Method of production of recombinant antigen g2 of hantavirus dobrava in cells of e coli

FIELD: biotechnology.

SUBSTANCE: method is characterised in that the DNA of the structure RNAb indicated on Figure 1, which encodes the fused protein of three parts, where N-terminal position is green fluorescent protein GFP, central - peptide of 73 amino acid residues with the amino acid sequence of SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEW KDPDGMLKDHLNILVTKDIDFDT, and C-terminal - light chain of double-stranded protein Kunitz-type inhibitor from potato tubers (PKPI-BI), are introduced into cells of E. coli. The cells transformed by this construction are cultured, the biomass is lysed, the insoluble fraction of the lysate is separated by centrifugation. The product of expression in the form of inclusion bodies is solubilised with the denaturant. Chromatography is carried out under denaturing conditions. The resulting product is used for detection of specific antibodies in serum of patients with hemorrhagic fever with renal syndrome.

EFFECT: invention enables to obtain the recombinant antigen G2 of Hantavirus Dobrava with increased yield.

6 dwg, 1 ex


The technical field of the invention: the invention relates to the field of biotechnology, in particular, to obtaining vaccines for the prevention of viral infections, namely infection, hemorrhagic fever with renal syndrome.

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]. Inactivated vaccine Hantavax [2] was developed for use in the Republic of Korea, dominated by species of Hantaviruses Canaan and Seoul. Currently, the vaccine under the brand name Hantavax is produced industrially and commercially available. It is approved for clinical use in the Republic of Korea.

Along with Hantavax in China and South Korea developed, licensed and actively used in the treatment of HFRS other variants of killed vaccine against hemorrhagic fever with renal syndrome:

1) In China is formalin inactivated vaccine based on virus Seoul, grown in primary cell cultures of Syrian hamster kidney; for inactivation of virus in the production of this vaccine is used β-propiolactone.

2) China is a vaccine on the basis of the virus Hantaan, grown in primary cultures of kidney cells Mongolian gerbils, inactivated by formalin.

3) Purified vaccine based on virus Hantaan obtained from the brain of experimentally infected rats suckling [3].

4) currently, China has also developed and is undergoing clinical trials of the bivalent vaccine based on virus Hantaan and Seoul obtained on transplantable cell cultures Vero-E6 (inactivation of β-propiolactone). According to the developers, this drug should replace the inactivated vaccine, since the latter is due to the low level of treatment cause serious side effects, not providing the maximum level of the synthesis of neutralizing antibodies.

However, 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 [4-10].

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 against bacterial the m cells.

Was previously obtained recombinant antigen G2 Hantaviruses NT-Δ12 [11]. However, the output of this protein did not exceed 20 mg per liter of culture, which together with the losses during the purification of the drug prevents the use of this antigen in immunological tests.

The aim of our work is to develop ways of increasing the number of product accumulation in the producer strain due to changes in the structure of the target protein.

Disclosure of the invention: the invention is a method of increasing the yield of recombinant antigen G2 Hantaviruses in E.coli cells by optimizing the structure of the antigen to trifunctional derived peptide NT.

Based on the design of pet-NT-Δ12 [11]that encodes the peptide HT in the isolated state, was designed and obtained design, where this peptide was part trifunctional fused protein. This N-terminal position occupied by the green fluorescent protein GFP, Central - peptide neurotensin and C-terminal light chain double-stranded protein inhibitor Konitza of potatoes (PKPI-B1). When assembling structures instead of vector RET based on the T7 promoter was used pQE30 containing less powerful T5 promoter. This decision was motivated by a desire to improve the conditions of the folding of the product by reducing the intensity of its accumulation. For the same purpose Gras on the Itza domains GFP, HT and PKPI-B1 design trifunctional fused protein were introduced artificial flexible peptide linkers, enriched in Gly, Ser and Ala encoded by synthetic oligonucleotides. In addition, when designing a new plasmid gene peptide NT was shortened by 40 BP from the 3'-end, which will not affect the immunochemical properties of the protein, but reduces to it the number of disulfide bonds and hydrophobic areas, which improves the solubility of the product and reduces its negative impact on cell viability producer.

A brief description of graphics:

1. Plasmid construction RNC, schematic diagram.

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

• The sequence of the GFP gene is underlined with a wavy line;

• The sequence of the peptide HT of the composition of the cDNA of the virus Dobrava

• light chain inhibitor Konitza of potatoes

• artificially introduced by linker sequences, providing the flexibility of articulation of the globular domains of different origin in italics.

3. Electrophoretic analysis of the products of expression of design RNC in soluble and insoluble fraction of the lysate is one E. coli TG1, detransforming: (1) the Vector pQE30, (2) Design RNC. Proteins are separated in denaturing 15% SDS page and stained for total protein with Coomassie Blue R-250. The arrow indicates the location of the protein NK6 (estimated weight 42 kDa).

Fig.4. Anion-exchange chromatography protein NK6 on a column of Sepharose-Q in the presence of 4M urea. Thin gray lines indicate the concentration of NaCl in the eluting buffer and the conductivity of the solution, and the black line is the optical density at λ=280 nm. Dashed lines indicate the range of fractions selected for electrophoretic analysis, black line - fractions selected for further work.

5. Electrophoretic analysis of protein fractions NK6 after ion-exchange chromatography under denaturing conditions. Proteins are separated in denaturing 15% SDS page and stained for total protein with Coomassie Blue R-250. The position of the target protein by mass of 42 kDa indicated by the dotted oval. For further work were selected fractions D6-F6.

Fig.6. The study of the antigenic activity of the protein NK6 by the method of enzyme-linked immunosorbent assay. Tablets activated protein-antigen NK6 and blocked with normal rabbit serum at a dilution of 100 times. Serum of patients with hemorrhagic fever with renal syndrome (virus Dobrava, Puumala) and healthy donors were practicability in a series of dilution from 20 to 1280 times in increments of 2 times. On the ordinate axis is specified As492each t is his minus background (control without the addition of human serum).

The implementation of the invention:

Research methodology:

1) obtaining design

2) expression and analysis of protein yield

3) preparation of test samples for chromatography

4) ion-exchange chromatography.

5) conduct immunochemical tests.

1) Obtaining design

Getting constructs was performed according to the following scheme:

1. Using a matrix design pET-Ht-Δ12 and primers Li4 (GGAGATCTCTAGCGGGGGCGCCGGAGGAATT) and NT (GGACTAGTATCAAAGTCAAGTGCCTT) conducted PCR. The product is treated with the restriction enzyme BglII.

2. On the matrix plasmids pQE-GFP performed PCR using primers ECFP1 (GGGGATCCATGGTGAGCAAGGGCGAGGAand GFP10 (GGAGATCTCTTGTACAGCTCGTCCATGCC). The product is treated with the restriction enzyme BglII.

3. The products of stages 1 and 2 combined and subjected to legirovanie.

4. The matrix product of ligation stage 3 performed PCR with primers ECFP1 and NT. The PCR product purified by separation from the gel and subjected to cleavage by restrictase BamHI and SpeI.

5. The design of the pQ-Knl representing gene PKPI-B1 (Tuberosum cultivar Istra, NCBI access number AY692479) in the vector pQE30-treated restrictase SpeI and BamHI.

6. Products of reaction of the enzyme with stages 4 and 5 combined, subjected to legirovanie and introduced into cells of E. coli TG1 by transformation.

7. The obtained intermediate design 6-based vector pQE30 were digested with restriction enzyme SpeI and ligated with a synthetic is NC-duplex, obtained by mixing in advance phosphorylated oligonucleotides CTAGCGGGGGCGCCGGAGGAATT and CTAGTTCCGGCGCCCCCG to introduce the linker sequence on the border areas of NT and easy C-terminal chain of the protein inhibitor PKPI-B1. The obtained expression design on the basis of the vector pQE30 was designated RNC (Fig.1 and Fig.2).

2) Expression and analysis of protein yield

Design RNC was introduced into the cells of the strain E. coli TG1 by taking ampicillin-resistant 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% peptone, 1.5% agar, 0.5% of NaCl)obtained by seeding primary transformants. The age of the transformants is not more than 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 did not.

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 cell lysate of each culture was collected in 100 μl. The lysate was subjected to centrifugation at 4000 G. within 15 min and was divided 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).

The analysis showed that trifunctionally protein-based peptide NT - design product RNC as free peptide NT, accumulated in the insoluble cell fraction, with a yield of about 40 mg/l of culture.

3) Preparation of test samples for chromatography

Coarse cell lysate culture TGl(pHK6) was subjected to centrifugation at 8000 g for 1 hour and separated soluble and insoluble cell fractions. The combined precipitate of insoluble cellular fractions washed three times in 25 ml of 4 M NaCl with addition of 1% Triton, 25 ml of saline with 1% Triton and 25 ml of water. Sediment suspended in 8 ml of buffer (Tris-HCl 10 mm, pH=8,6)containing 8 M urea and 0.1% β-mercaptoethanol. The solution was boiled on a water bath for 10 minutes and centrifuged. Received the clarified supernatant was applied to gelfiltration chromatographic column filled with Sephadex G-25 fine at a speed of 5 ml/min using a liquid chromatographic system low pressure "AKTA-Purifile" (GE Healthcare, USA). Separation was carried out in buffer (Tris-HCl 10 mm, pH=8,6)containing 4 M urea, in order to remove salts, excess recover the I and low-molecular cell impurities. The solubilized under denaturing conditions, the protein was subjected to purification using anion exchange chromatography.

Isolation and purification of target proteins from the resulting supernatant was performed using a liquid chromatography system "AKTA-Purifile" (GE Healthcare, USA).

4) Ion-exchange chromatography

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

Obtained after gel filtration the solution of denatured protein NK6 in the volume of 15 ml was applied to the anion exchange column of Sepharose-Q (XL) (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 10 mm, pH=8,6, 4 M urea, 1 M NaCl) at a rate of 8 ml/min (Figure 4).

Collected 20 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 salt precipitation which was ilizarova under denaturing conditions in 15 μl of buffer laemmli's method (with the addition of urea) (Fig.5).

Material protein fractions NK6 obtained by anion-exchange chromatography under denaturing conditions, was immunochemical clean and electrophoretic homogeneous, allowing it to be directly used for renaturation and immunochemical tests (Fig.6).

5) Conduct immunochemical tests

As the format of analysis was selected typhoid direct sorption of recombinant protein antigen NK6 on the surface of immunological tablet. Were used in the experiment chromatographically purified protein NK6 in denatured form. In the process of sorption protein preparation is stored in the form of concentrated solutions in the presence of 4 M urea, 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. A necessary step in the analysis was blocking the normal binding of antibodies present in the serum of healthy donors, at the expense of competition. To this end, the tablets are subjected to blocking with 1% BSA, optionally processed, contributing to each well of normal rabbit serum, rasb is run by PBS buffer at a ratio of 1:100.

In the next stage of analysis was conducted serial titration test sera of patients with HFRS (or healthy donors in the comparison group). In this study the specificity of the reaction in the sample comparison included a) HFRS patients infected with the virus Dobrava, b) HFRS patients infected with the virus, Puumala, C) healthy donors. 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=492 nm. The results of the determinations are shown in figure 6.

The results show high reactivity of antibody positive sera against antigen NK6. The signals of positive sera of patients in 2-2,5 times higher than the signal sera of healthy donors in the same breeding that allows you to reliably diagnose the presence in the blood of people of specific antibodies against the virus Dobrava and Puumala.

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 inactivatedvaccine 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 (Engi). - 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., Y.K. Chu, 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., Eigh 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.

11. Abelev, Mosnarbank, Mvelopes, Eueope, Oageoojs, Tchudakorova, Eaatem. A method of obtaining a recombinant antigen G2 Hantaviruses in E. coli cells. The patent application. Entrance. No. 060029. Reg. No. 2010141819. Date 13.10.2010.

A method of obtaining a recombinant antigen G2 Hantavirus Dobrava in cells of E. coli, features resouses fact, the DNA constructs RNC shown in figure 1, 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 SRKKCNFATTPICEYDGNMVSGYKKVMATIDSFQAFNTSYIHYTDEQIEWKD PDGMLKDHLNILVTKDIDFDT and C-terminal light chain double-stranded protein inhibitor type Konitza of potatoes (PKPI-BI), is introduced into E. coli cells, 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 include solubilizers using denaturant, spend chromatography under denaturing conditions and use the product for detection of specific antibodies in the serum of patients with hemorrhagic fever with renal syndrome.


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EFFECT: methods provide producing the high-yield transmembrane polypeptides either of native conformation, or in a soluble form.

53 cl, 28 dwg, 5 tbl, 10 ex

FIELD: medicine.

SUBSTANCE: invention describes nucleotide sequence (dwg.2), coding immunogenic polypeptide LcrV(G113), serving the base for construction of recombinant plasmid DNA pETV-I-3455, with the size 6538 bp, which codes immunogenic polypeptide LcrV(G113). Plasmid consists of plasmid pBR322 replicon, β-lactamase gene, determining resistance to ampicillin, T7-promoter, 1ac-operator, f1-replicon and DNA fragment, flanked by the sites for restrictases Ndel and Hindlll, coding synthesis of protein LcrV(G113), which starts from initiating codon ATG. Described is recombinant strain of bacteria E. coli BL21 (DE3)/pETV-I-3455 - producer of immunogenic polypeptide LcrV(G113) with amino acid sequence, represented on dwg.3, where tryptophan in position 113 (W113) is substituted with glycin. Described is method of obtaining said polypeptide by cultivation of strain E. coli BL21(DE3)/pETV-I-3455. Cells are destroyed in buffer solution by ultrasound and polypeptide is isolated successively by gel-permeation chromatography with application of carrier TSK HW-40, anion-exchanging and hydrophobic chromatography.

EFFECT: invention makes it possible to obtain product with high immunogenic and protective activity.

5 cl, 10 dwg, 2 tbl, 5 ex

FIELD: medicine.

SUBSTANCE: invention refers to recombinant plasmid DNA pER-Hir coding a hybrid protein capable to autocatalytic breakdown to form [Leul, Thr2]-63-desulphatohirudin, to Escherichia coli to an ER2566/pER-Hir strain - a producer of said protein and a method for producing genetically engineered [Leu 1, Thr2]-63-desulphatohirudin. The presented recombinant plasmid DNA consists of the SapI/BamHI fragment of DNA plasmid pTWIN-1 containing a promoter and a terminator of T7-RNA-polymerase transcription, an amplifier of phages T7 gene 10 translation, β-laktamase (Ap) gene, modified mini-intein Ssp DnaB gene, with an integrated sequence of a chitin-binding domain, and the SapI/BamHI-fragment of DNA containing a sequence of a gene of recombinant [Leul, Thr2]-63-desulphatohirudin-1 containing β-laktamase (Ap) gene as a genetic marker, and unique recognition sites of restriction endonucleases located at the following distance to the left from the site BamHI: Nrul - 186 base pairs, Ndel - 594 base pairs, Xbal - 882 base pairs, EcoRV - 2913 base pairs, Hpal - 2966 base pairs.

EFFECT: inventions allow producing said compound which is used as a drug applied to prevent blood hypercoagulation.

3 cl, 1 dwg, 4 ex

FIELD: medicine.

SUBSTANCE: what is offered is a new method for producing human methionine-free interferon-alpha2b. The method starts with recombinant plasmid DNA containing a human interferon-alpha2b gene which is foregone by an enteropeptidase proteolysis site, and used to transform Escherichia coli cells. The cells are cultured, and inclusion bodies of the synthesised predecessor are isolated. It is followed by partial renaturation of the isolated predecessor in the presence of dithioerythrole preventing closure of disulphide bonds. The predecessor is hydrolysed by the enteropeptidase enzyme with producing human methionine-free interferon-alpha2b. After completion of the predecessor hydrolysis reaction, complete renaturation of human interferon-alpha2b is carried out in the presence of a pair of cystine and cysteine compounds promoting closure of disulphide bonds. The produced protein is purified by a chromatography in a KM-sepharose.

EFFECT: higher yield.

5 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention can be used for producing recombinant human interleukin-11 (rIL-11). The recombinant plasmid DNA pET32M/mTrx-rhIL-11 is produced by a method which involves synthesis of four fragments of a recombinant human interleukin-11 gene from oligonucleotide primers, cloning of each fragment in a plasmid vector pGEM5z restricted by EcoRV endonuclease, transformation of each of 4 plasmids of E.coli cells of strain DH5a, selection of clones coding the related fragments of the recombinant human interleukin-11 gene, selection of plasmids with the recombinant human interleukin-11 gene without mutations, combination of all fragments of the recombinant human interleukin-11 gene in the plasmid vector pUC19, construction of the vector pET32M of the plasmid pET32b, recloning of the recombinant human interleukin-11 gene in an expression vector pET32M, coding thioredoxin 1 E.coli with substitution of Asn84/Gln. The produced DNA is used to transform the E.coli BL21 (DE3) strain cells to produce the E.coli BL21 (DE3)/pET32M/mTrx-rhIL-11 strain that is a producer of recombinant human interleukin-11 as an ingredient of soluble fused protein mTrx-rhlL-11.

EFFECT: effective production of recombinant interleukin-11 in Escherichia coli cells.

3 cl, 5 dwg, 1 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: described fused protein contains at least two amino acid sequences. The first amino acid sequence, having 90% sequence identity with an amino acid sequence represented in SEQ ID NO:2, is fused with a second amino acid sequence, having at least 90% sequence identity with an amino acid sequence represented in SEQ ID NO:4.

EFFECT: invention provides immunity against various clinically vital strains of group B streptococci.

9 cl, 5 dwg, 8 ex