DD292928A5 - PROCESS FOR THE PREPARATION OF THE RECOMBINANT PROTEIN FRCP-PRES2 + ADW THAT CONSTITUTES A FUSION FROM COAT PROTEIN OF PHAGEN FR AND THE PRES2 FIELD OF THE HEPATITIS B VIRUS, SUBTYP ADW - Google Patents

Abstract

The invention relates to a process for the preparation of the recombinant protein frCP-preS2 / adw, which is a fusion of the coat protein of the phage fr and the preS2 region of the hepatitis B virus, subtype adw. The essence of the invention consists in the fact that the fragment of the hepatitis B virus (HBV) S gene, subtype adw, which encodes the amino acids 4-55 of the preS2 region and 9 N-terminal amino acids of HBsAg, into the codon for the second amino acid of the coat protein is incorporated by the phage to form the recombinant gene frCP-preS2 / adw. The producer strain of the recombinant protein frCP-preS2 / adw is obtained by transformation of E. coli K802 cells with recombinant plasmid DNA pFRd58. This producer strain allows the production of the recombinant protein frCP-preS2 / adw. {Kapsid; Coatprotein; Phage fr; Hepatitis B virus; preS2 region; 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 frCP-preS2 / adw, which is a fusion of the coat protein of the phage fr and the preS2 region of the hepatitis B virus, subtype adw.

Characteristic of the known state of the art

The preS region of the hepatitis B virus is of great importance in the induction of the immune response:

Neurath, A.R., Kent, S.B.H .: In Advances in virus research (ed K. Maramorosh et al.) 1988, Vol. 65, Academic press, Orlando, Florida.

In the development of alternative diagnostics and vaccines with the help of genetically engineered proteins or structures must therefore include, among other things, the preS2 region of HBV.

Phage fr coat protein based fusion proteins have a number of advantages over traditional hybrid proteins based on β-galactosidase, β-lactamase, chloramphenicol aethyltransferase, etc.

(Kozlovskaja, T.M., et al. Dokl. Akk. NaukSSSR, 1986, 485-455).

Expression of fusion proteins from phage coat protein and foreign antigenic determinant can result in the formation of capsid-like structures in E. coli that are not different in morphology and diameter from native capsids (Kozlovskaja, TM, et al., Molecular Biol., 1988, 22, 731- 740).

Even in the absence of self-assembly, the coat protein and its derivatives are characterized by high immunogenicity.

The presence of selenium directed against various forms of the coat protein provides good opportunities for purification of fr coat protein fusions.

Object of the invention

The aim of the invention is the production of a recombinant protein having immunological activities of the fr coat protein and the preS2 of HBV, subtype adw.

Presentation of the essence of the invention

The object of the invention is to obtain a recombinant protein which has the immunological activities of HBV-coat protein and preS2, subtype adw, and to provide a producer strain for this protein. This object is achieved according to the invention by obtaining, as a result of the construction of the recombinant gene ffCP-p? S2 / adw, which codes for the synthesis of the corresponding protein, and the expression plasmid pFRd58, a producer strain E. coli (pFRd58) which inhibits the production of the recombinant protein frCP-preS2 / adw assures.

The recombinant plasmid pFRd58, whose design scheme is shown in Fig. 1, consists of the following elements:

DNA of the plasmid pFR 7-71, a variant of the plasmid pFR20 which possesses the complete coat protein gene of the phage fr, in which a unique restriction site for Asu II was incorporated in the codon for the second amino acid and which contains a deletion in the gene for the tetracycline Resistance possesses, length 5279Bp

Fragment of the HBV genome, subtype adw, cloned in plasmid pAE 10, which corresponds to the section between the restriction sites Eco Rl ₀ and Eco 1301, and the region of preS2 of amino acids 4 to 55 and 9 N-terminal amino acids of the

HBsAg encoded (length 180bp)

 The size of the plasmid is 5459Bp

 The DNA of the recombinant plasmid pFRd 58 contains the genes:

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

- Gene bla, which confers ampicillin resistance.

The gene frCP-preS2 / adw is under the control of a promoter P, _lp . The plasmid pFRd 58 is amplified non-conjugatively after addition of chloramphenicol in the medium. The essence of the construction of the plasmid pFRd58 is that the 180-bp fragment of the S gene from the HBV (subtype

adw), which encodes amino acids 4-55 of the preS2 and 9 N-terminal amino acids of HBsAg, into the codon for the

2. Amino acid of the coat protein of phage fr is incorporated to form the recombinant gene frCP-preS2 / adw.

The producer strain of the recombinant protein frCP-preS2 / adw is obtained by transformation of E. coli K802 cells

with recombinant plasmid DNA pFRd 58.

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, p-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 5238 deposited. embodiment

The invention will be illustrated in more detail below using an exemplary embodiment. The accompanying figure shows

FIG. 1: Construction diagram of the recombinant plasmid DNA pFRd 58 according to the invention. The method according to the invention is implemented in 2 methods:

1. Construction of Recombinant Plasmid DNA pFRd58

2 μg of the plasmid pFR7-71, obtained according to standard methods, are cleaved with 3 units of the restrictase Asu II in 25μΙ of

A solution (1 Omm Tris-HCF, pH 7.5; 5OmIvI NaCl; 10 mM MgCl ₂₎ for 2 hours at 37 ⁰ C. The restrictase is inactivated at 15 min

65 ⁰ C. The linearized plasmid DNA is isolated from a 0.8% agarose gel and resuspended in 20μΙ H ₂ O (DNAI).

20pg of the plasmid is pAEIOspaltet with 50 units of Eco RI and 50 units restrictase the restrictase Eco 1301 100 μΙder solution A16 hours at 37 ⁰ C. Following incubation, the fragment mixture loaded onto a 2% agarose gel is applied and

Fragment with a length of 180 bp, eluted from the gel by known methods. The fragment is dissolved in 2ΟμΙ H ₂ O

resuspended (DNA2).

The characterization of the recombinant protein according to the invention is carried out as follows: Determination of the synthesis rate of the fusion protein frCP-preS2 / adw by means of radial immunodiffusion (RID) The titer of recombinant protein frCP-preS 2 / adw is determined by means of radial immunodiffusion of Ouchterlony. This is done by preparing a dilution series of the cell digestions, which was carried out with the aid of a buffer to Laemmli. As standard

purified phage coat protein from recombinant bacteria (1 mg / ml) is used. The results of the titration of the frCP activity by RID are shown in Table 1.

Synthesis of frCP-preS 2 / adw in E. coli K802 cells carrying recombinant plasmid DNA pFRd58 Strain OD ₆₆ O titer in RID protein frCP yield of

mg / ml mg / ml product (%)

E.COÜK802

(pFRd58) 4.0 1: 256 20.0 1.0 5.0

Determination of preS2 and coat protein activity by immunoblotting

For immunoblotting, cells (6 mg) are resuspended in 100 μl of Laemmli sample buffer containing 2% SDS and 2% mercaptoethanol and lysed by heating in the boiling water bath for 2 minutes. The resulting lysates (2-4 mg / ml protein) are applied to a gradient polyacrylamide gel (12-23%) (size 150mm x 150mm x 0.75mm). The electrophoresis is carried out for 15 hours at a current of 7 mA.

0.05 μg of DNA2 and 0.1 μg of DNAI are mixed and the overhanging ends are mixed in 2 μl of solution B (50 mM Tris-HCl, pH 7.5, 10 mM MgCl ₂ , 10 mM DTT, 25 mM NaCl) by adding 100 μl each of daTP, dCTP , dTTP and dGTP and 5 units of E. coli DNA polymerase (Klenow fragment) were filled in at room temperature over 20 minutes. The DNAs are ligated with 10 units of T ₄ DNA ligase in 10μΙ of solution B with the addition of 1 mM ATP over 12 hours at 4 ° C. To the reaction mixture are added ΊΟΟμΙ E. coli RR1 cells previously treated with 10 μM CaCl ₂ (final concentration 5 × 10 'cells / ml) 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 pFRd58. 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 recombinant plasmid pFRd 58. The cells are incubated in M9 medium supplemented with 10 g / l casamic acid, 2 g / l glucose and 0.02 g / l ampicillin to an optical density of OD ₆₆₀ 4-5.

After electrophoresis, the proteins are transferred to nitrocellulose HAWP. The filters are incubated with the murine monoclonal antibody MAb E at a dilution of 1: 500 in TBS buffer (50 mM Tris-HCl, pH 7.8, 15 mM NaCl, 0.1% Triton X100 + 1%).

BSA) incubated for 1 0 hours at 2O ° C. After washing three times in TBS buffer, incubate the filters with 2O ⁰ C for 2 hours

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.

In the parallel approach to the filters are incubated with anti-rabbit FRCP antibodies at a dilution of 1: 200 in TBS buffer (+ 1% BSA) for 15 hours at 2O ⁰ C. After washing twice with TBS buffer to the filter 2 Hours with peroxidase conjugate of protein A from S. aureus.

The cell lysates of the producer strain exhibit the occurrence of specific staining zones which are associated with a protein

a molecular weight of 19.8kD (length 190 amino acids), which is in accordance with the design scheme in Figure 1. The control rates of E. coli K802 cells do not show such zones.

Claims (3)

1. A method of producing the recombinant protein frCP-preS2 / adw, which is a fusion of coat protein of the phage fr and the preS2 region (4-55th amino acid) and 9 N-terminal amino acids of the HBsAg of HBV, subtype adw , characterized in that in the. 2. Amino acid of the coat protein of the phage for the preS2 region (4-55th amino acid) and 9 N-terminal amino acids of HBsAg are inserted, and the recombinant protein has the antigenic properties of both proteins. 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pFRd58 with a size of 5459Bp, consisting of the DNA of plasmid pFR7-71, 5279 bp in length, containing the bla gene conferring ampicillin resistance and the complete fr-coat protein gene under the control of a P _trp promoter, and The fragment of the HBV genome, subtype adw, which corresponds to the sequence between the restriction sites Eco Rl ₀ and Eco 1301 ^ 0 with a length of 180 bp and the preS2 region (4-55 th amino acid) and 9N-terminal amino acids of the HBsAg, which allows the synthesis of the recombinant protein frCP-preS2 / adw. 3. The method according to claim 1 and 2, characterized in that one obtains the producer strain of the frCP-preS2 / adw by transformation of E. coli K802 cells with recombinant plasmid DNA pFRd 58.