigen of the hepatitis B virus with the surface-exposed epitope HIVgp41 (78-129). The essence of the invention is that the fragment of the env gene of HIV 1, designated HIVgp41 (78-129), with a length of 153 base pairs (bp) in the restriction site Cla I of the vector plasmid pHBc 1615 to form the recombinant gene HBcAgd-HIVgp41 (78-129) is incorporated. Due to a deletion in the vector plasmid, the coding sequence for the C-terminal part of the HBcAg is lost so that the termination of the translation takes place immediately after the epitope HIVgp41 (78-129). The producer strain of HBcAgd-HIVgp41 (78-129) is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pHIV 41-9. This producer strain makes it possible to produce the recombinant capsid structure HBcAgd-HIVgp41 (78-129), which represents the core antigen of the hepatitis B virus with the surface epitope HIVgp41 (78-129). {Kapsid; Core antigen; Hepatitis B virus; Human immunodeficiency virus; Transmembrane protein; 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 capsid structure HBcAgA-HIVgp41 (78-129), which represents the core antigen of the hepatitis B virus with the epitope HIVgp41 (7C 129) which is more highly exponentiated on its surface.

Characteristic of the known state of the art

Recombinant structures are known which carry epitopes of the transmembrane protein gp41 of HIV 1 and are derived on the basis of bacterial proteins but have no capsid-forming abilities (1-5). Their disadvantage consists in a low immunogenicity, which is caused by the fact that the inserted epitopes do not have their native conformation. Another obstacle to the use of such proteins as vaccines is the high proportion of bacterial i'rotein.

Recombinant capsid structures carrying HIV 1 protein epitopes, including gp41, are unknown.

Object of the invention

The aim of the invention is to inexpensively produce recombinant capsid structures with high immunogenicity and high yields.

Explanation of the essence of the invention

The object of the invention is to obtain recombinant capsid structures representing the core antigen of the hepatitis B virus with the epitope HIVgp41 (78-129) exposed on its surface, and to provide a produceront stem for such structures.

This problem is solved according to the invention, as a result of the construction of the rokomininnnten gene HBcAgA-HIVgp41 (78-129), which \ 's the synthesis of the corresponding protein, and the Proprssionsplasmids pHIV 41-9 a producer strain is obtained (E. coli / pHIV41 -9), which ensures the production of the recombinant capsid structure HBcAgA-HIVgp 41 (78-129) in a proportion of 0.4% of the total protein of E. coli.

The recombinant plasmid pHIV41-9, dosson construction scheme is shown in Fig. 1, consists of the following elements:

- DMA of the plasmid pHBc 1615 (6), a variant of the plasmid pHBc 3, which contains the gene of HBcAg with an optimized translation initiation region (7), and a mutant gene of HBcAg, which has a polylinker insert in the codon for the 144. has amino acid, which is intended for the incorporation of foreign parts in this area, has; Length 6700bp

Fragment of the HIV 1 genome isolated from the plasmid pBH 10 (8), which corresponds to the section between the restriction sites Sau 3A, and Hind III and encodes the region of gp41 (78-129); Length 153bp

The size of the plasmid is 6900 bp, the molecular weight is 4.41 MD.

The DNA of the recombinant plasmid pHIV 41-9 contains the genes:

Gene HBcAgA-HIVgp41 (78-129), which ensures the synthesis of the final product,

- Gene bla, which gives an ampicillin resist.

The gene HBcAgA-HIVgp41 (78-129) is under the control of a tandem promoter P _up . The Pias.'ι lid pHIV 41-9 is non-conjugative amplified after addition of chloramphenicol in the medium. The essence of the construction of the plasmid pHIV 41-9 is that the fragment of env-ion from KIV1, designated as HIVgp41 (78-129), 153 bp in length, into the restriction site CIa I of the vector plasmid pHBc 1615 to form the

Recombinant gene HBcAgA-HIVgp41 (78-129) is incorporated. Due to a deletion in the vector plasmid, the coding sequence for the C-terminal part of the HBcAg is lost, so that the termination of the translation immediately after the

Epitope, KIVgp41 (78-129) takes place. The producer strain of HBcAgA-HIVgp41 (78-129) is obtained by transformation of E. coli K802 cells

recombinant plasmid DNA pHIV 41 -9.

Morphological features: gram-negative rod-shaped cells, Cultural features: Cells grow on common nutrient media, form colonies of medium size, Physico-biochemical characteristics: optimal culture temperature37 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 peptone, 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 resistence, as well as recovery and analysis of plasmid DNA.

The cell strain is deposited in the collection of the Central Museum of Industrial Microorganisms of the USSR under the number WKPMW4971.

embodiment The invention will be illustrated in more detail below using an exemplary embodiment. The accompanying Figure 1 shows the construction scheme of the recombinant plasmid DNA of the invention pHIV41-9. The realization of the method according to the invention takes place in 2 process steps:

1. Construction of Recombinant Plasmid DNA pHIV 41-9

2μρ of the plasmid pHBc 1615, obtained according to standard methods, is cleaved with 4 units of the restrictase CIa I in 2SpI of

Solution A (1OmM Tris-HCl, pH 7.5, 5OmM NaCl and 1OmM MgCl ₂₎ for one hour at 37 ⁰ C. The restrictase inactivates one

15 minutes at C5 ° C. The overhanging endon is filled in 100 μl of solution B (50 mM Tris-HCl, pH 7.5; 10 mM

MgCl ₂ ), 10 mM DTT, 25 mM NaCl by addition of 100 μM each dATP, dCTP, dTTP and dGTP and 5 units of DNA polymerase

from E. coli (Klenow fragment) in the course of one hour at 12 ⁰ C. After purification in agarose gel (on DE-81 paper), according to known

Method, the DNA is dissolved in 20μΙ H ₂ O (DNA I).

20pg of the plasmid pSS 3.8, which carries the fragment SaI I _UH -Sac Igtsi dos HIV 1 genome, with 50 units of the

Restrictase BgI II in 100μΙ of the solution B (1OmM Tris-HCl, pH 7.5, 1OmM MgCl ₂ ) in the course of 2 hours at 37 ° C cleaved and

the fragment mixture is applied to a 1% agarose gel. The fragment Bgl H ₇₁₉₈ -Bgl Hm ₂₈ with the molecular weight of 0.51 MD (size 1430 bp) is extracted from the agarose gel in light melting agarose (DNA2).

5pg DNA2 was cleaved with 10 units of Sau 3A, and 10 units of Hind IM in 20μΙ the solution A in the course of 2 hours at 37 C. ⁹

The fragment sow 3A ₇₆ «-Hind ΙΙΙ _; Separate on slightly melting agarose and fill the overhanging ends as above

described on (DNA3).

0.1 μg of DNA I and 0.05 μg of DNA 3 are combined by means of 10 units of T ₄ DNA ligase in 10 μl of solution B with addition of 1 mM DTT and 5 mM ATP with an incubation time of 12 hours at 4 ° C.

To the reaction mixture are 100μΙ cells RR1, which had previously been treated with 10OmM CaCl ₂ (final concentration

5 x 10 ⁹ cells / ml) was added to transform the recombinant plasmid DNAs.

Dio selection of the clones is done by restriction analysis of the plasmid DNA, which had been isolated by rapid procedure. The plasmid with the expected P.estriklionsspaltmuster receives the name pHIV 41-9. FIG. 1 shows this Construction scheme of this plasmid.

2. Transformation of the Inventive Plasmid Into a Production Strain

Don producer strain is obtained by transformation of E. coli K802 with the recombinant plasmid pH IV41-9. Dio cells were cultured in M9 medium supplemented with 10 g / L casamine acids, 2 g / L glucose and 0.02 g / L ampicillin to an optical density of OD ₁₄₀ 4-5. The cells are collected by centrifugation and lysed in three volumes of a buffer containing 0.05 M Tris-HCl, pH 8.0, 0, 15 MNaCl, 0.1% Triton X100, 0.005 MEDTA and 3 mg / ml lysozyme. After 30 minutes incubation at 4 ^C C has been subjected to a three-times freezing / thawing dio cells. Subsequently Desoxyribonukleaso and MgCIj up to final concentrations of 20 mg / ml and 1OmM added and incubated for another 30 minutes at 4 ⁰ C. The lysate is sonicated for 30 seconds and then centrifuged (10000g, 4 ° C). The recombinant protein HBcAgA-HIVgp41 (78-129) is purified as follows:

The lysate is fractionated with ammonium sulfate. HBcAgA-HIVgp41 (78-129) is precipitated by 30% saturation with ammonium sulfate. The pellet is collected and dissolved in 0.04 M sodium phosphate, pH 8.0, 0, 15 M NaCl, 0.1% Triton X 100, to a final concentration of the proin of 25-50 mg / ml. 6-7 ml of this solution is applied to a column of Sepharose CI4B (2.1 x 75 cm), which was balanced with the same buffer (only without Triton X 100). The fractions with HBcAg activity (see below) are collected and used immediately or after concentration by repeated precipitation with 50% ammonium sulfate.

The characterization of the capsid structure according to the invention is carried out as follows:

Treatment of HBcAg Aktlvltat by means of radial immunodiffusion (RID)

The HBcAg titer is determined by radial immunodiffusion according to Ouchterlony. For this purpose prepare a dilution series (1: 2 \ η s 1) of the cell lysate and titrated against human anti-HBc antibodies.

As a standard, purified HBcAg from recombinant bacteria (1 mg / ml) is used. The results of the titration of HBcAg activity by RID are shown in Table 1.

Synthesis of HBcAgA-HIVgp41 (78-129) in E. coli K802 cells carrying recombinant plasmid DNA pHIV 41-9

Strain OD _iM titer in protein HBcAg yield of

RID mg / ml mg / ml product (%)

E.CONK802 4.0 1: 2 15.0 0.06 0.4

Determination of gp4Vactivation by immunoblotting

For immunoblotting, cells (6 mg) are resuspended in 100 μl Laemmli sample buffer containing 2% SDS and 2% β-mercaptoethanol and lysed by heating in the boiling water bath for 2 minutes. The obtained lysates (2-4 mg / ml protein) are applied to a gradient polyacrylamide gel (12-18%) (size 150mm χ 150mm χ 0.75mm). The electrophoresis is carried out for 15 hours at a current of 7 mA.

After electrophoresis, the proteins are transferred to nitrocellulose HAWP. The filters are incubated with the human monoclonal anti-gp41 antibody 3D6 at a dilution of 1: 500 in T8S buffer (50 mM Tris-HCl, pH 7.8, 15 mM NaCl, 0.1% Triton X 100.1% BSA). Incubated for 15 hours at 20 ⁰ C. After washing three times in TBS-buffer by incubating the filters for 2 hours at 20 ⁰ C with the peroxidase-labeled conjugate of antibodies gogen human Immungobulin (dilution 1:16).

The filters are rinsed 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 having a molecular weight of 21 kD (length 200 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)

A method of producing the recombinant capsid structure HBcAgA-HIVgp41 (78-129), which is the coreacting of the hepatitis B virus with the epitope HIVgp41 (78 to 129) exposed on its surface, characterized in that in the 144th amino acid of the HBcAg the sequence of the epitope HIVgp41 (78-129) with a length of 51 amino acids (153 base pairs) is inserted, wherein the 39 C-terminal amino acids of HBcAg are deleted. 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pHlV 41-9 with a size of 6900 bp (molecular weight 4.41 MD), consisting of the DNA of plasmid pHBc 1615, 6700 bp in length, containing the gene bla conferring ampicillin resistance and the HBc gene, under the control of a tandem promoter P, _fp , and the fragment of the HIV 1 genome which corresponds to the sequence between the restriction sites Sau 3 A ₇₅₆₅ and Hind _{HI 7718} with a length of 153 bp and encodes the region of gp41 from the 78th to the 129th amino acid and in the restriction site CIa I the DNA of the plasmid pHBc 1 615 is incorporated, allows the synthesis of the recombined capsid structure HBcAg-HIVgp41 (78-129). 3. The method according to claim 1 and 2, characterized in that one obtains the producer strain of HBcAgA-HIVgp41 (78-129) by transformation of E. coli K802 cells with recombinant plasmid DNA pHIV 41-9.