DD292927A5 - METHOD FOR THE PRODUCTION OF THE RECOMBINANT CAPSIDAL STRUCTURE HBCAG DELTA-PRES2 / ADW, WHICH CONSTITUTES THE COREANTIGEN OF HEPATITIS B VIRUS WITH THE EPITOPE HBVPRES2 / ADW EXPOSED TO ITS SURFACE - Google Patents

Abstract

The invention relates to a method for the production of the recombinant capsid HBcAgD-preS2 / adw, which represents the core antigen of the hepatitis B virus with the epitope HBVpreS2 / adw exposed on its surface. The essence of the invention is that the fragment of the S gene of the hepatitis B virus, subtype adw, referred to as preS2 / adw with a length of 180 base pairs (bp) in the restriction site Cla I of the vector plasmid pHBc 1615 to form the recombinant Gens HBcAgD-presS2 / adw is incorporated. The producer strain of HBcAgD-presS2 / adw is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pHBV 32-46. This producer strain makes it possible to produce the recombinant capsid structure HBcAgD-presS2 / adw, which represents the hepatitis B virus (HBV) core antigen with the preS2 / adw epitope exposed on its surface {Kapsid; Core antigen; Hepatitis B virus; preS2 region; epitope; recombinant DNA; genetic engineering; Escherichia coli; Diagnostic Virology; Vaccine}

Description

For this 2 pages drawings

Field of application of the invention

The invention relates to the recombinant capsid structure HBcAg Δ-preS2 / adw, which represents the core antigen of the hepatitis B virus with the epitope HBVpreS 2 / adw exposed on its surface.

Characteristic of the known state of the art

The possibility of effective synthesis of capsid-like recombinant particles in E. coli representing the core antigen (HBcAg) of human hepatitis B virus (Borisova, GP, et al., Dokl. Akad. Nauk SSSR, 1984, 279, 1245- 1249 Borisova, GP, et al., Biopolimery i kletka, 1985, 1-109-105) and exposing on their surface the complete amino acid sequence of the preS2 / subtype ayw has been shown: HBcAg-preS 2 (2-54) and HBcAg Δ -PreS 2 (1-55). The fusion proteins contain the preS2 region from the 2nd to the 54th and from the 1st to the 55th amino acid, respectively. The fusion protein HBcAg-preS 2 (2-54) contains the complete sequence of HBcAg, while the fusion protein HBcAgA-preS2 (1-55) lacks the 39 C-terminal amino acids of HBcAg. These proteins are produced as a result of the expression of recombinant genes in which the coding sequence of the preS2 region excised from the cloned HBV genome of the subtype ayw (Pumpen, PP, et al., Dokl. Akad. Nauk SSSR, 1981, 260, 1022-1 024) into which codon for the 144th amino acid of HBcAg is inserted. Such capsid-like structures expose on their surface preS2 epitopes of the HBV subtype ayw. They have the immunogenic properties of preS2, HBc and HBe antigens and can be used in diagnostics. In addition, the preS 2 antigen can induce HBV-neutralizing antibodies (Neurath, R.S., et al., Science, 1984, 224, 392, loth, Y.E., et al., Proc. Natl. Acad., U.S.A., 1986, 83, 9174). Thus, such recombinant proteins can serve as components of diagnostics and / or vaccines. HBcAg-based capsids that exhibit on their surface the preS2 region of HBV subtype adw have not previously been described.

Object of the invention

The aim of the invention is recombinant K ^r;. Sidstrukturen with high immunogenicity and high yields to produce.

Presentation 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 (HBV) with the epitope HBVpreS2 / adw exposed on its surface, and to provide a producer strain for such structures.

This object is achieved according to the invention by obtaining a producer strain E. coli (pHBV 32-46) as a result of the construction of the recombinant gene HBcAgA-preS2 / adw, which codes for the synthesis of the corresponding protein, and the expression plasma pHBV 32-46. which ensures the production of the recombinant capsid structure HBcAg A-preS2 / adw with a share of 13.2% in the total protein of E. coli.

The recombinant plasmid pHBV 32-46, the construction of which is shown in Fig. 1, consists of the following elements:

DNA of the plasmid pHBc1615, a variant of the plasmid pHBc3, which contains the gene of HBcAg with an optimized translation initiation region, and a mutant HBcAg gene having a polylinker insert in the codon for the 144 amino acid, which is known for the Incorporation of foreign sections in this area is provided; Length 6900bp

Fragment of the HBV genome, subtype adw, which corresponds to the section between the restriction sites Eco Rl ₀ and Eco 130I ₁₈₀ and encodes the region of the preS2 (4th to 55th amino acid) and 9 amino acids of the S antigen (length 180 bp). The size of the plasmid is 7080Bp.

The DNA of the recombinant plasmid pHBV 32-46 contains the genes:

- HBcAgA-preS2 / adw gene, which ensures the synthesis of the final product,

- Gene bla, which confers ampicillin resistance.

The gene HBcAgA-preS2 / adw is under the control of a tandem promoter P, _rp . The plasmid pHBV 32-46 is amplified non-conjugatively after addition of chloramphenicol in the medium. The essence of the construction of the plasmid pHBV 32-46 is that the fragment of the S gene from HBV, subtype adw,

referred to as preS2 / adw, having a length of 180 bp into the restriction site CIaI of the vector plasmid pHBc1615 to form the recombinant gene HBcAg A-preS2 / adw.

The producer strain of HBcAgA-preS2 / adw is obtained by transformation of E. coli K802 cells with recombinant Plasmid DNA pHBV 32-46. Morphological characteristics: gram-negative rod-shaped cells, cultural features: cells grow on common nutrient media,

form colonies of medium size,

Physico-biochemical characteristics: optical culture temperature 37 ⁰ C, pH optimum 7.0-7.4. As a carbon source Hydrocarbons, as nitrogen source both mineral salts and organic compounds in the form of peptone, tryptone

and amino acids used.

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, also by 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 WKPM W 5240 deposited. embodiment

The invention will be illustrated in more detail below using an exemplary embodiment. The corresponding pictures show>

Fig. 1: Construction scheme of the recombinant plasmid DNA of the invention pHBV 32-46 Fig. 2: Determination of preS2 activity by immunoblotting.

The realization of the method according to the invention takes place in 2 process steps:

1. Construction of recombinant plasmid DNA pHBV 32-46

2μρ of the plasmid pHBc1615, obtained according to standard methods, is cleaved with 2 units of the restrictase CIaI in 25μΙ of the solution A (1 OmM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl ₂ ) for 2 hours at 37 ⁰ C. Die Restrictase is inactivated for 15 minutes at 65 ° C. The linearized plasmid DNA, 6900 bp in length, is isolated from a 0.8% agarose gel and resuspended in 20μΙ H ₂ O (DNAI).

20mg of plasmid pAE 10, the adw the complete genome of the HBV genome, subtype contains, is cleaved with 50 units of Eco RI and 50 units restrictase the restrictase Eco 1301 100 μΙ of the solution A16 hours at 37 ⁰ C. After the Incubation, the fragment mixture is applied to a 2% Agaiosegel and the fragment with a length of 180 bp, which carries the epitope preS2 / adw eluted by known methods from the gel. The fragment is resuspended in 10μΙ H ₂ O (DNA2). 0.05 μg of DNA2 and 0.1 μg of DNAI are mixed and the overhanging ends are mixed in 20 μl of solution B (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 25 mM NaCl) by adding in each case 100 μM dATP, dCTP , dTTP and dGTP and 5 units of E. coli DNA polymerase (Klenow fragment) were filled in at room temperature over one hour. The DNAs are connected by means of 10 units T4 DNA ligase in 10μΙ of the solution B with the addition of 1 mM ATP in the course of 12 hours at 4 ⁰ C to the reaction mixture are ΙΟΟμΙ E. coli RR1 cells, previously with 10OmM CaCl ₂ (final concentration 5 × 10 ⁹ cells / ml) were 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 is designated pHBV 32-46. Figure 1 shows the construction scheme of this plasmid.

2. Transformation of the plasmid according to the invention into a producer strain

The producer strain is obtained by transformation of E. coli K802 with the recombiriant plasmid pHBV 32-46. The cells are cultured in M 9 medium with addition of 10 g / l casamic acids, 2 g / l glucose and 0.02 g / l ampicillin to an optical density of OD ₆ Gd 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 M NaCl; 0.1% Triton X100; 0.005 M EDTA and 3 mg / ml lysozyme. After 30 minutes incubation at 4 ⁰ C, the cells are subjected to three repeated freeze / thawing. Then deoxyribonuclease and MgCl ₂ are added to make final concentrations of 20 .mu.g / ml and 1OmM and incubated again for 30 minutes at 4 ⁰ C. The lysate is treated for 30 seconds with ultrasound and then centrifuged (1000Ox g, 4 ⁰ C).

The recombinant protein HBcAgA-preS2 / adw is purified as follows: The lysate is fractionated with ammonium sulfate. HBcAg A-preS2 / adw 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 X100 dissolved, up to one Final concentration of the protein of 25-50 mg / ml. 2-4ml of this solution is applied to a Sepharose CL4 B column

(2.1 cm x 75 cm) balanced with the same buffer (only without Triton X100). Collect the fractions with HBcAg activity (see below) and use them immediately or after concentrating by reprecipitation with 50%.

Ammonium sulfate. The characterization of the recombinant capsid structure according to the invention is carried out as follows: Determination of HBcAg Activity by Radial Immunodiffusion (RID) The HBcAg titer is determined by radial immunodiffusion according to Ouchterlony. For this one prepares one Serial dilution series (1: 2 ", η is 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 are in the Table 1 listed. Table 1 Synthesis of HBcAg A-preS2 / adw in E. coli K802 cells carrying recombinant plasmid DNA pHBV 32-46 Strain OD ₆₆₀ titer in the RID protein HBcAg yield of

mg / ml mg / ml product (%)

E.coli

(pHBV 32-46) 4.0 1: 128 15.0 2.0 13.2

Determination of preS2 activity by immunoblotting For immunoblotting, the cells (6 mg) are resuspended in 100 μl of Laemmli sample buffer containing 2% SDS and 2%. Contains mercaptoethanol, and lysed by heating in a boiling water bath for 2 minutes. The obtained lysates (2-4 mg / ml Protein) is applied to gradient polyacrylamide gel (12-23%) (size 150mm χ 150mm χ 0.75mm). 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 monoclonal with the murinon Antibody MAb E at a 1: 500 dilution in TBS buffer (50 mM Tris-HCl, jH 7.5, 15 mM NaCl, 0.1% Triton X100 + 1%). BSA) for 15 hours at 2O ⁰ C incubated. After washing three times in TBS-buffer by incubating the filters for 2 hours at 20 ⁰ C with

the peroxidase-labeled conjugate of the antibodies against mouse immunoglobulin (dilution 1: 200). The filters are rinsed with

TBS buffer (3-5x) and developed with diaminobenzidine. The cell lysates of the producer strain exhibit the occurrence of specific staining zones which are associated with a protein

corresponds to a molecular weight of 21.3 kD (length 205 amino acids), which is in accordance with the

Construction scheme in Figure 1 stands. The control rate of E. coli K802 cells (pHBc3) does not show such zones. Electron Microscopic Characterization of Recombinant Capsid Structures HBcAg A-preS2 / adw The electron microscopic characterization of recombinant capsid structures HBcAgA-preS2 / adw becomes as follows

performed:

The preparations of the purified protein HBcAg A-preS2 / adw (see above) are by negative contrasting under Using 1% uranium acetate and in an electron microscope at an acceleration voltage of 8OkV and

examined a 10000X magnification. The protein HBcAg A-preS2 / adw forms capsids with a diameter of

Claims (3)

1. Process for the preparation of the recombinant capsid structure HBcAg Δ-preS 2 / adw, which is the Coreantigen of the hepatitis B virus with the epitope exposed on its surface HBVpreS 2 / adw, characterized in that the 39C-terminal amino acids of HBcAg by the sequence of the epitope preS2 / adw (4-55th amino acid) and 9N-terminal Amino acids of HBsAg are replaced. 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pHBV 32-46 with a size of 7 080 bp, consisting of - the DNA of the plasmid phbC 1615 with a length of 6900 bp, which under control of a tandem promoter P _trp which contains the bla gene, which mediates the ampicillin resistance, and the HBc gene, and the fragment of the HBV genome, subtype adw, which corresponds to the sequence between, the restriction sites Eco Rl ₀ and Eco 1301 ₁₈₀ with a length of 180 bp and the region of the preS2 region from the 4th to the 55th amino acid and the 9 N-terminal amino acids of HBsAg encoded and incorporated in the restriction site Cia I of the DNA of the plasmid pHBc 1615, the synthesis of the recombinant capsid structure HBcAg Δ-preS 2 / adw allows. 3. The method of claim 1 and 2, dadu. ^ H characterized in that one obtains the producer strain of HBcAg Δ-preS 2 / adw by transformation of E. coli K802 cells with recombinant plasmid DNA PHBV36-46.