Method of obtaining recombinant antigen g2 of dobrava hantavirus in e.coli cells

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and a method of obtaining a recombinant antigen G2 of hantaviruses. The disclosed method is characterised by that the DNA structure pGHF, which encodes a fused protein of three parts, where N-terminal position is occupied by a green fluorescent protein GFP, central position is occupied by a peptide of 73 amino acid residues with the amino acid sequence SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and the C-terminal position is occupied by a mini-domain Foldon of coliphage fibritin JS98C3 (dwg 2), is introduced into E. coli cells; cells transformed by this structure are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifuging, the product of expression in the form of inclusion bodies is solubilised with methylated spirit, chromatography is carried out and the obtained product is used to detect specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: disclosed solution improves repeatability and sensitivity of immunoenzymatic assay when diagnosing hemorrhagic fever with renal syndrome.

7 dwg


The technical field of the invention: the invention relates to the field of biotechnology, in particular, to obtain recombinant antigen G2 Hantaviruses with the capability to improve the reproducibility and sensitivity of enzyme immunoassay in the diagnosis of HFRS.

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 anywhere in the world, due to their toxicity in relation to bacteria inim cells.

Was recently obtained recombinant antigen NT-Δ12, representing a fragment of a protein G2 Hantavirus Dobrava, preserving the immunogenicity of a full size product, but does not exhibit toxicity towards cells of the producer (U.S. Pat. application RU # 2010141819 from 13.10.2010). This protein was accumulated in E. coli cells as Taurus enable [8-10]. Consequently, when using antigen NT-Δ12 in immunological tests, it is necessary to pre-renaturation product, which in turn negatively affects the sensitivity and reproducibility of the immunological test.

The aim of our work is to develop ways of improving the quality characteristics of antigen NT-Δ12 by changing its structure.

Disclosure of the invention: the invention is a method of improving the quality characteristics of recombinant antigen G2 (HT-Δ12) for use in immunological tests in the diagnosis of HFRS due to the improved solubility of the target protein antigen G2 Hantavirus Dobrava by stabilizing its globular structure of beta-structural mini-domain foldon fibritin coliphage JS98C3. Thus, the present invention relates to a method for producing a protein-antigen G2 Hantavirus Dobrava with specified performance characteristics (protein GHF) to improve FOTS is izvedenosti and sensitivity of enzyme immunoassay in the diagnosis of HFRS. The method involves the following stages:

1) preparation of plasmid constructs pGHF carrying trifunctionally fused gene;

2) expression and analysis of protein yield GHF;

3) chromatography protein GHF on a column of Sepharose 6 FF;

4) conduct an immunochemical tests.

A brief description of graphics:

Figure 1. Plasmid construction pGHF concept.

Figure 2. The sequence trifunctional fused gene encoding a derivative of the peptide HT, in the structure of the pGHF.

The GFP gene sequence is underlined with a wavy line;

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

The gene sequence foldon fibritin highlighted by the dotted line;

Figure 3. Electrophoretic analysis of the products of expression of design pGHF soluble in (1) and insoluble (2) fraction of the cell lysate E. coli JM109, detransforming design pGHF. Proteins are separated in denaturing 15% SDS page. Figure (a) shows the total protein (gel stained Coomassie Blue R-250), figure (b) presents the glow of the protein in the gel under UV radiation.

Figure 4. Chromatography of protein GHF on a column of Sepharose 6 FF. The optical density at λ=280 nm are shown in the graph (1).

Dashed lines mark the range of fractions containing protein GHF (B1-B8).

Figure 5. The study of the antigenic activity of the protein GHF met the house enzyme-linked immunosorbent assay. Tablets activated protein-antigens GHF (a) and NK6 (b). Pools of blood sera of patients with hemorrhagic fever with renal syndrome (virus Dobrava, Puumala, Hantaan) and healthy donors were used in a dilution of from 50 to 3200 times in increments of 2 times. On the ordinate axis is specified As450each point minus background (control without the addition of human serum). The graph shows the reaction of protein antigens from the blood sera of patients with HFRS (1) and with sera of healthy blood donors (2).

The implementation of the invention:

Research methodology:

1) obtaining design pGHF,

2) expression and analysis of protein yield GHF,

3) chromatography protein GHF,

4) conduct an immunochemical tests.

1) Obtaining design

Getting constructs was performed according to the following scheme:

In the previously described construction of pet-NT-Δ12 (U.S. Pat. application RU # 2010141819 from 13.10.2010)that carries the gene peptide NT cloned gene foldon fibritin of design pET-cd-32a [11]. This vector construct pet-NT-Δ12 uncoupled sites SpeI and XhoI, and the insertion of the constructs pET-cd-32a removed the restriction sites XbaI and XhoI.

Later in the unique XbaI site of the resulting construction on the basis of the vector Rita have introduced a synthetic DNA duplex made up of pre-phosphorylated oligonucleotides:



Next, the resulting structure-based rate COI is litovali as template for PCR with a unique primer


and with a standard primer T7terminator.

The resulting PCR product length 600 BP were digested with restrictase BglII and XholI and cloned in the vector pGem3z, split across sites BamHI and SalI.

Insert "NT+ foldon fibritin from design pGem3z removed the restriction sites SacI and > PST and cloned in the expression vector pQE30-GFP, containing the gene for green fluorescent protein GFP-based vector pQE30 (Qiagen) [12] on the same site.

As part of the design (Fig 1 and 2) we can identify the following elements:

- expression vector pQE30 (Qiagen);

gene green fluorescent protein GFP (Evrogen);

- fragment of polylinker vector pGem-3z (Promega);

gene Ht from the construction of pet-HT-12;

- fragment of polylinker vector pGem-3z (Promega);

- gene foldon fibritin phage JS98C3 [11].

2) Expression and analysis of protein yield

Design pGHF was introduced into cells of E. coli strain JM109. Selection of colonies was carried out by two parameters: ampicillinampicillin and by the presence of green fluorescent staining of the colonies. 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 washed cell culture plates with agar medium (0.5% of yeast extract, 1% Pepto is, 1.5% agar, 0.5% of NaCl)obtained by seeding primary transformants. 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.

Assessment of the accumulation of the target product in E. coli cells was performed using electrophoretic analysis of total proteins recombinant producer according to laemmli's method. This of coarse cell 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 (figure 3). Fluorescence proteins soluble fraction in the gel was observed in the region of 47 kDa, which corresponds to the calculated mass of the protein GHF. Proteins insoluble fraction fluorescent glow had not.

3) Chromatography protein

Coarse cell lysate culture of JM109 (GHF) was subjected to centrifugation at 8000 g for 1 hour and separated soluble and insoluble cell fractions. Protein from the soluble fraction was subjected to initial purification by double vysalivaniya ammonium sulfate. The first precipitate, obtained by deposition of a 1.2% solution (NH) 4SO2were removed from the faction, the second residue (after deposition of 2% solution (NH)4SO2containing the target protein GHF, collected by centrifugation at 8000 G for 1 hour. Sediment suspended in 8 ml buffer (10 mm Tris-HCl, 130 mm NaCl, pH=7,8).

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 GHF in the volume of 15 ml was applied on the column Sepharos 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 (figure 4).

Fractions for further work containing the target protein GHF, were selected by evaluating the presence of fluorescent green color in solution. Selected fractions of 10 ml volume each were combined and subjected to concentration and purification on HisLink Protein Purification Resin (Promega). Material protein fractions GHF obtained, was immunochemical pure and homogeneous electrophoretic that made it possible to use it to conduct and monogenically tests.

4) conduct an immunochemical tests

As the format of analysis was selected typhoid direct sorption of recombinant protein antigen GHF on the surface of immunological tablet. Drug for comparison served as a protein-antigen NK6. In the process of sorption of protein preparations GHF and NK6 was dissolved in 20-50 times 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 the blocking of nonspecific binding to the substrate with 1% BSA in buffer KGB. In the next stage of analysis was conducted serial titration pools of blood sera of patients with HFRS and, as a comparison of healthy donors. When this pool of blood sera included HFRS patients, infected with the virus, Dobrava, Puumala, and Kuantan. Serum was taken in a dilution of from 50 to 3200 times in increments of 2 times. As a control sample used wells, in which, instead of diluted human serum contributed PBS buffer ("conjugate control).

In conclusion, all wells were treated individuum conjugates against human IgG labeled with peroxidase and TMB substrate in the presence of hydrogen peroxide. The signal was measured on a flatbed-spectrophotometer at λ=450 nm. Resulttemplate presented in figure 5.

The results demonstrate that increasing the solubility of the recombinant antigen by changing its structure leads to increased resolution immunoassay for the diagnosis of HFRS.

List of used sources

1. Maes P., Clement J., Van Ranst M. Recent approaches in hantavirus vaccine development. Expert Rev Vaccines. - 2009 - V.8, No. 1, Page 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, №2, R-184.

5. Choi Y. Ahn C.J., Seong C.M., M.Y. Jung, B.Y. Ahn 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 vacine combining expression of G2 glycoprotein and fused interleukin-2. - Genetic Vaccines and Therapy. - 2008 - V.6. - P.15-21.

11. Latypov O.R., Besides A.K., Litrov AV product Characteristics wac gene of bacteriophage JS98C3 closely related to phage JS98 // Bulletin of the KSU. 2007. T. S.112-122.

12. Shevelev A.B., Fomenkov VG, Kuznetsova T.V., Kovalev, L.I., Lagodna NV creating a producer of myostatin in the form of a fused protein with 6His-GFP-based E. coli and analysis of its immunogenicity. The collection of articles "biomedical technology to increase efficiency in the conditions of intense physical activity. The Ministry of education and science of the Russian Federation, Moscow, 2004, P.140-150.

Fig. 2

A method of obtaining a recombinant antigen G2 Hantavirus Dobrava in E.coli cells, characterized by the fact that DNA constructs pGHF encoding a protein of three parts, where the N-terminal position of the green fluorescent protein GFP, Central - peptide length 73 S.A. with the amino acid sequence SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEWKDPDGMLKDHLNILVTKDIDFDT and C-terminal mini-domain foldon fibritin coliphage JS98C3 (figure 2), is introduced into cells of E. coli cultured transformed this design cells are lysed biomass, separating the insoluble fraction of the lysate by centrifugation, the expression product in the form of Taurus VK is Uchenie solubilizing using denatured alcohol, spend chromatography, 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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11 cl, 4 dwg, 14 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

FIELD: medicine, genetics, biochemistry.

SUBSTANCE: invention relates to new NOS-variants or mutants that comprise structural modifications in site Akt-dependent phosphorylation. Modified NOS-proteins or peptides, in particular, human proteins or eNOS-peptides having change of amino acid residue corresponding to S/T in motif of the consensus-sequence RXRXXS/T of NOS-polypeptide of wild type and nucleic acid molecules encoding thereof can be used in genetic therapy and proteins and NOS-peptides can be used in screening methods of agents modulating activity of NOS. The advantage of invention involves the creature of new NOS-variants or mutants that can be used in genetic therapy.

EFFECT: valuable medicinal properties of mutants.

25 cl, 1 tbl, 9 dwg, 3 ex