Recombinant swine influenza virus h1n1 vaccine and method for preparing it
SUBSTANCE: engineered recombinant protein molecule for preparing a recombinant vaccine for an infection caused by swine influenza virus (HlNlv-2009). The molecule consists of a residue of methionine, a sequence of extracellular M2 protein domain of 2 to 24 amino acids and a sequence of a nuclear hepatitis B virus antigen of 4 to 149 amino acids. The molecule is able to form virus-like particles. There are also disclosed a recombinant nucleic acid coding such molecule, a vector for its expression, virus-like particles formed by such molecules and a vaccine based on the prepared virus-like particles.
EFFECT: prepared vaccine can be considered as a candidate recombinant swine influenza virus vaccine.
6 cl, 8 dwg, 3 tbl, 5 ex
The present invention relates to immunology and protein engineering, and in particular, to the creation of the immunogenic preparations and vaccines that can be used for prevention of swine influenza H1N1v-2009.
Influenza is one of the most common viral diseases of humans and animals. Influenza viruses type A differ in the degree of pathogenicity for humans and animals. In recent years, highly pathogenic strains of H5N1 caused local outbreaks with high mortality in the regions of Southeast Asia. The H1N1 virus of swine origin caused the flu pandemic of 2009-10, with an unexpectedly high mortality among high-risk groups and people of middle age. Many phenotypic properties and phylogenetic origin pandemic H1N1v-2009 close to the virus that caused the "Spanish flu" in 1918-1920, which confirms the possibility of return in circulation among the people of the viruses with high potential pathogenicity. Currently used influenza vaccines based on the produced in chicken embryos the influenza virus or its components [Nicholson K., Webster, R., Hay, A. (1998) Textbook of Influenza // Oxford, Blackwell Science]. High variability of surface proteins of the virus, hemagglutinin and neuraminidase leads to the emergence of a new epidemic strain every 1-2 years Webster et al., (1992), Environ. Rev.56. P.152-179], with the same frequency you want to make the "standard" strain-specific vaccines.
One of the potential sources of antigenic variability of the virus of human influenza is its recombination with influenza viruses of animals, which can lead to the emergence of new highly pathogenic recombinant virus, "unknown" on the human immune system and therefore capable of causing a pandemic. At the same time, creating a traditional vaccine for the new strain takes a long time (from 6 to 9 months), during which the emergence of a new pandemic strain of influenza can lead to the death of millions of people. This new highly pathogenic strain is caused the 2009 pandemic virus so-called "swine" flu related, sequences of its hemaglutinin and neuraminidase, a type H1N1.
One of the most promising ways to solve the problem of generating "pandemic" vaccines is to create a "universal" vaccines based on the use of conservative immunodominant peptides, such as the extracellular domain of the M2 protein of influenza virus, a sequence of almost invariant in all human strains. However, the virus of swine influenza H1N1v-2009 this sequence significantly different from that found in human strains.
So about the time, the task of creating a vaccine against the new influenza viruses, including H1N1v-2009, continues to be relevant. This vaccine can protect people from a new pandemic H1N1v virus-2009, as well as its derivatives, which can be the result of recombination between this strain and other avian and human viruses.
The level of technology.
1) Modern influenza vaccine.
Table 1 presents the list and characteristics of influenza vaccines, registered and used in Russia.
All vaccines can be divided into the following types:
From this list it is clear that for such topical vaccines like influenza, was not developed recombinant epitope vaccine. However, the advantage of genetic engineering technologies in addressing the efficacy and safety of flu vaccines is obvious and you can list them in the following order of importance:
1. Guaranteed complete absence of egg protein (ovalbumin) - the main allergen in modern vaccines.
2. Individuality favorite epitope - assurance of the absence of autoimmune reactions and complications. This danger is especially great for live and televizionnyh vaccines and I do not exclude the moved to the subunit.
3. The relative ease of production, the lack of dependence on natural raw materials, such as chicken embryos, and full compliance with safety requirements (absence of viral components). Good economic performance.
4. The ability to quickly deploy large-scale production in case of greater demand for vaccine in preponderancy and pandemic periods.
|Vaccines for preventing influenza in domestic and foreign production, used in Russia.|
|Vaccine||Drugs and their manufacturers||Age vaccinated, schema|
|Live influenza vaccine (live flu vaccine)||Live flu vaccine (for children and adults), FSUE "Microgen", Irkutsk||Children from 3 years and adults : 0.5 ml once intraline|
|Inactivated influenza vaccine (VVM), televisiona||The influenza vaccine inactivated, FSUE "Microgen" Ufa; RIPPOWAM, ENT. SPb NEWS||Children from 7 years of age, adolescents and adults : 0.5 ml twice intranasal is but with an interval of 3-4 weeks. Adults 18 years of age 0.5 ml once parenteral|
|Inactivated influenza vaccine (VVM), split||Fluarix, SmithKline Beecham (Germany)||Children from 6 months to 6 years 0.25 ml twice parenteral with an interval of 4-6 weeks. Children older than 6 years and adults : 0.5 ml once parenteral|
|Vaxigrip, Sanofi Pasteur (France)||The same|
|Begrivac, Chiron Behring (Germany)||The same|
|Fluvoxin (China)||The same|
|Inactivated influenza vaccine (VVM), subunit||Influvac, Solvay Duphar (Netherlands)||The same|
|Agrippal S1, Chiron Behring (Italy)||The same|
|Inflexal V (viralimalai) VEGA(Switzerland)||The same|
|Grippol, FSUE "Microgen", Ufa||Children from 6 months to 3 years 0, 25 ml, children over 3 years of age and adults in 0.5 ml of parenteral|
|Grippol plus LLC FC "Petrovax" Moscow||Children from 3 years and adults : 0.5 ml which has been many times parenteral|
|Grippol Neo, LLC FC "Petrovax" Moscow||Adult 0.5 ml once parenteral|
2) Biologically-active nanostructures on the basis of capsid animal viruses
The ability of biological macromolecules to the self-Assembly and self-organization is one of the hallmarks of living systems and provides countless opportunities for the use of biomolecules as "building blocks "molecular design" for directional create new nanoarhitektur with specified spatial and functional properties. One of the most striking examples of such structures, with a clear symmetry, extensive opportunities for directed modification of modern methods of genetic engineering are viral particles.
High stability of recombinant virusopodobnyh particles (the VLP), symmetric spatial organization, ease of access and cleaning of the VLP, security, opportunities for directed modification of the structural proteins of the virus by means of genetic engineering make these particles are ideal vectors vectors to represent alien functionally important peptides and whole proteins. Presentation of antigenic determinants of epitopes on the surface of the VLP provides high immunogenet is due to the use of the virion as an adjuvant in combination with high yield, ease of cleaning and storage stability. The effectiveness of this approach is illustrated by numerous examples of the VLP to combat communicable diseases such as malaria, rabies, viral hepatitis B, C and E, human papilloma virus infection (cervical cancer), AIDS, etc.
One of the most effective media antigenic determinant is nucleocapsid protein of hepatitis B virus human and closely related animal viruses, referred to collectively as UGB. The monomers of this protein consisting of 183-185 amino acids in infected cells gather in stable aggregates (virus-like particles, called HBc particles. There are two types of particles - particles with a diameter of about 30 nm, consisting of 180 monomers, and in a larger number of particles with a diameter of 34 nm, consisting of 240 subunits.
It is known that the HBc-particles can be used as a highly immunogenic carrier alien peptides that stimulate T-cell immune response. Two district HBc can be used for the presentation of foreign peptides on the surface of the HBc-particle - N-end of the protein and the so-called immunodominant loop, located between 75 and 85 amino acid residues of the protein. It was shown that the C-terminal arginine-rich region of the protein (150-183 amino acid residues) can be removed without compliance with the foster self-Assembly of particles, moreover, unlike the educated full-length protein, such particles when the Assembly does not include a nucleic acid of the host cell.
Chimeric HBc particles often have a less ordered structure than particles not containing epitopes [Schodel et al., (1994) J. Exp. Med., 180:1037-1046]. In many cases, the inserted epitope causes so strong destabilization that the hybrid particles or cannot be obtained, or be so unstable that it cannot be used as a component of vaccines [Schodel et al. (1994) Infect. Immunol., 62:1669-1676]. Therefore, shown below ordered structure and stability established in the present invention the recombinant HBc particles containing epitopes of influenza viruses of birds are not anticipated in advance.
The use of recombinant HBc particles to obtain influenza vaccines described in [Neirynck et al., (1999) Nature Med., 5(10):1157-1163] and subsequent papers of this group. The authors obtained a series of recombinant HBc particles containing the extracellular domain of M2 (M2E) protein of the virus of human influenza and shows their high immunogenicity and protection against infection by human viruses in experiments on laboratory animals. In these works was used M2E sequence SLLTEVETPIRNEWGCRCNDSSD present in virtually unchanged form in all human strains of the virus, since isolated the frame in 1933 strain A/WSN/33.
Protein M2 of influenza virus type A fundamentally important for the design of universal vaccines in connection with a number of undervalued its properties:
1. localization and exposure on the surface of the virion,
2. it is of critical importance in the processes of fugina and budding (early and late stage of infection),
3. the highly conserved among all subtypes of avian influenza virus A.
Selection of highly conserved antigen included the induction of antibodies capable of suppressing the reproduction of most subtypes A influenza viruses. This vaccine is called "universal" - assuming protection virtually all of the influenza virus type A.
Therefore these authors vaccine was "universal" in the sense of human influenza. However, in animal influenza viruses M2E sequence differs from those listed above. For example, M2e virus of swine influenza A/California/04/2009(H1N1) has the sequence SLLTEVET PTRSEWECRCSDSSD (highlighted differences from the consensus human sequence (Figure 1). Published data on the effectiveness of the vaccine, created based on human ME in respect of "swine" influenza H1N1v-2009 no. Moreover, obtained during the implementation of the present invention results indicate that M2E-HBc particles containing swine M2E, f is unbiased against swine influenza A/California/04/2009(H1N1), but not against human strain A/PR/8/34. Had no produktivnost against strain A/PR/8/34 we created earlier using similar approaches candidate vaccine against the virus of avian influenza (Ravin NV, I. Kiselev, Scriabin KG 2009. A universal vaccine against influenza virus in birds. RF patent №2358981).
Thus, current methods of creating influenza vaccines, with its basis in the design of recombinant M2E-HBc particles containing human M2E, do not solve the problem of creating a vaccine against the virus of swine influenza H1N1v-2009. In this regard, the aim of the invention was to develop approaches to the creation of a "universal" vaccines and, in particular, the vaccine provides effective protection against evolving viruses of swine influenza type H1N1, presenting a special risk for people in the pandemic period.
Disclosure of inventions
In the present invention the aim was to create a candidate recombinant vaccine against the virus of swine influenza a (H1N1v-2009). The basis of the solution of the task was the construction of recombinant virus-like particles based on the core antigen of hepatitis B virus (HBc), which on their surface a polypeptide extracellular domain of the M2 protein of the virus of swine influenza A/California/04/2009(H1N1).
Fakticheskaia was solved by:
a) the design for the expression of functionally important and accessible epitope of the most conservative protein of influenza virus type A,
b) synthesis of the gene encoding a hybrid protein comprising ME and devoid of the C-terminal region HBc antigen of hepatitis b virus,
C) create a strain of E. coli - producer of the hybrid protein M2Es-HBc
g) develop a Protocol for isolation and purification of recombinant M2Es-HBc particles,
d) testing-derived drugs in laboratory animals, the results of which indicate the high immunogenicity of virus-like M2Es-HBc particles and 100% protection of animals against lethal influenza infection.
The first aspect of the present invention associated with obtaining a recombinant nucleic acid (synthetic gene)that encodes a recombinant protein molecule (recombinant protein) M2Es-HBc amino acid sequence which, starting from N-Terminus consists of a methionine residue, sequence SLLEVETPTRSEWECRCSDSSD extracellular domain of the M2 protein of the virus of swine influenza A/California/04/2009(H1N1) from the 2nd to the 24th amino acids (M2Es), depicted in figure 1, and the sequence of the nuclear antigen of hepatitis B virus (HBc) from 4th to 149-th amino acid, relative to the first methionine in native HBc, which, if necessary, may be followed by one cysteine.
The second aspect of the invention is POPs is the use of recombinant expression vector and strain E. coli - producer of recombinant protein M2Es-HBc. With this purpose, a recombinant nucleic acid encoding a M2Es-HBc, cloned in the expression vector pQE60 under control RT-l promoter, LacI repressed and induced by IPTG, resulting recombinant expression vector.
Accordingly, a third aspect of the invention relates to optimization of the conditions of production of recombinant protein M2Es-HBc in the cells of E. coli and the development of methods for isolation and purification formed them like M2Es-HBc particles.
A fourth aspect of the invention is associated with the testing of the obtained virus-like drugs M2Es-HBc particles in laboratory animals. It was shown that (1) drugs M2Es-HBc particles are not toxic to laboratory animals (mice), (2) immunization of mice leads to the formation of their blood antibody specifically recognizes ME peptide in the composition M2Es-HBc particles, (3) immunization of mice with the drug M2Es-HBc particles provides 100% protection against lethal influenza infection. Thus, virus-like M2Es-HBc particles can be used as the basis of a vaccine against swine influenza H1N1v-2009.
Brief description of drawings
Figure 1 - comparison of the amino acid sequence of the extracellular domain of M2 protein of different strains of influenza type a in humans and animals. Differences from the consensus of the members is Telenesti Me human influenza virus are highlighted.
Figure 2 Amino acid sequence of the recombinant protein molecule M2Es-HBC. The amino acid sequence of the M2 protein is underlined.
Figure 3 - Structure of recombinant expression vector pQE-M2EsHBc.
Figure 4 - Expression and purification of recombinant protein M2Es-HBc.
Shows the results of analysis of protein drugs using SDS-PAGE.
1 and 5, molecular weight marker (shown as position markers 19 and 26 kDa)
2, is a protein preparation from strain DLT1270/pQE-M2EsHBc-l before the induction of the synthesis of the product
3, is the same, but after 16 hours after induction of the synthesis of the product
4, the fraction of soluble proteins extracted from the producer strain after induction of the synthesis product.
5, is a purified product M2EsHBc particles
Figure 5 - analysis of the structure of virus-like M2Es-HBc particles using electron microscopy. The scale corresponds to 100 nm.
6 is a Serum IgG antibodies immunized with virus-like M2Es-HBc particles mice, communicating with synthetic M2e peptides. (A) - serum IgG to G26; (B), serum IgG to G18; (C) - serum IgG to G11-1; (D) - serum IgG to G19.
7 - Dynamics of mass of the mice immunized with virus-like M2EsHBc particles after infection with influenza virus A/California/04/2009(H1N1).
Fig - Dynamics death of mice immunized with virus-like M2EsHBc particles after infection by various strains of influenza virus.<> The implementation of the invention.
Example 1. Recombinant protein molecule M2Es-HBc and coding its recombinant nucleic acid.
To obtain immunogenic virus-like particles used recombinant protein comprising the extracellular domain of the M2 protein of influenza virus (ME domain) and a fragment of HBc antigen UGB. Recombinant protein molecule, M2Es-HBcl has consistently included the following elements: N-terminal methionine, the amino acid sequence of SLLTEVETPTRSEWECRCSDSSD corresponding M2E domain from strain A/California/04/2009(H1N1) influenza virus amino acid sequence of HBc [Bichko, V., Pushko, P., Dreilina, D., Pumpen, and P. Gren, E. (1985) FEBS Lett. 185 (1), 208-212], starting with asparagine at position +4 to valine at position +149 (relative to the first methionine in native HBc), and cysteine on the C - end (Figure 2). The introduction of the C-terminal cysteine has been to improve the stability of recombinant M2E-HBc particles, but not necessarily to obtain high virus-like particles.
To obtain the above-described recombinant protein molecule was synthesized encoding its recombinant nucleic acid (gene). At the first stage portion of the sequence of this gene was obtained by PCR using primers M2sF1 (GAA ACC CCG ACC CGT GAA AGC TGG GAA TGC CGT TGC AGC) and HBC-R (AAG ATC TCA GCA AAC AAC AGT AGT CTC CGG AAG) and DNA previously obtained plasmid pQE-M2HBc-2 [Ravi is NV, Kiselev, I., Scriabin KG 2009. A universal vaccine against influenza virus in birds. RF patent №2358981] as a matrix. PCR was performed under the following conditions: (1) 94°C - 30 sec, (2) 50°C - 60 s, (3) 70°C 90 sec, steps 1-3 were repeated 30 times. In the next step, the obtained DNA fragment was used as template for PCR amplification with primers M2sF2 (CTC ATG AGC CTG CTG ACC GAA GTG GAA ACC CCG ACC CGT AGC) and HBC-R. PCR was performed under the following conditions: (1) 94°C - 30 sec, (2) 50°C - 60 s, (3) 70°C 90 sec, steps 1-3 were repeated 30 times.
After electrophoretic analysis was isolated PCR fragment size of 0.52 TPN, which is ligated with the DNA of the vector pUC19, pre-treated with restriction enzyme SmaI. The obtained plasmid, which was selected by restriction analysis, was designated pUC-M2EsHBc and used to obtain a fragment containing a recombinant nucleic acid encoding a protein M2Es-HBC. For this purpose it was treated with restrictase PagI and Bg1II, were isolated fragment 0,52 TPN representing the desired recombinant nucleic acid.
Example 2. Creating a recombinant expression vector and E. coli strain - producer of recombinant protein M2Es-HBc.
To create a recombinant expression vector recombinant nucleic acid that represents the DNA fragment size of 0.52 TPN cut from the plasmid pUC-M2EsHBc with the help of the of restricted PagI and Bg1II, cloned in the expression vector pQE60 sites NcoI and Bg1II. The obtained recombinant expression vector was designated as pQE-M2sHBc (Figure 3) and used in further work. In this vector the recombinant nucleic acid encoding a M2Es-HBc, operatively linked to a promoter PT5-lac and a transcription terminator t0 (Figure 3), which ensures its expression in the cell of the bacterium Escherichia coli (Figure 4). Absence due to PCR mutations in the synthesized gene was confirmed by DNA sequencing.
To obtain strain-producer of recombinant protein M2Es-HBc recombinant expression vector pQE-M2sHBc was introduced into cells of E. coli strain DLT1270 using transformation. Strain DLT1270 derived strain DH10B [Grant et al., Proc.Natl. Acad. Sci. USA. 1990. v.87(12), p.4645-4649], contains a gene repressor of Lac operon lacI, integrated into the chromosome. Culture of transformed cells DLT1270/ pQE-M2sHBc were grown in standard conditions suitable for expression of the recombinant protein, for example, in LB-broth to mid-logarithmic growth phase (OD600=0,5) at 37°C, IPTG was added to a concentration of 1mM (sufficient for full induction of the promoter) and cells were grown for 16 hours at 37°C. Samples for protein determination were collected at the initial moment of time, and 16 hours after induction. The level of expression of recombinant Bel is and M2Es-HBc was performed by analysis of total protein drugs, isolated from bacterial cultures using SDS-PAGE. The maximum production level of recombinant protein M2Es-HBC was about 30-40% of the total cellular protein (Figure 4).
Example 3. Isolation and purification of virus-like particles from recombinant protein M2Es-HBc
Cells of E. coli producer strain recombinant protein M2Es-HBc after induction (see above) was besieged by centrifugation 3000 rpm for 30 minutes and resuspendable in 50 mm Tris-HCl buffer pH 8.0 containing 0.5 M NaCl, 15 mm EDTA and 20% sucrose at a rate of 1 ml buffer to 50 ml of the culture fluid. The cell suspension was treated with lysozyme (1 mg/ml) for 15 minutes at +4°C, then the cells were destroyed by ultrasound. To the resulting lysate was added 1/20 volume of a solution of polyethylene glycol (50% weight/volume) and incubated for 30 minutes at +4°C. Then, centrifugation was performed for 10 minutes at 13,000 rpm To the supernatant was added 1/5 volume of a saturated solution of ammonium sulfate, stirred and left for 30 minutes at +4°C. Formed after centrifugation of the precipitated protein was dissolved in 1 ml of the same buffer and re-precipitated with ammonium sulfate in the same conditions. The precipitate was dissolved in 1 ml of a physiologically-acceptable carrier, 50 mm Tris-HCl buffer, pH 8.0, containing 0.15 M NaCl, 1 mm EDTA and 20% sucrose. The resulting recombinant protein M2Es-HBc contained according to SDS-PAGE, about 90% of the be the ka M2Es-HBc in a concentration of about 1.5 mg/ml
These conditions of isolation and purification were selected experimentally and may vary widely known to the average expert in the field values.
The fact of the formation of recombinant protein M2Es-HBc virus-like particles was shown by electron microscopy of selected drug as a control was used the drug HBc particles not containing MA polypeptide, but otherwise equivalent to real particles. Electron microscopic analysis (Figure 5) confirmed the self-Assembly of recombinant protein molecules M2Es-HBc in virus-like particles of 30-40 nm, similar to particles formed by the HBc antigen, not containing M2E polypeptide.
Example 4. The immunogenicity of the candidate vaccine based on M2Es-HBc particles.
The drug-like M2Es-HBc particles dissolved in a physiologically acceptable medium, was tested as a candidate influenza vaccine. As a physiologically-acceptable carrier can be used in a variety of aqueous solutions used to dissolve drugs, well known to the average expert in this field.
To characterize the immunogenicity and protective actions an experiment was conducted by immunization of mice with purified virus-like drug M2Es-HBc particles. A pilot group of 60 animals of immunities is whether subcutaneously using for the first injection of the vaccine adjuvant TiterMax Gold Adjuvant (Sigma), and for the two subsequent immunization is incomplete adjuvant's adjuvant (Sigma). Scheme of immunization are shown in table 2.
To determine the immunogenicity of the candidate vaccine were studied in the serum of mice after immunization M2Es-HBc. Serum was collected 2 weeks after the 1st and after the 3rd immunization, antibody titers were determined in pooled sera of mice of each group (3-5 mice). The negative control serum is not immunized mice, positive control monoclinal antibodies to M2e Potsdam (D2).
ELISA to determine the titer of specific antibodies was performed by standard method. 96-well tablets with high sorption capacity (Greiner, Germany) were coated with synthetic peptides G-11-1 (Me virus avian influenza strain A/Chicken/Kurgan/05/2005 (H5N1)), G19 (M2e virus avian influenza strain A/Duck/ Potsdam 1402-6/1986 (H5N2)), G26 (M2e virus of swine influenza strain A/California/7/2009 (H1N1)), G18 (M2e virus human influenza strain PR/8/34) at a concentration of 5 μg/ml and 3M2eHBc at a concentration of 3 µg/ml in carbonate buffer, pH of 9.5 and 9.6), kept overnight at 4°C. the Tablets were treated with blocking buffer (0.01 M FSB, pH 7.2 to 7.4 with 5% ETS) for 1 hour at room temperature, washed 3 times the FSB with twin. A pool of sera from mice of each group were tested in duplicates. In wells tablet was added 100 μl of 2-fold dilutions of sera (starting with 1:400) in Blo is IRowset buffer, incubated 1 hour at room temperature. As conjugate used rabbit polyclonal antimurine IgG (Abcam, UK) at a dilution of 1:8000 labeled with horseradish peroxidase. As the substrate used TMB. Accounting the reaction was carried out at a wavelength of 450 nm. The title took the highest serum dilution that gives an optical density of at least 2 times higher than the serum is not immunized mice in the same breeding.
Data IFA presents figure 6 and summarized in Table 3.
|Titers of IgG antibodies to synthetic peptides Me.|
|Serum samples||The titers of antibodies to synthetic peptides M2e|
|M2Es-HBc with adjuvant||after the 1st immunization||1600||1600||800||800|
|after the 3rd immunization||51200||51200||6400|
|The positive control (monoclonal antibodies to peptide G19-clone D2)||>51200||>51200||>51200||1600|
|The negative control (serum not immunized mice)||<400||<400||<400||<400|
Thus, after immunization of mice candidate vaccine based on virus-like M2Es-HBc particles are formed at high titers of serum antibody isotype IgG, communicating with a synthetic peptide G-26 "swine" influenza A/California/7/2009 and synthetic peptides, the sequences of which correspond to the "bird" (A/Chicken/Kurgan/05/2005 and a/Duck/ Potsdam 1402-6/1986) and "human" (PR/8/34) flu viruses. The highest antibody titer was observed after 3-fold immunization.
Example 5. The protective effect of the candidate vaccine based on M2Es-HBc particles on the model of lethal influenza infection.
To infect animals immunized with candidate vaccines have been used the following influenza viruses adapted to mice: A/Duck/Potsdam/1402-6/1986 (H5N2), A/California/04/2009 (H1N1) and A/PR/8/34 (H1N1). Viruses were injected intranasal is but a dose of 5 LD/ 5050 μl/mouse under light ether anesthesia (1 LD/50- the dose corresponding to 50% mortality).
After infection conducted daily observation of animals. The protective effect of the candidate vaccine was evaluated using two parameters:
1. Determine the dynamics of drop body weight of mice after infection;
2. Survival of mice after infection.
Figure 7 shows the dynamics of the falling weight of animals after infection, which may be an indicator of the severity of the disease. After infection, the weight of the immunized animals was reduced (up to 90% of initial), but to a much lesser extent than in mice of the control group (70% of initial weight). These results show that immunization with candidate vaccine does not prevent influenza infection, but facilitates its flow.
On Fig shows the results of the main indicator which assesses the effectiveness of protective actions M2Es-HBc particle - dynamics death of mice after infection 5 LD/50 of the virus of swine influenza A/California/4/2009 (H1N1). The obtained data clearly indicate 100% protective action of the candidate vaccine based on virus-like particles M2Es-HBc after three immunizations. For the entire observation period, all animals in this group were alive. In the control group of mice under these conditions, the infection remained the ü alive only 12% of the animals. Partial protection from infection was observed against strains of influenza, in which the amino acid sequence of M2e peptide differs from that used for immunization drug. Thus, during infection of mice with virus avian strain A/Duck/Potsdam/1 402-6/1986 (H5N2) observed a 60% survival of immunized animals against 13% in the control group (statistical significance P<0,006 test Fisher). During infection with human influenza A/PR/8/34 survival of animals was 40% in the experimental group and 20% in the control. These results demonstrate the functional significance of the differences of the sequences Me peptides of influenza viruses of humans and animals that must be considered when developing a "universal" influenza vaccines based on M2e.
The data on the efficacy of the candidate vaccine against swine influenza in mice can extrapolate the achieved effect and, because the mechanisms of functioning of the immune system in all mammals basically similar, and laboratory animals are the standard model for developing vaccines. Consequently, the resulting preparation can be considered as a candidate for a vaccine against swine influenza a H1N1v 2009 is designed for a person.
1. Recombinant protein molecule for the preparation of recombinant vaccinates infection, caused by the virus of swine influenza a (H1N1v-2009), amino acid sequence which, starting from N-Terminus consists of a methionine residue, the sequence of the extracellular domain of the M2 protein (ME) virus of swine flu from the 2nd to the 24th amino acids presented in figure 1, and the sequence of the nuclear antigen of hepatitis b virus (HBC) from 4th to 149-th amino acid, relative to the first methionine in native HBC, which are capable of forming virus-like particles size of about 30-40 nm.
2. Recombinant nucleic acid for the preparation of recombinant vaccines against infections caused by the virus of swine influenza a (H1N1v-2009), encoding a recombinant protein molecule according to claim 1 and consisting of consecutive parts: a triplet of nucleotides encoding a methionine, a nucleotide sequence corresponding to the amino acid sequence of a fragment ME, and the nucleotide sequence corresponding to the amino acid sequence of a fragment of HBC.
3. The recombinant expression vector containing the nucleic acid according to claim 2, operatively linked with regulatory elements ensuring expression in the cell of the bacterium Escherichia coli.
4. A method of obtaining a recombinant vaccine against infections caused by a virus of swine influenza a (H1N1v-2009), involving the introduction of a nucleic KIS is the notes according to claim 2 in a cell of the bacterium Escherichia coli, production of recombinant protein molecules in a given cell with the formation of virus-like particles, their separation and mixing with physiologically acceptable medium.
5. Virus-like particle formed of recombinant protein molecules according to claim 1, intended for use as a component of vaccines against infections caused by a virus of swine influenza a (H1N1v-2009).
6. Vaccine against infections caused by a virus of swine influenza a (H1N1v-2009), including virus-like particles according to claim 5.
SUBSTANCE: what is offered is a flat-bottomed well applicable for purposes of a diagnostic technique for viruses. The well consists of a primary chamber and a secondary chamber. The primary chamber has a base, a liquid sample inlet and side walls extending from the inlet to the base of the chamber. The secondary chamber extends from the base of the primary chamber, and has a flat bottom. Thus the primary chamber has a square cross section, and the secondary chamber has a round cross section to decrease or eliminate a meniscus. What is also disclosed is a method of virus detection with using such wells.
EFFECT: technique improvement.
21 cl, 4 dwg
SUBSTANCE: present invention relates to biotechnology and genetic engineering. The method of producing a target virus or one or more target proteins different from adenovirus involves culturing an expression spinal cell and extracting the end product. The cell is a spinal cell containing a gene which is stably integrated into its genome, where said gene codes PIX adenovirus or its functional version, as heterologous regulatory protein. The cell stably expresses said regulatory protein or its functional version via a target virus which is not adenovirus or via a vector carrying nucleic acid sequences which code said virus or via a vector carrying nucleic acid sequences which code said one or more target proteins.
EFFECT: disclosed method enables to increase output of the end product.
24 cl, 18 dwg, 15 ex
SUBSTANCE: invention relates to biotechnology and virology. Compositions and methods used to induce immune response against the influenza virus in canines using novel strains, polynucleotides or polypeptides thereof are described. The invention can be used in veterinary.
EFFECT: versions of an influenza virus, which can infect canines and cause a respiratory disease in canines are disclosed.
38 cl, 14 dwg, 25 tbl, 16 ex
SUBSTANCE: invention relates to veterinarian virology and biotechnology. A novel industrial strain NADL-ARRIAH of bovine viral diarrhoea is obtained. The strain is deposited in the microorganisms used in veterinary and animal husbandry of FGU VGNKI under registration number - industrial strain NADL-ARRIAH-DEP bovine viral diarrhoea. The strain is reproduced in passaged cultures of calf kidney cells, saiga finite cell line, intestinal mucus finite cell line, intraspecific swine embryo kidney hybrid finite cell line with swine splenocyte (A4×C2) at 37°C and in 72-96 hour of incubation, the virus accumulates therein in amount of 6.0-7.0 lg TCD50/cm3. The strain is stable and retains its properties for 10 passages. The strain is used to obtain hyperimmune blood serum to bovine viral diarrhoea on rabbit and guinea pigs, used for agent diagnosis and treating animal diseases.The invention can be used in veterinary.
EFFECT: inactivated emulsion vaccine derived from said strain protects immunised animals from direct infection by a virulent virus.
1 dwg, 10 tbl, 7 ex
SUBSTANCE: chimeric adenovirus genome comprises the nucleotide sequence SEQ ID N0:1 provided a transcription site E2B therein comprises the nucleotide sequence SEQ ID NO:3.
EFFECT: adenovirus presented under the present invention shows a high therapeutic index.
9 dwg, 1 tbl, 8 ex
SUBSTANCE: all the stages of host cell culture cultivation are implemented in multilayered HYPERFlask culture vessels. Also, the present invention provides an recombinant adenovirus preparation produced by the method according to the present invention.
EFFECT: method provides intensifying production of the recombinant adenovirus drug preparation, considerably reducing time and space resources required for manufacturing and storage, as well as considerably lowering the ratio of physical and infectious viral particles that leads to higher quality of the drug preparation.
12 cl, 2 tbl, 4 ex
FIELD: biology, medicine, nanotechnology.
SUBSTANCE: group of inventions is referred to the area of biology, medicine, nanotechnology and involves processing of new platform carriers for formation of complexes with biologically active compounds. The proposed platform carriers are of spherical shape and are made by the way of thermal reconfiguration of structure of tobacco mosaic virus (TMV) or another tobamovirus or another plant virus with spiral structure or their fragments. The platform carriers are also produced by the way of thermal reconfiguration of coat protein of TMV group virus (tobamoviruses) or another phytovirus with spiral structure or their fragments. There were also proposed the compositions containing the platform-carrier of said spherical carrier connected with the alien protein or another biologically active compound. The advantages of new platform carriers include the simplicity and quickness of production, possibility to get spherical particles of regulated sizes, long term storage, absence of aggregation and preservation of spherical shape during its storage, centrifugation and resedimentation, the convenience of spherical particles shape for formation of complexes with target compound.
EFFECT: important feature of a new carrier platform is the capability to adsorb on the surface and to form compositions with different structurally and functionally alien proteins/antigens.
3 cl, 6 ex, 10 dwg
SUBSTANCE: complex antigen of measles virus used as a component of immunoenzymometric test system for diagnostics of antibodies to measles virus was produced from culture liquid containing measles virus stain Leningrad 16 with titer no less than 105.0 50% tissue cytopathic dose/ml, Edmonston - no less than 106.0 50% tissue cytopathic dose/ml, NovO/96 no less than 108.0 50% tissue cytopathic dose/ml by inactivation of its infectious activity by detergent and separation of protein from cellular lysate by chromatographic purification in amount no less than 2 mcg/ml with the purity no less than 70%. Culture virus-containing liquid was processed using separate cultivation of measles virus stains Leningrad 16, Edmonston and NovO/96 on monolayer of Vero cells culture with subsequent mixing of culture virus containing liquid in proportion 1:1:1 v/v.
EFFECT: Usage of invention provides for increased sensitivity and specificity of a complex antigen possessing the property to detect antibodies in blood serum.
2 cl, 4 tbl, 6 ex
SUBSTANCE: group of inventions is referred to the area of medicine, namely to the area of virusology, and is related to virosomes containing hemagglutinin extracted from influenza virus produced in the cell lines, compositions, containing said virosomes, means of manufacturing and applications. The essence of the invention including virosomes containing hemagglutinin extracted from influenza virus produced in the bird cell lines, compositions, containing said virosomes, application of virosome as a vessel, set, methods of vaccination, methods of treatment and methods of virosome production.
EFFECT: production of virosomes having improved merging capability and increased immunogenicity.
21 cl, 3 tbl, 5 dwg
SUBSTANCE: what is described is a composition containing an ordered antigen pattern where antigen represents IL-1, mutein IL-1 or fragment IL-1. There is also offered a based vaccine. The compositions offered in the invention can be applied for producing vaccines for inflammatory diseases and chronic autoimmune diseases, transmittable diseases and cardiovascular diseases.
EFFECT: compositions effectively induce immune responses, particularly humoral immune responses; compositions are the most suitable for effective induction of autogenic immune responses.
46 cl, 2 dwg, 3 tbl, 14 ex
SUBSTANCE: what is offered is an expression construct for expression of single- or multipass transmembrane polypeptides in a bacterial host cell. Said construct contains a protein-coding polynucleotide, a strictly sensitive promoter of lower basal activity in the host-cell, and a leader sequence comprising a translation initiation enhancer. The strictly sensitive promoter comprises at least one positive control element and at least one negative control element. One or more positive and negative control elements represent a heterologous control element. Besides, what is offered is a method for producing the expressed transmembrane polypeptide and a method of recovering it form the host cell.
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
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
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
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
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
SUBSTANCE: polypeptide contains extracellular and transmembrane domains of a human tissue factor. A DNA fragment coding this polypeptide, and a plasmid pET28a(+) are used to produce a genetic make-up enabling biosynthesis of the recombinant human tissue factor. Also, the E coli BL21 [DE3]/p6E-tTF strain that is a producer of the recombinant human tissue factor is offered.
EFFECT: high-yield recombinant protein and simplified recovery and purification procedures.
2 cl, 4 dwg, 3 ex
SUBSTANCE: stable inheritance effect is independent of an insertion position and orientation. The introduction of said sequence essentially reduces the loss frequency of both low-copy, and high-copy vectors. The given invention can find application in plasmid-bearing strain selection. And the cultivation of said strains does not require a selective medium to be used.
EFFECT: higher stability of vector inheritance.
1 dwg, 1 tbl, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biotechnology and deals with recombinant plasmid DNA, which contains sequence of gene of mature staphylokinase Staphylococcus aureus with replacement of codons K74, E75 and R77 with triplets, which code Ala, of strain Escherichia coli MZ09 and method of obtaining recombinant protein, which contains sequence of gene of mature staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets. Essence of method includes recombinant plasmid DNA, which codes sequence of gene of mature protein of staphylokinase from Staphylococcus aureus staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets, which has nucleotide sequence, given in dwg.1. Invention also includes strain Esherichia coli MZ09, producent of recombinant staphylokinase, with sequence, given in dwg.1, as well as method of its obtaining.
EFFECT: invention makes it possible to obtain staphylokinase protein which possesses high fibrinogenic activity, with output of recombinant protein to 20% from total amount.
3 cl, 2 dwg
SUBSTANCE: agent contains mutant luciferase of glowworms Luciola mingrelica (SEQ ID No:2), recovered from recombinant cells E.coli transformed by plasmid pETL7, luciferin, magnesium sulphate, tris-(oxymethyl)-aminomethane, acetic acid, sodium ethylene aminotetraacetate, dithiotreitol, bovine serum albumin, sucrose and water.
EFFECT: higher sensitivity of ATP test, lower enzyme consumption.
2 cl, 3 dwg, 1 tbl, 4 ex
SUBSTANCE: recombinant plasmid pETPHOFus enabling synthesis of an anthrax lethal factor substratum as a part of one polypeptide with a mutated reporter enzyme of alkaline phosphatase Escherichia coli. Said fused polypeptide represents a new high-sensitivity anthrax lethal factor substratum. Besides, an Escherichia coli BL-PHOFus strain being a producer of the fused polypeptide is offered. The strain provides a no-fermentation high-yield synthesized protein not less than 30 mg in 1 litre of a liquid culture. The prepared anthrax lethal factor substratum shows hypersensitiveness and high specificity to LF splitting and is able to detect proteolytic activity of the anthrax lethal factor in concentration up to 1 pM.
EFFECT: invention can find application in clinical recognition of anthrax disease; for detecting hot spots of anthrax, for screening the compound libraries and searching anthrax lethal factor activity inhibitors.
2 cl, 4 dwg, 4 ex
SUBSTANCE: invention relates to biotechnology and virology. Compositions and methods used to induce immune response against the influenza virus in canines using novel strains, polynucleotides or polypeptides thereof are described. The invention can be used in veterinary.
EFFECT: versions of an influenza virus, which can infect canines and cause a respiratory disease in canines are disclosed.
38 cl, 14 dwg, 25 tbl, 16 ex