Method of obtaining recombinant enzyme-labelled antigen g2 of hantavirus in e. coli cells aimed at its application in immunosorbent assay in hfrs diagnostics

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to obtaining recombinant enzyme-labelled antigen G2 of hantavirus Dobrava. Essence of invention consists in the following: claimed method of obtaining antigen G2 of hantavirus Dobrava consists in expression of antigen in cells of E.coli in form of enzyme-labelled antigen of G2 hantaviruses based on HT protein antigen. Beta-galactosidase, which is highly active stable enzyme, serves enzyme label for protein antigen. Invention can be used to increase specificity and reproducibility of immunosorbent assay in HFRS diagnostics.

EFFECT: presence of commercially available chromogenic substrate of beta-galactosidase (X-gal) makes it possible to quickly estimate result of immunosorbent reaction visually or by change of optic density of solution in visible area.

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The technical field to which the invention relates.

The invention relates to the field of biotechnology, in particular the production of recombinant enzyme-labeled antigen G2 Hantavirus Dobrava with the purpose of its use to improve the specificity and reproducibility of enzyme-linked immunosorbent assay in the diagnosis of HFRS immunoassay methods.

Level of technology: hemorrhagic fever with renal syndrome (HFRS) virus retransmissions zoonotic disease is one of Russia's leading position in the number of cases among the natural focal infections.

The most effective method of dealing with HFRS is specific prevention, i.e. vaccination of the population in endemic regions [1-3]. Currently, the commercial vaccine against hemorrhagic fever with renal syndrome produced only in China, but none of these vaccines cannot be applied in the European regions of Russia, because it does not provide protection from virus, Puumala and Dobrava - causative agents of HFRS in these regions [4-7].

For production and control of vaccines against the virus, Puumala and Dobrava necessary to solve the problem of developing an effective serological methods based on proteins of the outer shell - envelope of Hantaviruses. To date, however, the problem of obtaining a full-sized commercial preparations of proteins envelope G1 and G2 of Hantaviruses not solved nig is in the world, due to their toxic to bacterial cells.

In recent times it has become urgent to develop immunological tests of the fourth generation, which allow you to diagnose various diseases with high specificity in the early stages.

Previously obtained recombinant antigen NT-Δ12 (U.S. Pat. application RU # 2010141819 from 13.10.2010) is a fragment of a protein G2 Hantavirus Dobrava, however, although he retains the immunogenicity of a full size product and is not toxic to the cells of a producer, the possibility of its use as antigen in immunological tests (especially the last generation) is difficult, as this protein accumulates in the cells of the producer in the form of Taurus include [8-10].

The aim of our work is to obtain recombinant enzyme-labeled antigen protein-based antigen-HT in cells of E. coli with the aim of increasing the specificity and reproducibility of the diagnostic analysis through the use of this antigen in immunological tests fourth generation. Enzymatic label to the protein antigen will serve as β-galactosidase, which is highly stable enzyme that will allow you to quickly visually assess the immunological reaction.

Disclosure of the invention:

The essence of the image is the shadow is a method of obtaining a peptide HT (derived surface antigen G2 Hantavirus Dobrava) in E. coli cells in the form of enzyme-labeled antigen for future use in enzyme-linked immunosorbent assay. Enzymatic label to the protein antigen is beta-galactosidase, which is highly stable enzyme in combination with a chromogenic substrate X-gal, which enables visual (qualitative) or spektrofotometricheskoe (quantitative) evaluation of the immunological reaction.

The method involves the following stages:

1) preparation of plasmid constructs pQL-Ht,

2) expression and analysis of protein yield,

3) chromatography protein Ht-LacZ on a column of Sepharose 6 FF,

4) conduct an immunochemical tests.

A brief description of graphics:

Fig.1. Plasmid construction pQL-Ht, is a concept.

Fig.2. The sequence of the bifunctional fused gene encoding a derivative of the peptide HT, in the structure of the pQL-NT.

The sequence of the peptide HT of the composition of the cDNA of the virus Dobrava grayed out;

Sequence of the gene beta-galactosidase underlined with a wavy line.

Fig.3. Chromatography of protein Ht-LacZ on a column of Sepharose 6 FF. On the graph (1) shows an optical density at λ=280 nm. Dashed lines mark the range of fractions selected for electrophoretic analysis and further work.

Fig.4. Determination of the activity of beta-galactosidase in the fractions after chromatography. On the ordinate axis is specified As620.

Fig.5. Electrophoretics the rd analysis of protein fractions Ht-LacZ after chromatography. Proteins are separated in denaturing 15% SDS page and stained for total protein with Coomassie Blue R-250.

Fig.6. The study of the antigenic activity of the protein Ht-LacZ by the method of enzyme-linked immunosorbent assay. Tablets activated protein-antigen NK6. The protein blend Ht-LacZ and serum of patients with hemorrhagic fever with renal syndrome (virus Dobrava) and healthy donors were practicability in a series of breeding from 3 to 192 times in increments of 4 times. On the ordinate axis is specified As450(620)each point. Black represents the reaction with the serum of people with HFRS, gray - reaction with the blood sera of healthy people. The results were determined spectrophotometrically at two wavelengths: (A) at 620 nm (detection of the enzymatic activity of beta-galactosidase). - At 450 nm (the enzymatic activity of horseradish peroxidase).

The implementation of the invention:

Research methodology:

1) preparation of plasmid constructs;

2) expression and analysis of protein yield;

3) chromatography of recombinant protein;

4) conduct an immunochemical tests.

1) preparation of plasmid constructs

Obtaining design pQL-Ht was performed according to the following scheme:

1) Gene beta galactosidase (LacZ) in the composition of the PCR product length 3080 p. N. cloned from the genome of E. coli C600 and was introduced in the composition of the vector pQE30 sites SalI-HindIII. For PCR with the matrix genomic DNA of E. coli C600 was used primers Lac-fo1 (GGGTCGACACCATGATTACGGATTCACTG) and Lac-rev1 (GGAAGCTTATTTTTGACACCAGACCAACTG). The resulting construct was designated pQE-LacZ.

2) Gene peptide NT of construction pet-NT-Δ12 was visiplan by flanking sites polylinker Cloned and introduced into plasmid pQE-LacZ, also pre-split site kpni restriction sites. Thus, there was obtained a design pQL-HT (Fig.1 and 2).

2) Expression and analysis of protein yield

Design pQL-Ht was injected into the cells of the strain E. coli JM109. Selection of colonies was carried out by two parameters: resistance to ampicillin and by the presence of blue color of colonies with growth of the indicator medium with X-gal. The resulting product was cultured at 30°C in liquid medium (0.5% of yeast extract, 1% peptone and 0.5% NaCl) supplemented with 100 μg/ml ampicillin in Erlenmeyer flasks with a volume of 750 ml (30 ml medium per flask) for 14-18 hours. Seed material was a washout with cups cell culture obtained by seeding primary transformants with agar medium (0.5% of yeast extract, 1% peptone, 1.5% agar and 0.5% NaCl) containing X-gal and ampicillin. The age of transformants about 40 hours since the end of the transformation, the cultivation temperature of 30°C. the Dose of inoculation with 5×108cells per flask. Induction of the promoter in the cells of the producer spent IPTG.

Evaluation of the yield of the target product was performed using electrophoretic analysis of total proteins recombinant producer according to laemmli's method. This of coarse glue the face-to-face lysate of each culture was collected in 100 μl. The lysate was subjected to centrifugation at 14000 g for 15 min and separated soluble and insoluble cell fractions. Proteins were solubilizers in 30 ál of buffer laemmli's method and analyzed the composition of proteins by denaturing SDS-electrophoresis in 15% SDS page. After staining the gel with Coomassie Blue R-250 in the soluble fraction of cell lysate was watching the band corresponding to the calculated mass of the protein Ht-LacZ (about 120 kDa).

3) Chromatography of recombinant protein

Coarse cell lysate culture of JM109 (Ht-LacZ) was subjected to centrifugation at 8000 G for 1 hour and separated soluble and insoluble cell fractions. Protein from the soluble fraction was then subjected to chromatographic purification.

The main purpose of the GC protein - derived peptide NT under denaturing conditions was the removal of impurities nucleic acids, forming colloid involving proteins and preventing the effective application of other methods of chromatography. With the removal of drug impurities cellular proteins E. coli was considered only as a side task.

The resulting protein solution Ht-LacZ was applied to a column of Sepharose 6 FF (GE Healthcare, USA, height 24 cm, volume of 48 ml), equilibrated with buffer A. the Elution was performed with a linear gradient of NaCl from 0 to 0.5 M in chromatography buffer (Tris-HCl, 100 mm) at a rate of 8 ml/min (Fig.3).

The fractions obtained, to the each of 10 ml, were analyzed for the presence of activity of beta-galactosidase. The reaction mixture consisted of Tris-glycine buffer containing the substrate X-gal. The signal was measured on a flatbed-spectrophotometer at λ=620 nm. The result is shown in Fig.4.

The collected fractions of 10 ml each were analyzed using denaturing SDS-electrophoresis in 15% SDS page. For electrophoresis were selected by 50-100 ál from each fraction. Proteins were subjected to deposition of 1 ml of a mixture (500 ál of 100% acetone, 100 ál of 70% THU 1 ml). The obtained precipitation was solubilizers under denaturing conditions in 15 μl of buffer laemmli's method (with the addition of urea). The gel was progressively total protein Coomassie Blue R-250 (Fig.5).

Material protein fractions Ht-LacZ obtained by chromatography under denaturing conditions, was homogeneous electrophoretic, allowing it to be directly used for immunochemical tests.

5) Conduct immunochemical tests

As the format of analysis was selected indirect variant of typhus. On a die barbirolli protein-antigen NK6. In the process of sorption protein preparation was diluted in 20 to 50 times the carbonate-bicarbonate buffer (KGB), bringing the concentration of total protein in the solution to 10 μg/ml of the resulting solution was made in immunological tablets for 1 day, then spent blocking n the specific binding to the substrate with 1% BSA in buffer KGB. In the next stage of the review, the tablet made the complex enzyme-labeled protein antigen Ht-LacZ with the analyzed blood serum of patients with HFRS and blood sera of healthy donors in the comparison group. A mixture of the antigen-antibody rascaroli from 3 to 192 times in increments of 4 times. As a control sample used wells, in which, instead of diluted human serum contributed PBS buffer ("conjugate control). Next, wells were treated with the substrate X-gal and produced the measurement signal on the spectrophotometer at λ=620 nm. At the next stage of analysis wells were washed and treated individuum conjugate against human IgG labeled with horseradish peroxidase and TMB substrate in the presence of hydrogen peroxide. The signal was measured on a flatbed-spectrophotometer at λ=450 nm. The result is shown in Fig.6.

The results show that the use of protein-antigen enzyme-labeled Ht-LacZ, allows to distinguish between healthy donors and from patients with hemorrhagic fever with renal syndrome with high specificity, which makes possible the use of this antigen in the creation of modern immunological tests for the diagnosis of HFRS.

Sources of information

1. Maes P., Clement J., Van Ranst M. Recent approaches in hantavirus vaccine development. Expert Rev Vaccines. - 2009 - V. 8, №1, P. 67-76.

2. Cho H. W., Howard C. R., Lee H. W. Review of an inactivated vaccine against hantaviruses. - Intervirology. -2002 - V. 45, no. 4-6, p. 328-333

3. Song G. Epidemiological progresses of hemorrhagic fever with renal syndrome in China. - Chin Med J (Engl). - 1999 - V. 112, No. 5, P. 472-477.

4. Oya A. Japanese encephalitis vaccine. - Acta Paediatr Jpn. - 1988 - V. 30, No. 2, P. 175-184.

5. Choi Y. C. J. Ahn, Seong K. M., Jung, M. Y., Ahn, B. Y. Inactivated Hantaan virus vaccine derived from suspension culture of Vero cells. - Vaccine. - 2003 - V. 21, No. 17-18, P. 1867-1873.

6. Lee H. W., Chu, Y. K., Woo Y. D. Immune responses after two or three doses of Hantavax vaccination against Hantaan virus // Proc. fifth international conference on hemorrhagic fever with renal syndrome, hantavirus pulmonary syndrome and hantaviruses. Lion, Franch - 2001. - P. 234-242.

7. Ruan Y., Xu X., Liu W., Deng X, Weng S, Zhou W, Wang Q, Chen L, Fang L, Xu Z, Yan Q, Liu W, Dong G, Gu H, Yu Y, Xu Z. A study on immunogenicity and safety of left-hand drive vehicles inactivated vaccine against hemorrhagic fever with renal syndrome. - Zhonghua Yu Fang Yi Xue Za Zhi. - 1999 - V. 33, No. 6, P. 340-342.

8. Hooper J. W., Kamrud, K. I., Elgh F., D. Custer, C. S. Schmaljohn DNA vaccination with hantavirus M segment elicits neutralizing antibodies and protects against seoul virus infection. - Virology. - 1999 - V. 255, No. 2, P. 269-278.

9. Kallio-Kokko, H., Leveelahti R., Brummer-Korvenkontio, M., Lundkvist, A., Vaheri, A., Vapalahti O. Human immune response to Puumala virus glycoproteins and nucleocapsid protein expressed in mammalian cells. J Med Virol. - 2001 - V. 65, no. 3, p. 605-613

10. Huang H., Li X., Zehua Z. Genetic immunization with Hantavirus vaccine combining expression of G2 glycoprotein and fused interleukin-2. - Genetic Vaccines and Therapy. - 2008 - V. 6. - P. 15-21.

A method of obtaining a recombinant antigen G2 Hantavirus Dobrava in the cells of E. coli for use in the manufacture of diagnostics of hemorrhagic fever with renal syndrome, characterized by the fact that DNA constructs pQL-HT that encodes a protein of two parts (N-terminal position of the peptide length 99.about. with the follower of the awn amino acids RGSASGGAGGINSSRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQSIN TSTMHFTDERIEWKDPDGMLRDHINILATKDIDFDTSGGAGGTRGSPGT, and C-terminal - enzymatic label (gene beta-galactosidase) (the decree. Fig.2)), introduced into E. coli cells, which were cultured in a liquid growth medium, are lysed biomass, separating the soluble fraction of the lysate by centrifugation, hold gel chromatography on a column with sorbent Sepharose-6FF, and used the product for the detection of specific antibodies in the serum of patients with hemorrhagic fever with renal syndrome.



 

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