DD286818A5 - METHOD OF GENERATING THE RECOMBINANT CAPSIDAL STRUCTURE HBCAG DELTA-BLVGP 51 (56-103), WHICH SHOWS THE COREANTIGEN OF THE HEPATITIS B VIRUS WITH THE EPITOPLE BLVGP51 EXPOSED TO ITS SURFACE (56-103) - Google Patents

Abstract

The invention relates to a process for the production of the recombinant capsid structure HBcAgd-BLVgp51 (56-103), which represents the coreanting of the hepatitis B virus with the epitope BLVgp51 (56-103) exposed on its surface. The essence of the invention is that the fragment of the BLV env gene, designated BLVgp51 (56-103) of 143 base pairs (bp) in the Eco RV restriction site of the vector plasmid pHBc 1615, forms the recombinant HBcAgd gene. BLVgp51 (56-103) is inserted, deleting the coding sequence for the 39 C-terminal amino acids of HBcAg. The producer strain of HBcAgd-BLVgp51 (56-103) is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pBLV 51-3. This producer strain makes it possible to produce the recombinant capsid structure HBcAgdBLVgp51 (56-103), which represents the hepatitis B virus core (HBV) with the epitope BLVgp51 (56-103) exposed on its surface (numbering of amino acids of gp51 refers to the mature protein, without signal peptide). {capsid; Core antigen; Hepatitis B virus; Bovine Leukemia Virus; coat 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-BLVgp51 (56-103), which is the core antigen of the hepatitis B virus with the epitope BLVgp51 (56-103) exposed on its surface.

Characteristic of the known state of the art

Recombinant structures are known which carry epitopes of the outer envelope of the bovine leukemia virus and are obtained on the basis of bacterial proteins but have no capsid-forming abilities (.1). Their disadvantage is 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 protein.

Recombinant capsid structures carrying BLV protein epitopes, including gp51, 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 which represent the coreaning of the hepatitis B virus (HBV) with the epitope 3LVgp51 (56-103) exposed on its surface, and to provide a producer strain for such structures.

This problem is solved according to the invention in that, as a result of the construction of the recombinant gene HBcAgA-BLVgp51 (56-103), which encodes the synthesis of the corresponding protein, and of the expression plasmid pBLV 51 -3, a producer strain E. coli (pBLV 51-3) which ensures the production of the recombinant capsid structure HBcAgA-BLVgp51 (56-103) with an antoile of 26.7% of the total protein of E. coli.

The rokomininnnte plasmid pBLV51-3, whose design scheme is shown in Fig. 1, consists of the following elements:

DNA of the plasmid pHBc1615 (2), a variant of the plasmid pHBc3, which contains the gene of HBcAg with an optimized initiation area for translation (3), 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 6900bp

Fragment of the BLV genome (4) which corresponds to the section between the restriction sites BgL Hy ₃₈₃ and Bam HI ₅₂₂ S and encodes the region of the gpM (56th-103rd amino acid of the mature gp51) (length 143 bp).

The size of the plasmid is 7000bp, the molecular weight is 4.48md.

The DNA of the recombinant plasmid pBLV51-3 contains the genes:

- gene HBcAgA-BLVgp51 (56-103), which ensures the synthesis of the final product,

- Gene bla, which confers ampicillin resistance.

The gene HBcAgA-BLVgp51 (56-103) is under the control of a tandem promoter P ₁ , _p .

The plasmid pBLV 51-3 is amplified non-conjugatively after addition of chloramphenicol in the medium.

The essence of the construction of plasmid pBLV 51-3 is that the fragment of the env gene from BLV, designated BLVgp51 (56-103), with a length of 143 bp in the Eco RV restriction site of the vector plasmid pHBc 1615 to form dec recombinant gene HBcAgA-BLVgp51 (56-103) is inserted, deleting the coding sequence for the 39C-terminal amino acids of HBcAg.

The producer strain of HBcAgA-BLVgp51 (56-103) is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pBLV51-3.

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 source

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 resistance, 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 number WKPM W4969.

Ausführungsbelsplel

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

Fig. 1: Construction scheme of the recombinant plasmid DNApβLV 51-3 according to the invention Fig. 2: Electron micrograph of the capsid 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 pBLV51-3

2μρ of the plasmid pBHc 1615, obtained according to standard methods, is cleaved with 2 units of the restrictase .icoRV in 25μΙ of the solution AdOmM Tris-HCl, pH 7.5; 5 mM NaCl and 10 mM MgCl ₂ ) 30 minutes at 37 ° C. The Restraktase is inactivated for 15 minutes at 65 ° C. The linearized plasmid DNA, 6900 bp in length, is isolated from a 0.7% agarose gel and resuspended in 2OmIH ₂ O (DNAI).

20 μg of the plasmid pI IM 19-3 / 4 are cleaved with 50 units of the restriction enzyme Bgl II and 50 units of the restrictase Bam HI in 100 μl of the solution A for 2 hours at 37 ° C. After incubation, the fragment mixture is applied to a 1.5% agarose gel and the fragment having a molecular weight of 91 kO (length 143 bp) ₁ which carries the epitope BLVgp51 (56-103), according to known methods (6) from Geleluiort. The fragment is resuspended in 10μΙ H ₂ O. The overhanging ends are filled in 20 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 μl dATP, dCTP, dTTP and dGTP each of which contains 5 units of DNA polymerase from E. . coli (Klenow fragment) for one hour at 12 ⁰ C (DNA 2).

0,25pgDNAIund0,1 Mg 2 DNA is associated with aid of 10 units T ₄ DNA ligase in 10μΙ the solution B with the addition of 1OmM DTT and 50μΜ ATP in the course of 12 hours at 4 ^C C.

To Reak'.ionsions ΙΟΟμΙ cells E. coli RR1, which had previously been treated with 10OmM CaCl ₂ (final concentration 5 × 10 ⁹ cells / 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 is named pBLV51-3. 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 cam recombinant plasnide pBLV51-3. The cells are cultured in M9 medium supplemented with 10 g / L casamic acids, 2 g / L glucobium and 0.02 g / L ampicillin to an _OD6i0 4-5 optical density. The cells are collected by centrifugation and lysed in three volumes of a buffer containing 0.05M Tris-HCl, pH 8.0, 0, 15 M NaCI, 0.1% Ti 'ton X100, 0.005MEDTA and 3 mg / ml lysozyme. After 30 minutes incubation at 4 "C, the cells are subjected to three times freeze / thawing. Thereafter, deoxyribonuclease and MgCl ₂ are added to final concentrations of ΣΟμρ / ΓηΙ or 1OmM added and again incubated for 30 minutes at 4 ⁰ C, the lysate for 30 seconds with ultrasound. treated and then centrifuged (1000Ox g, 4 ⁰ C) The recombinant protein HBcAgA-BLVgp51 (56-103) is purified as follows.:

The LysiM is fractionated with ammonium sulfate. HBcAgA-BLVgp51 (56-103) is precipitated by 30% saturation with ammonium sulfate. The pellet is collected and dissolved in 0.04M Natrimphoshpat, pH8.0, 0.15M NaCl, 0.1% Triton X100, to a final protein concentration of 25-50mg / ml. 2-4 ml of this solution is applied to a column of Sepharose CL4B

(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 (RIO) The HBcAg titer is determined by radial immunodiffusion according to Ouchterlony. For this one prepares one Dilution series | 1: 2 ", of the cell stock 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 HBcAgrActivity by RID are listed in Table 1. Synthesis of HBcAgA-BLVgp51 (56-103) in E. coli K802 cells carrying recombinant plasmid DNA pBLV 51-3 Strain C ¹ De ₆₀ titer in protein HBcAg yield of RID mg / ml mg / ml product (%)

E.COÜK802 4.0 1: 256 15.0 4.0 26.7

pBLV51-3

Determination of gp51 activity by immunoblotting For immunoblotting, the cells (6 mg) are resuspended in 100 μl of Laemmli sample buffer containing 2% SDS and 2% β-buffer. 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 a gradient polyacrylamide gel (12-18%) (size 150mm χ 150mm x 0.75mm). Electrophoresis

is carried out for 15 hours at a current of 7mA.

After electrophoresis, the proteins are transferred to nitrocellulose HAWP. The filters are labeled with the murine monoclonal

anti-gp51 antibody mAb 14 at a 1: 500 dilution in TBS buffer (50 mM Tris-HCl, pH 7.8, 15 mM NaCl, 0.1% Triton

X100.1% BSA) for 15 hours at 20 ° C incubated. After washing three times in TBS buffer, incubate the filters for 2 hours

20 ⁰ C with the peroxidase-labeled antibody conjugate to 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 21.2 kD (length 200 amino acids), which is in accordance with dom

Construction scheme in Figure 1 stands. The control rate of E. coli K802-Zellon (pHBc3) does not exhibit such zones. Elastic Tetragonal Characterization of Recombinant Capillary Structures HBcAgA-BLVgp51 (56-103) The electron microscopic characterization of recombinant capsid structures HBcAgA-BLVgp51 (56-103) is as follows

performed:

The preparations of the purified protein HBcAgA-BLVgp51 (56-103) (see above) are removed by negative contrasting Use of 1% uranium sulfate Mat and in the electron microscope at an accelerating voltage of 8OkV and

examined a 400,000-fold magnification. The protein HBcAgA-BLVgp51 (5G-103) forms capsids with a diameter of 25nm (Figure 2).

Determination of gp51 Activity In the capsid structure HBcAgA-BLVgp51 (56-103) by SLISA

The solid phase used for the immune reactions is a 96-well polystyrene microtiter plate. The purified HBcAgA-BLVgp51 (56-103) (see above) is diluted to a concentration of 10 μg / ml in 50 mM Tris-HCl, pH 8.0; 0.15M NaCl and pipetted into all wells of the microtiter plate 100 μ I. After an incubation of one hour at 37 ⁰ C, the solution is removed from the wells, 4-5 times with distilled water. washed and dried. The wells of the plate are filled with murine monoclonal anti-gp51 antibody mAb 14 (see above). After one hour of incubation at 37 ⁰ C throw the microtiter plate with distilled water. rinsed. Subsequently, each ΊΟΟμΙ conjugate of horseradish peroxidase with antibodies to mouse immunoglobulin in a dilution of 1: 500 applied. After one hour, the microtiter plate is washed and the color reaction is carried out. In the wells are each filled ΙΟΟμΙ a solution of 0.25% Orthophenyldiamin-2HCI in 0.1 M sodium, pH5.0 and 0.02% H ₂ O ₂ and incubated for 30 minutes at 2O ⁰ C, the reaction is stopped by Add 100 μΙ0.1 NHCI. The appearance of browning indicates the presence of gp51 activity. For the quantitative determination of the activity one measures the four density at 492 nm (Tab. 2). The absence of a reaction in the negative control (HBcAg) indicates the specific binding of the anti-gp51 antibodies with the recombinant capsid structures HBcAgA-BLVgp51 (56-103) (Table 2).

Immunological reactivity of recombinant antigens in the ELISA Bound antigen Immunological reactivity (OD ₄ ä ₂ / ml)

anti-gp51 (mAb 14) anti-HBc (C1-3)

HBcAgA-BLVgp51 0.722 (56-103) HBcAg 0.016 (Control)

0.670 0.535

Claims (3)

1. Process for the production of the recombinant capsid structure HBcAgA-BLVgp51 (56-103), which represents the coreaning of the hopatitis B virus with the epitope BLVyp51 (56-103) exposed on its surface, characterized in that cjie 39 C- terminal amino acids of the HBcAg are replaced by the sequence of the epitope BLVgp51 (56-103) with a length of 47 amino acids (143 base pairs, amino acid positons refer to the mature gp51). 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pBLV 51-3 with a size of 7000bp (molecular weight 4,48MD), consisting of the plasmid pHBc 1615 DNA of 6900 bp in length containing the bla gene conferring ampicillin resistance and the HBc gene under the control of a tandem P _trp promoter, and the fragment of the BLV genome which corresponds to the sequence between the restriction sites Bgl H ₅₀ M and Bam HI ₅₂₂ B with a length of 143 bp and encodes the region of the mature gp51 from the 56th to the 103rd amino acid and in the restriction site EcoRV the DNA of the plasmid pHBc 1615 is incorporated, allows the synthesis of the recombinant capsid structure HBcAgA-BLVgp51 (56-103). 3. The method according to claim 1 and 2, characterized in that one obtains the producer strain of HBcAgA-BLVgp51 (56-103) by transformation of E. coli K802-ellen with recombinant plasmid DNA pBLV51-3.