DD286821A5 - METHOD OF GENERATING THE RECOMBINANT KAPSID STRUCTURE HBCAG-HIVGP41 (78-129), WHICH REPRESENTS 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 HBcAg-HIVgp41 (78-129), which is the coreantigen of the hepatitis B virus with the exposed on its surface epitope HIVgp41 (78-129). The essence of the invention is that the fragment of the env gene of HIV 1, designated HIVgp41 (78-129) of 153 base pairs (bp) in the restriction site Cla I of the vector plasmid pHBc 1615 to form the recombinant gene HBcAg -HIVgp41 (78-129) is incorporated. The producer strain of HBcAg-HIVgp41 (78-129) is obtained by transformation of E. coli RR1 cells with recombinant plasmid DNA pHIV 41-22. This producer strain makes it possible to produce the recombinant capsid structure HBcAg-HIVgp41 (78-129), which represents the hepatitis B virus (HBV) core antigen with the epitope HIVgp41 (78-129) exposed on its surface {Kapsid; Core antigen; Hepatitis B virus; Human immunodeficiency virus; Transmembrane protein; 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-HIVgp41 (78-129), which is the coreantigen of the hepatitis B virus with the epitope H! Vgp41 (78-129) exposed 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 obtained on the basis of bacterial proteins but have no capsid-forming abilities (1-5). 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 protoins as a vaccine is the high Antoi! of bacterial protein.

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 rokombinante capsid structures with high immunogenicity and high yields.

Presentation of the essence of the invention

The object of the invention is to obtain recombinant capsid structures representing the core antigen of the hapatitis B virus (HBV) with the epitope HIVgp 14 (78-129) exposed on its surface, and the production of 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 HBcAg-HIVgp41 (78-129), which encodes the synthesis of the corresponding protein, and the expression plasmid pHIV 41-22, a producer is obtained (E. coli / pHIV 41-22), which ensures the production of the recombinant capsid structure HBcAg-HIVgp41 (78-129) at a level of 3.3% of the total protein of E. coli.

The rokombinante plasmid pHIV 41-22, whose structural parts ^ in Fig. 1 is geschollt, consists of the following elements:

DNA of the plasmid pHBc 1615 (6), a varianto of the plasmid pHBc 3, which contains the gene of HBcAg with an optimized initiation of its translation (7), and a mutant gene of HBcAg, a Polylinker E in thrust in the codon for the 144ste amino acid, which is intended for de, ι incorporation of foreign parts in this area, has; Length 6900bp

Fragment of the HIV 1 genome isolated from the plasmid pBH 10 (8), which corresponds to the section between the restriction sites Sau 3A _{, Mi} and Hind III, and corresponds to the region of gp-11 (78-129) ( Length 153bp)

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

 The DNA of the recombinant plasma pHIV 41-22 contains the genes:

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

- gene b! a, which confers ampicillin resistance.

The gene HBcAg-HIVgp41 (78-129) is under the control of a Tn expression prp. P " _p . After addition of chloramphenicol, the plasmid pHIV 41-22 is amplified non-conjugatively into the medium. The essence of the construction of the plasmid pHIV 41-22 is that the fragment of the env gene of HIV 1, designated as HIVgp4i (78-129), 153 bp in length, into the restriction site CIaI of the vector plasmid pHBc 1615 to form the

recombinant gene HbcAg-HIVgp41 (78-129) is incorporated.

The prodrug strain of HBcAg-HIVgp41 (78-129) is obtained by transformation of E. coli RR1 cells

recombinant plasmid DNA pHIV 41-22.

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 temperature 37 "C, pH optimum 7.0-7.4

Hydrocarbons are 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 the number WKPM W 4972.

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

1: construction diagram of the recombinant plasmid DNA according to the invention, pHIV 41-22 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 pHIV 41-22:

2pg of the plasmid pHBc 1615, obtained according to standard methods, is cleaved with 4 units of the restrictase CIa I in 25μΙ 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 65 ° C. The overhanging ends are filled in 100 μl of solution B (50 mM Tris-HCl, pH 7.5;

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

after 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).

20 pU of the plasmid pSS 3.8, which carries the fragment SaI I "" - Sac I _{9153 of} the HIV 1 genome, with 50 units of the

Restrictase Bgl II in 100 μl of the solution BdOmM Tris-HCl, pH 7.5; 1OmM MgCl ₂ ) in the course of 2 hours at 37 ⁰ C cleaved and the Fragment mixture applied to a 1% agarose gel. The fragment BgI M ₇₁₉₁ -OgI II _M29 with the molecular weight of

0.51 MD (size 1430 bp) is extracted from the agarose gel in a light melting agarose (DNA 2).

5pgDNA 2 was cleaved with 10 Einheiton Sau 3A and Hind III 10 Einheiton pider in 20 solution Aim Laufo of 2 hours at 37 ⁰ C.

The fragment Sau 3 A _76W -Hind Ill ^ n is separated on a slightly melting agorose and fills the overhanging ends, as above

described on (DNA 3).

0, ^ g DNA 1 and 0.05μΙ DNA 3 are combined with 10 units of T "DNA ligase in 10μΙ of solution B with addition of 1 mM DTT and 60μΜ ATP with an incubation time of 12 hours at 4 ° C.

To the reaction mixture are 100μΙ cells E. coli RR1, which had previously been treated with 10OmMCaCl ₂

(Final concentration 5x10 "inchon / 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 called pHIV 41-22. FIG. 1 shows this Construction scheme of this plasmid.

2. Transformation (Jos plasmids according to the invention into a producer strain:

The producer delay is obtained by transformation of E. coli RR1 with the recombinant plasmid pHIV 41-22. The cells are cultured in M9 medium supplemented with 10 g / L casamic acids, 2 g / L glucose and 0.02 g / L ampicillin to an optical density of OD ₆ Jo 4-5. The cells are collected by centrifugation and in three volumes of an Iy siort buffer containing 0.05 M Tris-HCl, pH 8.0, 0, 15 M NaCl, 0.1% Triton X, 100, 00, 5 M EDTA and 3 mg / contains lysozyme. After 30 minutes incubation at 4 ^C C, the cells are subjected to three repeated freeze / thawing. Subsequently Desoxyribonukleaso and MgCl ₂ are added to make final concentrations of 20 pg / ml and 1OmM and incubated again for 30 minutes at 4 ⁰ C. The Lyset is treated 30 Sekundon with ultrasound and then centrifuged (1000Ox g, 4 ⁰ C). The recombinant protein HBcAg-HIVcp41 (78-129) is purified as follows:

The lysate is fractionated with ammonium sulfate. HβcAgHIVgp41 (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 protein concentration of 25-50 mg / ml. 6-7 ml of this solution is applied to a Sepharose CI <1B column (2.1 × 75 cm) which has been equilibrated with the same buffer (only without Triton X 100). The fractions with HBcAg activity (see below) are collected and used s ^; o immediately or after concentration by repeated precipitation with 50% ammonium sulfate.

The characterization of the recombinant capsid structure according to the invention is carried out as follows: Determination of the HBcAg AktlvKIt mitte's Radial Immunodeficiency (RID) The HBcAg titer is determined by radial immunodiffusion according to Ouchterlony. For this one prepares one Dilution series (1: 2 ", η 2: D cell lysate and titers against human anti-HBc antibody. As a standard, purified HBcAg from recombinant bacteria (1 mg / ml) is used. The results of the titration HBcAg activity by RID are listed in Table 1.

Teb.l.

Synthesis of HBcAg-HIVgp41 (78-129) in E. coli RR1 cells carrying recombinant plasmid DNA, pHIV 41-22 Strain OD _SM titer in protein HBcAg yield of RID mg / ml mg / ml product (%)

E. coli RR1 4.0 1:32 15.0 0.5 3.3

(PHIV41-22)

Determination of gp41 Aktlvltit by Immunoblottlng For immunoblotting, resuspend the cells (6 mg) in emmμΙ sample buffer according to Laemmli, containing 2% SDS and 2%.

ß-mercaptoethanol, and lyslert by heating 2 minutes in a boiling water bath. The obtained lysates (2-4 mg / ml

Protein) is applied to a gradient polyacrylamide gel (12-18%) (size 150 χ 150 χ 0.75mm). The electrophoresis becomes 15 Hours at a current of 7 mA. After electrophoresis, the proteins are transferred to nitrocellulose HAWP. The filters are with the human

monoclonal antibody 3D6 (antigp41) in a dilution of 1: 500 in TBS buffer (5OmM Tris-HCl, pH 7,8,15OmM NaCl, 0.1% Triton X 100.1% BSA) for 15 hours at 20 ° C inc <; biert. After washing three times in TBS buffer, incubate the filters 2

Hours at 20 ⁰ C with the peroxidase-labeled conjugate of antibodies to human immunoglobulin (dilution 1:16). The filters flush with TBS buffer (3-5x) and developed with di-minobonzidine. The cell lysates of the producer strain exhibit the occurrence of specific staining zones which are associated with a protein

corresponding to a molecular weight of 25.5 kD (length 241 amino acids), which is in accordance with the

Construction scheme in Figure 1 stands. The control rate of E. coli K802 cells (pHBc 3) does not exhibit such zones. Electron Microscopic Characterization of Recombinant Capsid Structures H8cAg-HIVgp41 (78-129) The electron microscopic characterization of recombinant capsid structures HβcAg-HIVgp41 (78-129) is as follows

performed:

The preparations of the purified protein HBcAg-HIVgp41 (78-129) (see above) are negatively contrasted under negative contrast Using 1% uranium acetate and in an electron microscope at an acceleration voltage of 8OkV and

examined at 400,000 times magnification. The protein HBcAg-HIVgp41 (78-129) imaged capsids with a diameter of 25nm (Figure 2).

Determination of the gp41 act in de? Kapkid structure HBcAg-HIVgp41 (78-129) by ELISA

The solid phase for the immune reaction is a 96-well polystyrene microtiter plate. The purified HBcAg-HIVgp41 (78-129) (see above) is diluted to a concentration of 10 μg / ml in 50 mM NaHCOj-Na ₂ CO], pH 9.5 and pipot'.ert in all wells of the microtiter plate in each case 100μΙ. After incubation for 16 hours at 37 ^° C., the solution is removed from the wells and the microtiter plate is dried. The wells of the plate are filled with the human monoclonal antibody 3D6 (anti-gp41; see above) at a dilution of 1: 5000 in TBS pulper. After 2 hours of incubation boi 37 ⁰ C, the microtiter plate 5-6 times with distilled water. abgospült. Subsequently, per 100μΙ conjugate of horseradish peroxidase with S. aureus protein A (dilution 1: 5000) are applied. After one hour, the microtiter plate is washed and the color reaction is carried out. Each well is filled with ΙΟΟμΙ of a solution of 0.25% orthophenyldiamine-2HCl in 0.1 M sodium citrate, pH 5.0 and 0.02% HjOj, and incubated for 30 minutes at 20 ° C. The reaction is stopped by adding ΙΟΟμΙ 0.1 NHCI. The appearance of browning indicates the presence of gp <51 activity. For the quantitative determination of the activity one measures the optical density boi 492 nm (Tab. 2). The absence of a reaction in the negative control (HBcAg) indicates the specific binding of the anti-gp41 antibodies with the recombinant capsid structure HBcAg-HIVgp41 (78-129) (Table 2).

Tab. 2

Immunological reactivity of recombinant antigens in the ELISA

Tied antigen Immunological reactivity of recombined antigens (OD _4i2 / ml) human anti-HBc anli-gp41 (3D6) 1,315 0.93 pounds HBcAg-HIVgp41 (78-129) HBcAg (control) 0.393 0.018

Claims (3)

A process for the preparation of the recombinant capsid structure HBcAg-HIVgp41 (70-129) which is the coreactivity of the hepatitis B virus with the epitope HIVgp41 (78-129) exposed on its surface, characterized in that in the 144.Amino acid of the HBcAg, the sequence of the epitope HIVgp41 (78-129) with a length of 51 amino acids (153 base pairs) is incorporated. 2. The method according to claim 1, characterized in that the recombinant plasmid DNA pHIV 41-22 with a size of 7000bp (molecular weight 4.48 MD), consisting of animal DNA of the plasmid pHBc 1615 of 6900 bp length containing the gene bla conferring ampicillin resistance and the HBc gene under the control of a tandem promoter P " _p , and the fragment of the HIV 1 genome which corresponds to the sequence between the restriction sites Sau 3 A ₇₅₆ S and Hind IH ₇₇ I ₈ with a length of 153 bp and encodes the region of gp 41 from the 78 th to the 129 th amino acid and in the Restriction site CIa I of the DNA of the plasmid pHBc 1615 is incorporated, allows the synthesis of the recombinant capsid structure HBcAg-HIVgp41 (78-129). 3. The method according to claim 1 and 2, characterized in that one obtains the producer strain of HBcAg HIVgp41 (78-129) by transformation of E. coli RR 1 cells with recombinant plasmid DNA pHIV 41-22.