Method of obtaining peroral vaccine against rabies virus
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biotechnology of veterinary medications. Vaccine against rabies virus represents allantoic fluid of chicken embryos. Fluid contains glycoprotein of rabies virus, as well as mixture if recombinant adenoviruses of birds, which carry gene of surface glycoprotein of rabies virus, one of which contains secreted form of surface glycoprotein of rabies virus, and second - membrane-bound one. Interaction of glycoproteins with animal organism is reached by peroral introduction of medication.
EFFECT: invention can be applied in veterinary.
3 dwg, 1 tbl, 6 ex
Rabies is an acute infectious disease of zoonotic origin. Cases of rabies in wild and domestic animals are regularly recorded in many countries, posing a serious threat to the population. In veterinary practice is widespread live and inactivated televisionnye vaccines vaccine strains of rabies virus (Rabies, 2nd edition. Alan .Jackson, William H.Wunner, 2007). Traditional vaccines against rabies derived from nervous tissue of infected animals. The use of such vaccines often leads to undesirable side effects (polyneuritis, encephalomyelitis), associated with the presence of impurities myelin in preparation (J. Postgrad Med. 1970 16(3): 132-4). Less reactogenicity have a vaccine based on the neural tissue of newborn animals (mice, lambs), which is associated with poor development of the myelin sheath in the nervous tissue of these animals. However, these vaccines are unsafe. The first vaccine against rabies, based not on nervous tissue vaccines were produced in duck embryos (Mil Med. 1964; 129:960-5). These vaccines were soon replaced by the modern culture vaccines based on diploid human cells, Vero cells (Dev Biol Stand. 1981; 50:173-82), cells of the kidneys of newborn hamsters (Appl Environ. 1973 Dec; 26(6):858-62), chicken embryo fibroblasts (Monogr Ser World Health Organ. 1966; 23:124-31). Despite effective is Yunosti use of live attenuated strains of rabies virus for vaccination, their use is associated with risk of release to the environment of a live infectious virus, because it does not exclude the possibility of reversion to a virulent form.
Currently, the widely used culture vaccine containing attenuated strains of rabies virus, inactivated various chemical agents (phenol, beta-propiolactone) or physical stress (UV radiation) (Jpn J Med Sci Biol. 1953; 6(6):577-86). However, the receipt of such vaccines is associated with the need to work directly with a live pathogen. In addition, the process of attenuative virulent strain with the purpose of reception of a preparation suitable for mass immunization, is a tedious and a chore, requiring the expenditure of a large amount of time and money.
Advances in the field of cloning and gene expression has led to the development of recombinant vaccines against rabies, which is easy to manufacture, stable in the environment and induce intense immunity. The use of recombinant virus eliminates the ingress of the external environment potentially dangerous genome vaccine rabies virus. The most studied and promising for use in the vaccination of rabies are recombinant vaccines based on cowpox virus (Vaccine. 2009 27; 27(51):7198-201) and adenoviruses (Virology. 2065-20; 356(1-2):147-54).
Recombinant viral vaccine against rabies on the basis of vaccinia virus expressing the glycoprotein of rabies virus used to immunize animals in the wild (J. Wildl. Dis., 1998, 34, 752-763). However, human contact with the cowpox virus may be accompanied by serious side effects that were shown during the mass vaccination of soldiers of the American army (Comp Immunol Environ Infect Dis, 2003, 26, 423-430).
Recombinant adenoviruses in comparison with other vector systems are highly efficient expression of the target transgene in different types of cells, the security of the vector to humans and animals, the accumulation of recombinant viruses in cells producing high titer, the induction of both humoral and cellular immune response to the transgene product, great packing capacity vector (Curr Top Environ Immunol. 2004, 273, 335-57). Recombinant adenoviruses as vaccine can be used for both parenteral and oral administration.
The known method of oral immunization of mice with recombinant replication-defective adenovirus 5 (adenovirus) and 68 serotype (chimpanzee adenovirus), bearing glycoprotein gene of rabies virus (U.S. Pat. 2004019603).
The disadvantages of the described method applies the high cost of the process poluchenierazreshenija for immunization of the preparation of recombinant adenovirus using cell lines of human rights.
The most secure among modern vaccines are split split vaccine containing virus particles - isolated surface and internal proteins. Manufactured vaccine by cleavage of viral particles using organic solvents or detergents, also viral components can be obtained in the laboratory using genetic engineering technology (J Immunol. 1996 15; 156(10):3579-82). Split vaccines are characterized by a significantly lower risk of adverse reactions, in connection with the removal of reactive lipids that make up the shell of the virus. Since the production of neutralizing antibodies against rabies is the surface glycoprotein, this structural component is used to create reiseplaner vaccines. The use of genetic engineering techniques to obtain isolated glycoprotein rabies virus, excluding work with the pathogen, increase safety received vaccines.
A method of obtaining the rabies vaccine, containing insulated surface glycoprotein of rabies virus, expressed in eukaryotic cells (US. Pat. 9411112). The coding DNA sequence of the glycoprotein of rabies virus is produced by reaction of reverse transcription on the matrix RNA virus or artificially synthesized, and then she can is to be embedded in commercial expressing vector. For expression of the glycoprotein of rabies virus in cells of S. cerevisiae developed commercial plasmid vector pYES2 (hivitrogen, San Diego, CA). Receiving glycoprotein of rabies virus in insect cells is provided baculovirus expression system (Virology. 1989; 173(2):390-9), for example, a commercial set Mahvash (bivitrogen, Sail Diego, CA). To obtain the glycoprotein of rabies virus in mammalian cells (cells of the Chinese hamster ovary) can be used plasmid vector pcDNA I bivitrogen, San Diego, CA). High level expression of the gene of interest is provided by the presence of promoter sequences, enhancers, polyadenylation signal. Thus, the receiving surface glycoprotein of rabies virus in prokaryotic and eukaryotic expression systems, however, the use of eukaryotic cells is preferred because they provide the necessary post-translational modification that leads to the formation of functionally active product.
Recombinant glycoprotein of rabies virus is administered orally to the animals as bait with attractive taste and smell in the amount of from 100 to 300 micrograms. Oral immunization as described glycoprotein of rabies virus, once in the body of animals associated with the epithelium of the oral cavity and pharynx, which then leads to the development of mucosal immunity.
The method described as the closest to the claimed chosen for the prototype.
However, the shortcomings of the prototype is rapid degradation in the body due to the action of proteolytic enzymes and cells of the immune system, the short half-life of a foreign protein in the body (approximately 2-3 hours), the need for ingestion of large doses of vaccines to achieve a positive effect; effective oral immunization with isolated viral protein requires the use of the adjuvant; the high cost of the process to obtain preparative amounts of the recombinant glycoprotein of rabies virus associated with the need for the cultivation of eukaryotic cells to obtain preparative amounts of recombinant protein.
The purpose of the proposed invention to provide a multicomponent recombinant vaccine accumulated in allantoine fluid of chicken embryos, a single oral immunization of animals against rabies virus.
The essence of the invention is the use of a mixture of recombinant glycoprotein of rabies virus and adenovirus birds CELO expressing membranosvazanny and secreterial forms of the glycoprotein of rabies virus accumulated in allantoine fluid of chicken embryos. Recombinationally birds CELO, expressing secreterial the form of the glycoprotein of rabies virus ensures the presence of this component directly in vaccine preparation, and recombinant adenovirus birds CELO expressing membranosvazanny glycoprotein of rabies virus allows efficient and prolonged expression of the target gene in the body of immunized animals, which leads to the formation of potent humoral and cytotoxic immune response against rabies.
As a carrier for the active ingredient of the vaccine and the adjuvant used recombinant adenovirus birds CELO. Vector CELO provides a high level of expression of a target gene in chicken embryo cells (Protein Expr Purif. 2009; 65(1): 100-7), which leads to the accumulation of recombinant glycoprotein of rabies virus in allantoine fluid in concentrations sufficient for immunization (up to 100 µg/ml). Vector CELO able to deliver reporter genes and genes of interest in mammalian in vivo and provides a prolonged expression (30 days), which provides an extension of the period of circulation of the antigen in the body (Mol Gen Mikrobiol Virusol. 2008; (4):26-30). This system viral gene delivery is safe for mammals, because the CELO virus is unable to replicate in cells of the data owners. The main advantage of adeno is yushnogo vector CELO is the ability to obtain preparative amounts of recombinant CELO virus in chicken embryos. The number of recombinant adenovirus CELO derived from one chicken embryo, is more than 1012viral particles. The titer of virus in allantoine fluid is 5×108PFU/ml Recombinant glycoprotein of rabies virus and adenovirus particles present in allantoine fluid, a relatively stable during the time required for immunization, therefore, an additional stabilization of the drug is not required.
To solve the problems associated with limited efficiency transduction of mammalian cells by vectors based on adenovirus birds, the developed approaches in the genetic modification process of Penton (fiber). These approaches include the construction of "chimeric" filerow (replacement globular domain with the appropriate domain of alternative serotypes of adenoviruses) or the introduction of various heterologous receptor-binding sequences on the C-end or HI-loop 3 globular domain of the fibre (J Virol., 2007 81(18); 9641-9652.). These modified vectors are able to provide efficient delivery of genetic information into mammalian cells normally resistant to infection by adenoviruses.
Through the ability to interact with receptors of innate immunity adenoviral vector can act as an adjuvant (J. irol., 2002, R-135).
The rabies virus vaccine strain TC-80 propagated in cell cultures of kidney saiga. The titer of the resulting virus is determined in accordance with the recommendations of the who Laboratory techniques in rabies. 4thedition. WHO, Geneva, 1996). The titer of the virus is 108PFU/ml
The standard strain of rabies virus CVS-24 for infection with a lethal dose of the virus obtained from the brain tissue of infected newborn mice (J Exp Med. 1977 1; 145(6):1617-22).
Gene glycoprotein G is obtained by amplification of cDNA synthesized by RT-PCR (Reverse Tpanscription System "Invitrogene" No. 12236-014, USA) on the matrix RNA isolated from rabies virus vaccine strain TC-80 using TRIZOL ("Invitrogene" No. 15596-018, USA). For PCR using oligonucleotides flanking the complete glycoprotein gene 5'-ggatccaggaaagatggttcctcaggctctcctgtttg and 5'-gctgcagcaaggggaggtgatcttcagacttggatcgt, selected according to the gene sequence of the glycoprotein of rabies virus (gene bank AB518487.1). The DNA fragment carrying the gene for glycoprotein G of rabies virus (sequence No. 1), clone into plasmid vector pGEM-T Easy Vector (Promega, A1360, USA). Gene glycoprotein G of rabies virus subcloning comprising expressing cassette consisting of the CMV promoter with enhancer and polyadenylation site BGH in vector pCBEdlRV containing a fragment of the genome of adenovirus CELO with a deletion of the not essential for virus replication about the Asti. Recombinant adenovirus CELO-glRb carrying the glycoprotein gene of rabies virus, produced by the method of homologous recombination in LMH cells. Soluble form of glycoprotein G of rabies virus (ΔglRb) is produced by site-specific mutagenesis using the oligonucleotides: 5'-aggagcatgcaaactcaag, 5'-ggtggcggccgctcaatgcac, making a substitution in amino acid sequence at position 417: Pro417 stop codon. Variant gene glycoprotein G of rabies virus with a deletion of the transmembrane domain (sequence No. 2) subcloning comprising expressing cassette consisting of the CMV promoter with enhancer and polyadenylation site BGH in vector pCBEdlRV containing a fragment of the genome of adenovirus CELO with a deletion of the not essential for virus replication region. Recombinant adenovirus CELO-ΔglRb carrying the glycoprotein gene of rabies virus with a deletion of the receive method of homologous recombination in LMH cells. Schematic representation of the genomic DNA of recombinant adenovirus birds CELO-glRb carrying the gene for glycoprotein G of rabies virus presented on figure 1A
Plasmid construction, carrying a full-sized genome of adenovirus birds CELO-HIRGD with modifications and structural proteins capsid (fiber) and expressing the cassette with the gene of the glycoprotein of rabies virus, produced by the method of homologous recombination in E. coli strain BJ5183 between dps is amidami pCELO-HIRGD, and Shuttle vector pCShCMV-glRb, bearing expressing cassette (CMV promoter, transgene, the polyadenylation signal), planbureau sequences CELO. Shuttle vector pCShCMV-glRb linearized the restriction enzyme AscI. Plasmid pCELO-HIRGD linearized by PacI. Shuttle vector with plasmid pCELO-HIRGD transformed into cells of E. coli strain BJ5183. The preparation of recombinant clones was confirmed by PCR, restriction mapping and sequencing. Schematic representation of the genomic DNA of recombinant adenovirus birds CELO-HIRGD-glRb, with modifications and structural proteins of the capsid, which carries the gene for glycoprotein G of rabies virus presented on figure 1B.
Gene expression of glycoprotein G of rabies virus in chicken embryos infected with adenovirus CELO-ΔglRb, CELO-glRb, CELO-HIRGD-glRb, determined by the method of Western blot turns. Chicken embryos infect recombinant adenovirus CELO-ΔglRb, CELO-glRb, CELO-HIRGD-glRb. As a control use adenovirus CELO wild type. 72 hours after infection selected allantoin liquid which is analyzed by the method of Western blot turns. In the reaction using monoclonal antibodies to rabies virus strain TC-80. Specific interaction of complex antigen-antibody reveal individuum immunoperoxidase conjugate (Goat-antimouse IgG-HRP, Amersham, 1:5000). Coloring, make use of a Hemi is eminiscences of a detection system (ECL-plus Detection System, Amersham). The result of Western blot turns presented in figure 2.
The result of the analysis shows specific interaction of antibodies and recombinant protein corresponding to the electrophoretic mobility of the glycoprotein G of the virus.
The obtained culture viral drug CELO-glRb infect chicken SPF embryos, at the rate of 100 μl of the culture of the drug on the embryo. At 72 h after infection selected allantoin liquid. Similarly receive recombinant adenovirus birds CELO-HIRGD-glRb and recombinant adenovirus birds, bearing comprising expressing the gene cassettes of glycoprotein G of rabies virus without transmembrane domain - CELO-ΔglRb. For immunization allantoine the recombinant CELO virus-glRb and CELO-HIRGD-glRb mixed in a 1:1 ratio with recombinant adenovirus birds CELO-ΔglRb. The resulting combination is used for oral immunization of mice in the amount of 1 ml per animal.
The first group outbred mice orally once imposed a mixture of recombinant adenovirus CELO-glRb/CELO-ΔglRb as allantoinase of the drug. The second group is administered a mixture of recombinant adenovirus CELO-HIRGD-glRb/CELO-ΔglRb. A third group of mice twice subjected to immunization cultural inactivated vaccine, containing the th strain of rabies virus TC-80. As a negative control group of mice once injected adenovirus CELO wild type. 14 days after immunization mice intracerebral enter 100 BILLION50virus CVS-24. In the group of mice immunized once with recombinant adenovirus CELO-glRb and CELO-HIRGD-glRb, survived 91% and 90% of animals, respectively. When using standard culture inactivated vaccine only 82% of mice were protected from subsequent infection with a lethal dose of rabies virus strain CVS-24 (table).
|The level of protection against rabies virus of mice immunized with recombinant adenoviruses birds CELO-glRb and CELO-HIRGD-glRb, with modifications and structural proteins of the capsid|
|The group of animals||The number of animals infected with a lethal dose of rabies virus||The number of animals that died from rabies virus||The number of surviving animals||Survival, %|
|Immunization vaccine strain of rabies virus TC-80||11||2||9||82|
|Immunization with a mixture of adenovirus CELO-HIRGD-glRb/CELO-ΔglRb||10||1||9||90|
|Immunization with adenovirus CELO wt (negative control)||11||11||0||0|
Thus, it is shown that the maximum level of protection of mice from vnutricerepnogo infection with rabies virus strain CVS-24 - 91% - detected after a single immunization with recombinant adenoviruses birds CELO-glRb and CELO-HIRGD-glRb oral.
Specific antibodies to rabies virus in the serum of mice immunized with recombinant vaccine CELO-glRb or CELO-HIRGD-glRb, determined by indirect enzyme immunoassay.
Sensitization panels conduct normal and specific antigens that contribute diluted in the working dilution in 0.01 M phosphate-buffered saline (FSB), pH 7,2-7,4 in odd and even rows, respectively. Panel incubated over night at 4°C.
Available adsorption sites of polistina is and blocking with 1% BSA, in the volume of 0.15 ml /well, inquira for 30 minutes at 37°C. After further washing plates FSB with tween-20 (FSS T) subjects serum contribute to the wells of the panels in a dilution of from 1:3 to 1:729 1% BSA in FSB. Plates are incubated for 40 minutes at 37°C, followed by three times washing in the FSB-T, make a working dilution (1:1000) antimisting peroxidase conjugate and incubated for 40 minutes at 37°C.
After that, the plate is washed six times the FSB-T and make a solution of a chromogenic substrate (ABTS). Records of the hold after 30 minutes incubation at room temperature.
Accounting reactions carried out photometrically at a wavelength of 405 nm. The reaction is considered positive and specific, if the optical density of the chromogenic substrate solution into the wells, which are pre-incubated test serum, and in the holes, which made a positive control serum, 2 or more times greater than the mean optical density of the substrate in the hole, which made the control (negative) normal serum.
The result of the titration of specific antibodies to the rabies virus in the serum of mice immunized with recombinant CELO virus-glRb presented on figure 5. The antibody titer in the serum of mice immunized with recombinant adenovirus CELO-glRb and CELO-HIRGD-glRb correlates with the titer of antibodies detectionin in the blood serum of mice after immunization culture vaccine TC-80. Recombinant CELO virus-glRb and CELO-HIRGD-glRb when administered to mice induce the synthesis of high levels of specific antibodies against the glycoprotein G of rabies virus.
Thus, the resulting vaccine containing a mixture of the glycoprotein of rabies virus, and recombinant adenoviruses birds, one of which (CELO-glRb) expresses secreterial the shape of the surface glycoprotein of rabies virus, and the second (CELO-glRb or CELO-HIRGD-glRb) - membranosvazanny, single oral administration which leads to effective protection against a lethal dose of rabies virus.
The method of obtaining oral vaccine against rabies virus in animals, including the production of recombinant glycoprotein of rabies virus for oral immunization, characterized in that get allantoin fluid of chicken embryos containing the recombinant glycoprotein of rabies virus, its producer - recombinant adenovirus birds CELO, as well as recombinant adenovirus birds CELO, which is the producer of membrane-associated glycoprotein of rabies virus, or recombinant adenovirus birds CELO, with modifications and structural proteins of the capsid and which is the producer of membrane-associated glycoprotein of rabies virus.
SUBSTANCE: invention can be used in laboratory diagnostics of Yersinia enterocolitica strains with using moderate bacteriophages FK-99, FK-100, FK-101 for intraspecific differentiation of a yersiniosis agent. The Yersinia enterocolitica 2012 strain is recovered from human in 1966 and deposited in the State collection of pathogenic bacteria of Russian Research Antiplague Institution "Microbe", No. KM-206. When used as a mentor strain, it enables the particle concentration of the phage preparations being equal to n·10-n·10 particles/ml.
EFFECT: use of the recovered phages in Yersinia enterocolitica on the indicator KM-206 offered under the invention provides the additional differentiation of lysogenic cultures from non-lysogenic and ensures the effectiveness of laboratory diagnostics of Yersinia enterocolitica.
1 tbl, 1 ex
SUBSTANCE: disclosed is a conditionally defective particle of influenza virus with the absence of a nucleic acid segment of influenza virus selected from a group of segments, mainly coding acid polymerase (PA), basic polymerase 1 (PB1) and basic polymerase 2 (PB2). The conditionally defective particle of influenza virus is used for induction of influenza virus defence. Also, a method of producing such particles, a pharmaceutical composition containing such particles, its application and a method of induction of influenza virus defence are described.
EFFECT: higher efficacy.
27 cl, 4 dwg, 4 tbl, 2 ex
SUBSTANCE: it is described that immunogenicity of hemagglutinin (HA) molecule of influenza virus can be increased by substituting amino acids in the HA sequence. Substituting specific residues in HA such as asparagine introduction in position 223 in HA H5 allows ensuring more sensitive hemagglutination inhibition (HI) test provided by changing receptor specificity and/or ability to antibody-antigen linkage. The HA molecules having such substitutions can find application in creating diagnostic prototype viruses.
EFFECT: improved influenza virus vaccines.
9 cl, 3 dwg, 7 tbl, 7 ex
SUBSTANCE: disclosed is a plasmid for producing a viral vector transporting multiple expression cassettes to a target. The plasmid contains genome nucleotide sequences packed into a polyvalent capsid and a number of cassettes to be transported. A method for producing a viral vector, a viral vector and an immunogenic composition are described besides.
EFFECT: group of inventions can be used for wide-ranging expression of target antigens.
23 cl, 3 dwg
SUBSTANCE: modified virus-like particle (VLP) includes hybrid protein which consists of AP205 bacteriophage protein and any antigen. Also, a composition including VLP, being a derivative of RNA-containing AP205 bacteriophage is described. Besides, the invention describes a method for producing said VLP. Modified VLP of the present invention is applicable for producing compositions for inducing immune response for prevention or treatment of diseases, disorders, including infectious diseases, allergies, cancer and drug addiction.
EFFECT: offered group of inventions can be used in medicine, immunology, virology and molecular biology.
35 cl, 3 dwg, 1 tbl, 26 ex
SUBSTANCE: recombinant plasmid pFastBac-B17R DNA carries a cowpox virus genome fragment. BvB 17RG recombinant baculovirus strain is produced with the use of recombinant plasmid pFastBac-B 17R DNA and deposited in the Microorganism Cultures Collection of the Federal State Research Institution 'State Research Centre for Virology and Biotechnology 'Vector' of Federal Service for Supervision of Consumer Rights Protection and Human Welfare' (FGUN GNC VB 'Vector' of Rospotrebnadzor) under No. V-388, characterised as a producer of a soluble analogue protein of cowpox interferons type 1 cell receptor.
EFFECT: extended spectrum of preparations of new generation.
2 cl, 5 dwg, 8 ex
SUBSTANCE: viral particle consists of non-alphavirus structural elements and contains an alphavirus vector replication-defective by deletion or substitution by at least one transgene. Besides said particle contains structural elements not coded by an alphavirus vector genome. A method for preparing said particle according to the invention implies the expression in trans of the genes coding the non-alphavirus structural elements, and the alphavirus vector, in a cell line and then recovery of the viral particles found in the cell culture supernatant.
EFFECT: invention allows preparing the viral particles with lower recombination risk.
24 cl, 5 dwg, 3 tbl, 2 ex
FIELD: chemistry; biochemistry.
SUBSTANCE: invention relates to biotechnology. Recombinant adenovirus expresses a protein selected from a group comprising E1B and E4 proteins and their combination, and then a second protein which is E1A. Recombinant adenovirus consists of an expression cassette which codes E1B and E4 proteins under the control of a promoter which is different from the promoter which regulates expression in the wild type adenovirus. The recombinant adenovirus optionally contains nucleic acid which codes YB-1, expression cassettes which contain promoters and nucleic acids which code different genes. Disclosed also is a nucleic acid which codes such a recombinant adenovirus and use thereof.
EFFECT: recombinant adenovirus can be used in medicine for treating tumoural diseases.
30 cl, 26 dwg, 19 ex
SUBSTANCE: recombinant virus containing human gene p53, its application, manufacture method and pharmaceutical composition are offered.
EFFECT: invention can be used for gene therapy in human malignant neoplasm treatment and prevention.
8 cl, 14 dwg, 7 ex
SUBSTANCE: invention is related to preparation of protein, binding tumour necrosis factor (TNF), and may be used in medicine. Strain-producer of baculovirus BvG2RIgG is created with the help of recombinant plasmid DNA pFastBac-G2R-IgG with size of 6444 p.n. and molecular mass 4.18 mDa, which bears fragment of smallpox virus genome of strain India-1967, which codes protein that binds TNF, and fragment of human genome, which codes fragment of heavy chain of human antibody G. Produced strain produces soluble chimeric protein, which consists of smallpoz virus protein, which binds TNF, and fragment of heavy chain of human antibody G.
EFFECT: wider spectrum of new generation preparations intended for treatment of human diseases related to hyperproduction of tumour necrosis factor.
2 cl, 3 dwg, 1 tbl, 8 ex
SUBSTANCE: there are developed infectious clones having a nucleotide sequence identical to PRRS viruses such as VR-2332, Lelystad or others, and additionally optionally containing a deletion in the ORF1 region which codes nsp2 polypeptide.
EFFECT: applicability of the invention in veterinary science.
34 cl, 27 dwg, 6 tbl, 2 ex
SUBSTANCE: synthetic oligonucleotides for indentifying DNA of Torque teno virus of all known genotypes are disclosed. Primers are combined in a set for DNA identification in blood and other biomaterials of the infectious agent of the latent viral infection Torque teno virus of Circoviridae family by polymerase chain reaction.
EFFECT: invention allows reliable identification of said virus in a biological material.
SUBSTANCE: invention refers to synthetic oligonucleotide primers, complementary to high conservative VP60 gene region of a genome of rabbit viral hemorrhagic disease virus, to a method for identifying rabbit viral hemorrhagic disease virus and to a test system for identifying RNA of rabbit viral hemorrhagic disease virus. The offered invention can be used in veterinary virology. The method for identifying rabbit viral hemorrhagic disease virus involves sample preparation, RNA recovery from the biological material. It is followed with conducting a polymerase chain reaction with using primers 5'-caa cgt get cca gtt ttg gta cg-3', 5'-att ctg tct ggt tgg ggc gtg t-3'. Further, viral RNA is amplified. Then, the reaction is assessed by agarose gel electrophoresis, with a reaction result considered as positive if the PCR product corresponds to the size of 398 base pairs.
EFFECT: invention allows higher sensitivity of the method, as well as reduced time of diagnostic manipulations with organ and blood samples of the infected animals.
3 cl, 4 tbl, 4 ex
SUBSTANCE: invention refers to synthetic oligonucleotide primers for detecting 1a and 1b virus subgenotypes of viral diarrhoea - bovine mucosal disease (VD - BMD) and to a method of applying said synthetic oligonucleotide primers for detecting 1a and 1b virus subgenotypes of viral diarrhoea - bovine mucosal disease. The method can be used in veterinary virology for subgenotype differentiation of strains and isolates of genotype 1 virus. The offered method involves a PCR with pairs of primers SEQ ID NO:1 5'-tcgacgccttaacatgaaggt-3' and SEQ ID N0:3 5'-ccatgtgccatgtacag-3' for subgenotype la, SEQ ID NO:2 5'-tcgacgctttggaggacaagc-3' and SEQ ID NO:3 5'-ccatgtgccatgtacag-3' for subgenotype lb. It is followed with detecting a DNA fragment sized 186 bp in each reaction. Further, the presence in an analysed material of 1a and lb subgenotype virus of viral diarrhoea - bovine mucosal disease is concluded.
EFFECT: invention allows express subgenotype differentiation of genotype one VD - BMD virus strains.
2 cl, 2 dwg, 2 tbl, 4 ex
FIELD: medicine; molecular biology.
SUBSTANCE: invention concerns molecular biology, genetic engineering and can be used in medicine. Quantitative express identification of the HIV-1 genome in assay is detected with the help of oligonucleotide primers, complementary to a conservative site 5'-LtR-part of the HIV-1 genome, fluorescently marked probes and the RNA-modified fragment of a conservative site of 5'LTR area of the HIV-1 genome as the internal quantitative standard. The analysis is performed in real time in "the closed test tube" format by means of the calibration curves received with the help plasmides pVarl5-HIV-LTR and pBluSK-HIV-LTR mod, containing the native and modified kdnk-fragment virus genome from conservative 5'-LTR-pocledovatelnocti accordingly. Plasmide pVar15-HTV-LTR it is received on the basis of the pVar15 plasmides. The pBluSK-HIV-LTR mod plasmides is received on the basis of pBluKSM plasmide.
EFFECT: possibility of determination of HIV-1 presence in assay irrespective of type of a virus, a combination of types and presence of related kinds.
4 cl, 10 dwg, 9 ex
FIELD: medicine diagnosis, molecular biology.
SUBSTANCE: invention relates to method for detection of DNA of human Lyme borreliosis excitant in peripheral blood, cell culture and mites. Claimed method includes DNA isolation from biological material; synthesis of primers; PCR with using synthesized primers and evaluation of obtained data. In method BBs14.F/BBs14.R - 5'-AAGAATACATTAAGTGCGATANN-3', 5'-CAATCCACTTAATTTTGTGTTAT-3' primers to gene site encoding 16S rRNA are used. Kit for this method also in disclosed.
EFFECT: high-specific, sensitive and simplified method.
2 cl, 2 dwg, 5 tbl
FIELD: veterinary virology.
SUBSTANCE: invention relates to 5 strains of II type porcine circovirus (PCV II) which represents causative agent of porcine post-wealing multy-systemic wasting syndrome (PMWS). Disclosed are various immunogenic compositions and vaccine based on said strains for PMWS prophylaxis and/or treatment. Also disclosed are vectors, viral preparations, cell extracts, cell culture supernatants, containing PCV II or nucleotide or protein components thereof; method for PCV II diagnosis, as well as diagnostic composition and kit.
EFFECT: new agent for treatment of porcine PMWS.
178 cl, 7 dwg, 5 tbl, 19 ex
FIELD: virology, biotechnology, molecular biology.
SUBSTANCE: invention proposes oligonucleotides, primers and probes based on thereof designated for a detection method and/or quantitative determination of nucleic acids of adenoviruses in biological sample. Method involves amplification by polymerase chain reaction (PCR) in real time by using the proposed primers HEX1 and HEX2 wherein product is detected by using probe HEX. Also, invention proposes a set of reagents for realization of such method and methods for diagnosis and detection based on thereof. Proposed inventions provide carrying out detection of different serotypes of adenoviruses for a single reaction and quantitative determination of their small amounts.
EFFECT: improved methods for detection, diagnosis and selection.
7 tbl, 1 dwg, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: present invention refers to cell biology, molecular biology, cancer biology and medicine and represents a pharmaceutical composition for targeting to the cells bearing BLyS receptor, containing an effective amount of fused protein consisting of BLyS polypeptide fused with cytotoxic polypeptide where said cytotoxic polypeptide is located on the N-end of fused protein, and BLyS polypeptide is located on the S-end of fused protein, and a pharmaceutically acceptable carrier.
EFFECT: invention provides treatment and prevention of B-cell proliferative disorder.
11 cl, 13 ex, 4 tbl, 20 dwg