Oligonucleotide primers for production of library of full-length genes encoding immunodominant proteins gn, gc and n of rift valley fever virus
SUBSTANCE: proposed primers comprise endonuclease cleavage sites, flanking genomic regions encoding the glycoproteins Gn and Gc, and the nucleoprotein N, for obtaining libraries of genes encoding glycoproteins Gn and Gc and N nucleoprotein of Rift Valley fever virus.
EFFECT: invention can be used in creating a bank of nucleotide sequences encoding immunodominant proteins of Rift Valley fever virus Gn, Gc and N, which can be used for creation of diagnostic and vaccine preparations based on recombinant technologies.
The invention relates to biotechnology and relates to a library of nucleotide sequences, comprising klonirovanija plasmid vector.
Rift valley fever (RVF) is an acute vector-borne zooantroponoznyh arbovirus disease included in the group of diseases subject to international health regulations 2005 on national and regional level . The pathogen belongs to the family Bunyaviridae genus Phlebovirus.
Rift valley fever is causing enormous socio-economic damage, accompanied by large losses of livestock during the epidemic, diseases and deaths, the cost of quarantine and preventive measures and also hospitalization and treatment of people.
In parts of Africa animals vaccinated with a live vaccine (strain Smithburn"), but in non-endemic regions, the use of live vaccines is prohibited. Currently in development, or in the testing stage, a number of candidate live, recombinant and DNA vaccines. For use among people developed inactivated vaccine. However, this vaccine is not licensed and is not for sale. It is used for experimental purposes to protect veterinarians and laboratory workers at high risk of infection LDR .
The emergence of RVF outside the African contin�that poses a real threat for Europe and Asia, where there are competent vectors. Given the danger of the emergence of the pathogen in new areas and the uncertainty of vaccination, there is a need to develop new scientific approaches for the prediction, diagnosis and prevention of this disease.
So, in the work of foreign researchers L. Liu, Cr. Celma and P. Roy "Rifit Valley fever virus structural proteins: expression, characterization and assembly of recombinant proteins described a process of obtaining recombinant proteins Gn, Gc and N, and their subsequent characterization and analysis.
Some success and there are design and recombinant DNA vaccines. So N. Bhardwaj et al. show the induction of high titers of neutralizing antibodies after inoculation of mice with plasmid expressing chimeric protein Gn-C3d .
G. Lorenzo al (2010) have received protection, immunosera different DNA constructs. Positive results received N. Boshra al (2012) who have achieved protection from the control of infection with virulent virus after DNA immunization design expressing ubiquitination nucleoprotein N virus LDR.
In the works for the construction of recombinant vaccines with genes encoding protective proteins LDR, cloned in a safe viral vectors. Similar designs obtained Elena Lopez-Gil and co-authors (2013), based on modified virus vacciniaAnkara (rMVA), on the basis of Newcastle disease virus Kortekaas J, with co-authors (2010).
For this research, as well as address various theoretical problems (e.g., determination of the nucleotide sequence of the genome of strains, to study the structure, function and regulation of individual genes, structure and functions of proteins) receive a library of genes, kourouma immunodominant proteins of the virus rift valley fever, which can be used to create diagnostic and vaccine products based on recombinant technology. Inserts from the library due to the restriction sites to allow perekodirovat coding sequence under different promoters (T5, T7, CMV, etc.) and vectors (plasmids, phages, viruses).
Therefore, the aim of the invention is to design oligonucleotide primers for obtaining a full-sized library of genes encoding immunodominant proteins Gn, Gc and N, of the virus of rift valley fever.
This goal is achieved by the synthesis of specific primers flanking the sites of the nucleotide sequences of the genome of LDR with the library.
In table.1 shows the sequences of specific primers for amplification of full-sized copies of the cDNA encoding the immunodominant proteins of the virus LDR.
|name and �connected site restriction||the nucleotide sequence of*||long, nt.||flanked plot|
|F-RVFV-Gn EcoRI||5'-CTCGAATTCATGCCTCATCTCAGAAACAGACCA-3'||33||M segment 474...2067 nt nt 1626|
|F-RVFV-Gc EcoRI||5'-CGCGAATTCATGTGTTCAGAACTAATTCAGGCA-3'||33||M segment 2079...3474 nt 1428 nt|
|F-RVFV-Np Sail||5'-CCCGTCGACTTAGGCTGCTGTCTTGTAA-3'||28||S segment 906 1635...757 nt nt|
|* the restriction site is indicated by underlining.|
The technical result consists in the creation of a Bank of nucleotide sequences encoding the immunodominant proteins of the virus rift valley fever Gn, Gc and N that can be used when �the create diagnostic and vaccine preparations based on recombinant technology.
The essence of the invention consists in the fact that the specific primers designed to produce libraries of genes encoding immunodominant proteins of the virus rift valley fever Gn, Gc and N and contain restriction sites for further manipulation of nucleotide sequences of the inserts.
Examples of specific performance
Example 1. Obtaining a library of genes comprises the following steps:
1. Refreshment and life viral material (Mouse-sucker and/or sensitive cell culture).
2. The allocation of RNA from virusdatabases material (Methods using 4M GTZ, Trizol reagent©, or using commercial kits: GeneJET RNA Purification Kit (Thermo Scientific), etc.).
3. Statement FROM PCR on the matrix RNA pool (using a random hexameric or specific primers).
4. Amplification of full-sized copies of the cDNA encoding corresponding proteins.
5. Cloning amplification in klonirovana vector (plasmids, phages, etc.).
As the source can be virussecurity material (infected culture cells, pathological material, lyophilized tab) vaccine or epizootic of rift valley fever virus strain. If necessary, the virus refresh and improve:
The virus titre is not less than 3.0 lg refresh the intracerebral Sara�of diurnal mice pigs: the dose of 0.1 ml per head. 48-72 hours away pups with obvious clinical signs: photophobia, cyanotic mucous membranes, paralysis and other nervous phenomena. These specimens aseptically taken away the brain, and prepare a 10% suspension in physiological solution and transferred to a -40°C.
The virus titer is not below 4.0 lg refresh sensitive cell culture: kidney saiga "PS", green monkey kidney CV-1", kidney Syrian hamster "VNK", etc., the Dose of infection is 10-3-10-4TCID50/CL. 3-5 a day, when you reach the JRS about 40-50% of the culture fluid decanted, and the monolayer pour THE buffer pH 7.0 and transferred to a -40°C.
The allocation of the total pool of RNA from virusdatabases of material is carried out by standard techniques using the 4M GTZ, Trizol reagent, etc., or with the use of commercial kits GeneJET RNA Purification Kit (Thermo Scientific), etc. Firostefani the above materials were prepared samples.
Statement FROM PCR is performed according to the selected passport revertase, using a random or specific primers as given in table.1. As RNA-dependent DNA polymerase may make AMV and M-MLV of revertase different manufacturers (Promega, Fermentas, α enzyme). The obtained cDNA can be stored at -40°C during the month.
The PCR on cDNA obtained. As a DNA-dependent DNA polymerase m�gut to speak polymerases from different manufacturers, making a small number of nucleotide substitutions when the amplification of long sections.
Formulation was conducted using specific primers as given in table. 1.
The PCR recipe mix
matrix to DNA 0.5 ál
10X Buffer (with 2.5 mM MgCl2) To 2.0 ál
10 mM dNTP mix - 0,4 ál
10 PM F-primer and 1.0 μl
10 RM R-primer - 1,0 ál
Pfu polymerase and 0.5 µl (1,25 u)
DNAse-free H2O - 14,6 ál
Time-temperature profiles of the reaction
For amplification of full-sized copies of the cDNA encoding the glycoproteins Gn and Gc are:
95°C - 2 min.
95°C - 30 sec.
55°C - 30 sec.
72°C 3 min 20 sec.
72°C - 7 min.
For amplification of full-sized copies of the cDNA encoding the nucleoprotein N:
95°C - 2 min.
95°C - 30 sec.
72°C 1 min 35 sec.
72°C - 3 min.
Detection and confirmation of the molecular mass of the PCR products is carried out in a 1.2% TAE or TBE agarose gel with EtBr as intercalating dye.
The molecular weight of PCR products obtained after the PCR FROM the genome matrix LDR strain "1974 Vniivvim":
Amplificare Gn - 1626 bp;
Amplificare Gc - 1428 bp;
Amplificare N - 757 bp.
A library of genes of the virus rift valley fever, is an incomplete library of nucleotide sequences that encode the major immunodominant proteins: �glycoprotein Gn and Gc, as well as the nucleoprotein (N) in the composition klonirovannikh vectors.
The essential difference data klonirovannikh primers is that in the sequence of the primers included sites endonuclease cleavage with annealing temperature for glycoproteins 57°C, for nucleoprotein - 55°C.
Example 2. Cloning amplification Gn and Gc, the nucleoprotein N virus PI strain "1974-Vniivvim" in the plasmid vector
Due to the amplification of cDNA copies with the use of Pfu polymerase and the ends of the fragments remain "dumb", i.e., the polymerase does not put additional nucleotides at the ends.
Cloning is carried out in the plasmid vector pJET1.2, using a commercial kit CloneJET PCR Cloning Kit. (Thermo Scientific). The feature of cloning in this vector is that the cloning was carried out by the "blunt" ends, with negatively lethal screening (box disturbs reading frame of the lethal gene ESOC in the composition of plasmids).
Cloning is carried out according to the standard recipe set. To do this, 1 ál of crude Reaction Buffer, PCR product made in a mixture consisting of 10 µl 2X Buffer, 1 ál of pJET1.2/Blunt, DNAse-free H2O - 7 ál. Mix on vortex for 3-5 seconds then make a 5u of T4 DNA Ligase. Incubated at 22°C for 10 min.
The transformation is carried out using reagents TransformAid Bacterial Transformation Kit (thermo Scientific). The competent cells of E. Coli you�RAS strain KRX (Promega).
Further screening is carried out in the ratio of colonies grown on Petri dishes in medium with addition of ampicillin.
The easiest method is PCR analysis. For this single, isolated colony is divided in two, half to make 2 ml of medium SOB with the addition of ampicillin, the other half into the prepared PCR mixture:
10X Buffer (with2,5 mM MgCl2) To 2.0 ál
10 mM DNTP mix - 0,4 ál
10 PM F-primer and 1.0 μl
10 RM R-primer - 1,0 ál
Tag polymerase and 0.5 µl (2.5 u)
DNAse-free H2O - 15,0 ál
The time-temperature profile of the reaction:
For screening clones containing the insert of glycoproteins Gn or Gc:
98°C - 2 min.
95°C - 30 sec.
57°C - 30 sec.
72°C 1 min 45 sec.
72°C - 4 min.
For screening clones containing the insert of nucleoprotein No. 98°C 2 min.
95°C - 30 sec.
57°C - 30 sec.
72°C - 48 h.
72°C - 2 min.
Detection and control of molecular weight of the PCR products is carried out in a 1.2% TAE agarose gel with the addition of EtBr.
The clones in the samples which are PCR products of a given molecular weight are being generated.
After outstanding plasmid and sent for sequencing using pJET1.2 sequencing primers". According to the results of sequencing selected clones carrying plasmids with intact reading frame, this clonetech lay aliquots.
Example 3. �primenenie library of genes of the virus PI strain "1974-Vniivvim"
Due to the presence of sites endonucleases splitting (in the case of glycoproteins Gn and Gc - Sail and EcoRI, the nucleoprotein N - Sail and StuI) these libraries can act as sources of specific inserts with sobraniem reading frames in plasmid vector rate (nucleoprotein is in pET32b), pET20b, pCI-neo, etc.
The data of the nucleotide sequence of the inserts can be used for producing recombinant proteins, in particular, work on obtaining chromatographically purified preparation of recombinant nucleoprotein N virus LDR [methods in chromatographic purification of recombinant nucleoprotein N virus rift valley fever (RVF). Malysheva V. I., Kazakov A. S., Imaginou I. R., 2013].
As a producer performs cell culture of E. Coli KRX strain with expressing the plasmid pET32b/RVFV/Np/3/9b. For perchlorovinyl insert plasmid library containing the nucleotide sequence encoding nucleoprotein N, restrictively sites endonucleases StuI cleavage and Sail. Vector 32b restricion Sail and EcoRV.
Ligation, the metamorphosis and screening of clones were performed according to standard procedures (M. R. Green and J. Sambrook, Molecular Cloning A Laboratory Manual, 4th Edition, 2012) and can be performed by any known to the researcher.
The data of the nucleotide sequence of the inserts may be required�us to create a candidate DNA vaccines.
The prospects for regarding DNA immunization with LDR showed in his works a number of foreign researchers (W. Sheng, W. HLW, 2007; N. Lagerqvist, J. Naslund, 2009; Lorenzo G, R. Martin-Folgar, 2010).
To generate constructs expressing in a eukaryotic system, was selected plasmid vector pCI-neo (Promega). This vector contains "strong cytomegalovirus promoter that ensures expression of a target protein in various cell cultures.
For perchlorovinyl glycoproteins Gn and Gc, plasmids from the library containing the nucleotide sequence encoding the glycoprotein glycoprotein Gn and Gc, respectively, were restrictively sites endonucleases splitting EcoRI and Sail. Vector pCI-neo restricion Sail and EcoRI.
Ligation, the metamorphosis and screening of clones was carried out according to standard methods (M. R. Green and J. Sambrook, 2012, (Molecular Cloning, A Laboratory Manual, 4th Edition) and can be performed by any known to the researcher.
After immunization of mice isotonic plasmid pCI-neo/Gn/1 and pCI-neo/Gc/1 for 14 days observed an increase in the titer of specific antibodies, which was 1:160-1:320.
Sources of information
1. The world health organization. International health regulations (2005). - 2nd ed. - Switzerland, 2008. 82 p. - ISBN 978 92 4 458041 7.
2. Newsletter No. 207. Who. "Fever Rift Valley". http://www.who.int/mediacentre/factsheets/fs207/ru/index.htl.
3. Project Epidemiologist.ru. "Fever Rift Valley". http://view-source:www.epidemiolog.ru/prof/1582.html.
4. Public Health England. "Rift Valley Fever Advice for Health Professionals, 23 May 2011". http://www.hpa.org.uk/web/HPAweb&Page&HPAwebAutoListName/Page/1274087829165.
5. Bhardwaj N, Pierce BR, Ross TM (2012) Immunization with DNA Vaccine Expressing Gn Coupled to C3d Prevents Clinical Symptoms of Infection and Protects Mice against an Aerosol Rift Valley Fever Virus Infection. J Bioterr Biodef S3:006. doi: 10.4172/2157-2526.S3-006.
Specific oligonucleotide primers enabled sites endonuclease cleavage flanking areas of the genome that encode glycoproteins Gn and Gc, nucleoprotein N, to obtain libraries of genes encoding glycoproteins Gn and Gc, nucleoprotein N virus rift valley fever, which had the following nucleotide composition and restriction sites:
F-RVFV-Gn (EcoRI) 5'-CTCGAATTCATGCCTCATCTCAGAAACAGACCA-3'
R-RVFV-Gn (SalI) 5'-CCGGTCGACTTATGATGCATATGAGACAATCAA-3'
F-RVFV-Gc (EcoRI) 5'-CGCGAATTCATGTGTTCAGAACTAATTCAGGCA-3'
R-RVFV-Gc (SalI) 5'-CCGGTCGACTTACTTAAGAGGCCCTCCAAACCA-3'
F-RVFV-Np (SalI) 5'-CCCGTCGACTTAGGCTGCTGTCTTGTAA-3'
R-RVFV-Np (StuI) 5'-CCGAGGCCTAATGGACAACTATCAAGAG-3'
SUBSTANCE: characterised oligonucleotide primers are complementary to a specific region of the mig-gene of Mycobacterium avium and have the following base composition: 5'-CGT CAA AAG CGA ACT GCA-3' and 5'-TAA TTC GTT GCC CGA CTC-3'. The method of detecting DNA of Mycobacterium avium comprises DNA isolation, DNA amplification using oligonucleotide primers, transfer of amplification product on the gel followed by detection of the analysis results on the transilluminator. In case of positive reaction a fragment is synthesised, corresponding to the size of 157 bps.
EFFECT: inventions can be used in veterinary diagnostic, scientific and practical laboratories for detection of genetic material of Mycobacterium avium in samples.
2 cl, 1 dwg, 4 ex
SUBSTANCE: set is used to recognise mutations of a coding part of NKX2.5, CFC1, GATA4 genes associated with an orphan single-gene pathology underlying familiar congenital heart disease. The mutations are recognised by identifying a nucleotide sequence of the coding part of NKX2.5, CFC1, GATA4 genes. The coding part of NKX2.5, CFC1, GATA4 genes is amplified by means of 15 synthetic base pairs at the same temperature and annealing time; that is followed by sequencing the amplification products by means of one pair of universal primers.
EFFECT: invention enables recognising the mutations of the above genes sensitively and specifically, reducing the amplification reaction time, the number of manipulations, the agent addition time for the sequencing reaction and decreasing a probability of the reaction error.
3 cl, 1 dwg, 4 tbl
SUBSTANCE: pair of primers has the following structure - PF: 5'-TGGAGAAGGGAATTTCAGATGGG-3' and PR: 5'-TTTGGGTCAAGTGCTACATATTGTGG-3'. The method includes performing of PCR with detection of obtained results by a horizontal electrophoresis method in 1.5% agarose gel. Amplification is performed in the following mode: total denaturation at 95°C for 5 min., denaturation cycle at 95°C for 20 or 60 sec., annealing at 56°C for 20 or 60 sec., elongation at 72°C for 40 or 60 sec. The cycle denaturation - annealing - elongation is repeated 35 times. The final elongation is performed at 72°C for 5 min. In case of existence of an amplificated fragment of nucleotide sequence with the length 366 p.n. the presence of FIV in a cat organism is obvious.
EFFECT: invention allows to perform diagnostics of a virus immunodeficiency of cats with high efficiency.
2 cl, 6 dwg, 3 tbl, 3 ex
SUBSTANCE: invention relates to fabrication of oligonucleotide primers and fluorescence-tagged probe for identification of inverse transcriptase polymerase chain reaction in real time.
EFFECT: higher efficiency of identification.
2 dwg, 2 tbl, 2 ex
SUBSTANCE: method provides DNA extraction, conducting of PCR with the use of synthesized primers for amplification of fragments of intergenic segments wzyE-dapF and YPDF_0064-YPDSF_0065 of the studied strain. Differentiation is conducted by comparison of sizes of produced amplicons of the studied strain with the sizes of similar fragments, typical for strains of the basic (151 p. n. and 374 p. n.), Caucasian (185 p. n. and 434 p. n.), Altai and Hissar (202 p. n. and 374 p. n.), and also Ulegey (185 p. n. and 374 p. n.) subspecies.
EFFECT: invention allows to conduct effectively subspecies differentiation of strains and considerably to reduce time of the analysis execution.
SUBSTANCE: invention relates to the field of biotechnology, in particular to the lentiviral delivery of apoptin into tumour cells, and can be used in medicine. The method includes obtaining a lentiviral construct, expressing modified apoptin, fused with a sectretory signal of lactotransferrin and a transduction signal (ST-CTP-apoptin), with the further obtaining of recombinant lentiviral particles, defective by replication and carrying the modified apoptin, which are later introduced into T-lymphocytes (TILs), obtained in the surgical ablation of the tumour or in the process of obtaining a biopsy, possessing the ability to penetrate into tumour cells. After that, obtained TILs are autotransplanted to the said patient.
EFFECT: invention makes it possible to increase the ability of apoptin to penetrate into tumour cells and produce an oncolytic effect with respect to all the tumour cells.
2 dwg, 3 ex
SUBSTANCE: method comprises amplification of fragments of genes EGFR, KRAS and BRAF using LNA-blocking multiplex "nested" PCR in which the template for amplification is used as DNA tumour sample. The single-stranded fluorescently labelled PCR product is obtained. The biochip for identification of somatic mutations in the genes EGFR, KRAS and BRAF is obtained. Hybridisation of fluorescently labelled PCR-product in the biochip is carried out. The results of hybridisation on the biochip are recorded and interpreted.
EFFECT: invention enables to carry out analysis in a short period of time and with low material costs.
4 cl, 2 dwg, 4 tbl, 4 ex
SUBSTANCE: invention refers to medicine and aims at diagnosing vaginitis in females of a reproductive age. Expression levels of mRNA of TNF, IL18, GATA3 and TLR4 genes are measured in vaginal smears. Expression levels of mRNA of TLR4/GATA3 and TNF/IL18 genes are related, and the derived threshold cycles are used to predict a probability of vaginitis by formula. If P≤57% for nonpregnant females or P≤68.5% for pregnant females, the absence of vaginitis is stated. If P≥57% for nonpregnant females or P≥68.5% for pregnant females, vaginitis is diagnosed.
EFFECT: invention provides the effective diagnosis of vaginitis in the females of a reproductive age.
3 tbl, 5 ex
SUBSTANCE: group of inventions relates to the field of biotechnology, in particular to micro-array based genomic selection (MGS). A device contains: (a) at least one reaction zone, containing a micro-array, one or more units of temperature control and regulation for the control and regulation of the temperature inside the reaction zone; (b) at least one non-reaction zone, containing one or more units of temperature control and regulation for the control and regulation of the temperature inside the non-reaction zone, which is connected with the reaction zone with the possibility of liquid transfer; and (c) at least one means for transfer, capable of generating and/or regulating a liquid flow between the said reaction zone (a) and the said non-reaction zone (b).
EFFECT: device can be used for the specific selection of nucleic acids, with an increase of binding and fluorescence intensity.
14 cl, 13 dwg, 1 ex
SUBSTANCE: invention refers to a method of detection of Mycobacterium tuberculosis isolates resistant to pyrazinamide by detection of mutations in pncA gene, associated with evolving resistance to pyrazinamide, by PCR in real time mode using HRM analysis. It involves amplification in a mixture of equal amounts of tested DNA and wild type DNA using primers: Pnc18U: 5'-TACGCTCCGGTGTAGGCAC-3' and Pnc15R: 5'-GAAGCGGCGGACTACCATC-3', formation of heteroduplexes due to simultaneous co-amplification of wild type DNA and test DNA, where the first PCR stage includes concentration equalisation by quantitative PCR using the same pair of primers (Pnc18U/Pnc15R) with calibration curve generation and use of regression equation.
EFFECT: reliable and fast detection of mutations in pncA gene associated with evolving resistance to pyrazinamide, due to high specificity and sensitivity.
SUBSTANCE: invention relates to the field of biotechnology and can be used for the identification of heteromultimeric ubiquitins possessing an ability to bind with a ligand-antigen. The method includes the contact of a totality of heterodimeric modified ubiquitins, including two ubiquitin monomers, bound to each other by a head-to-tail scheme, with the potential ligand in a display way. Each of the said monomers is modified in a different way and contains 5-8 substitutions in positions 2, 4, 6, 8, 62, 63, 64, 65, 66 and 68 SEQ ID NO:1. After that, a heterodimeric modified protein, which has bound with the ligand with the binding affinity Kd in the range of 10-7-10-12 M and the monovalent binding activity. Claimed are DNA-libraries, responsible for obtaining a population of the said heteromultimeric ubiquitins, as well as libraries of proteins, obtained by the expression of the said DNA-libraries.
EFFECT: invention makes it possible to obtain the novel bonding proteins based on heteromultimeric ubiquitin, capable of specific high affinity binding with the selected ligands.
8 cl, 17 dwg, 4 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: there are presented a composition and a method for producing it. The characterised composition contains: an effective amount of a viral antigen, which represents a live attenuated rotavirus pre-processed in 0.1% human serum albumin, and a pharmaceutically acceptable buffer. A method for producing a composition involves growing Vero cell culture pre-cultured in the presence of 5% foetal calf serum and 0.1% human serum albumin, infecting the above Vero cell culture with the live attenuated rotavirus, propagating the virus in the cell culture and adding a pharmaceutically acceptable buffer to the above virus.
EFFECT: presented inventions can be used to prevent rotavirus infection and/or rotavirus gastroenteritis.
11 cl, 17 dwg, 4 tbl, 2 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: inventions deal with infectious molecule of nucleic acid, coding infectious porcine Torque teNO viruses (PTTV), which contains at least one copy of genome sequence, selected from the group, consisting of sequences, corresponding to genotypes or subtypesPTTV1a-VA, PTTV1b-VA, PTTV2b-VA and PTTV2c-VA, as well as to biologically functional plasmid or viral vector, containing such infectious nucleic genome sequence, and host-cell, containing such plasmid or vector. In addition claimed inventions include live, attenuated expressible with vector application and purified recombinant capsid subunit or killed viral vaccines for protection against PTTV infection, as well as methods of immunisation of pigs against PTTV viral infection by said vaccine introduction.
EFFECT: characterised inventions can be used to prevent infection, caused by porcine Torque teNo virus.
23 cl, 53 dwg, 5 tbl, 24 ex
SUBSTANCE: characterised strain was isolated from diseased pigs and produced by serial passages on sensitive hetero- and homologous cell cultures and deposited in the collection of the FSBI "Federal Animal healthcare centre" under the registration number of FMD virus strain A No.2187/Kuti/2013 (production). The presented strain is reproduced in monolayer cell culture of porcine kidney (PK), passaged cell cultures of kidney of Siberian mountain ibex (SMIK-30), VPK-21 and IB-RS-2. During 18÷24 hours of incubation the virus yield in the said cell cultures reaches the values of 6.0÷7.0 lg TCD50/cm3. At high multiplicity of infection (1÷10 TCD/cell) causes TCID50 after 5 hours, while maintaining the original characteristics when passaging in cell cultures for 5 passages.
EFFECT: invention can be used to monitor the antigenic and immunogenic activity and for producing biological products for diagnostics and specific prophylaxis of FMD of type A.
6 tbl, 6 ex
SUBSTANCE: invention refers to medicine, namely to oncology. The subject of the invention is a new strain of the Sendai virus Sen293nsk1 adapted to effective replication in the human cell culture HEK293. The produced strain of the Sendai virus possesses lower virulence for laboratory mice and higher ability to destroy human tumour cells. The strain is supposed to be used experimentally as a therapeutic oncolytic preparation for treating malignant diseases. The invention can be used in treating oncologic diseases.
EFFECT: invention enables providing higher clinical effectiveness in oncologic diseases by using the murine Sendai virus non-pathogenic for humans, possessing an increased oncolytic activity and intensifying anti-tumour immunity.
FIELD: medicine, pharmaceutics.
SUBSTANCE: claimed invention relates to such compositions and pharmaceutical compositions, which include poxviruses, and namely to those, which include extracellular enveloped viruses. Claimed invention also relates to such method, which is intended for production of poxviruses, as well as poxviruses, obtained in accordance with claimed invention. In addition, claimed invention also relates to application of claimed poxviruses and said composition for medication preparation.
EFFECT: obtaining pharmaceutical compositions, which include poxviruses.
11 cl, 3 dwg
SUBSTANCE: method of detection and differentiation of a genome of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates comprises: extraction of the DNA from the biological material, posing a multiplex polymerase chain reaction using synthesised primers complementary to regions of genes M130R and M151R of the myxoma virus of rabbit, and having the following nucleotide composition: MF 5'TGG-AGC-TTT-TCA-AGC-ATT 3', MR 5'ATA-TCT-CGG-CTC-TAG-GGC-GAG 3', MZ 5' [FAM]-AG-CGT-CGG-ACG-TCT-TCG-TT-[RTQ1] 3', VF 5'AGC-CCT-ATA-AAC-CCG-TAG-ACG-AAC 3', VR 5'CAA-GCT-TTT-TTT-TAT-CCT-CGT-CCG 3', VZ 5' [R6G]-TCG-ACG-GTT-TCG-TCC-GCC-TTC-TTG-[BHQ2] 3', DNA amplification of the virus and evaluation of the reaction. The present inventions may be used in veterinary virology for the detection and differentiation of the vaccine strain B-82 of the myxoma virus of rabbits from the field isolates.
EFFECT: increase in accuracy.
2 cl, 6 tbl, 6 ex
SUBSTANCE: invention refers to biotechnology, virology and immunology. Particularly, the present invention refers to a new avian astrovirus; to antibodies and their fragments targeted to the above new virus; to antigen preparations, proteins and DNA molecules of new avian astrovirus; to vaccines based on the above new virus or to its antigen preparations, protein or DNA; to methods for producing such vaccines and to diagnostic kits. The present invention can be used in veterinary science.
EFFECT: preparing the new avian astrovirus.
11 cl, 5 dwg, 4 tbl, 12 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for preparing influenza A or B viruses in a cell culture, and a composition of the cell culture for preparing influenza A or B viruses.
EFFECT: invention provides the serum-free culture medium, avoids the need for the stage of cell culture medium replacement, prepares the influenza viruses with the high live virus recovery and can be used for the active immunisation of individuals and for producing the antibodies for various applications, including passive immunisation and diagnostic immunoassays.
22 cl, 24 dwg, 47 tbl, 1 ex
SUBSTANCE: invention refers to biotechnology and virology. What is presented is a method for producing viral-like influenza virus particles (IVP) in a plant or a part thereof. The method involves the expression of a new influenza virus protein HA in plants and purification thereof. The invention also aims at IVPs containing the influenza virus protein HA and herbal lipids. The invention also refers to nucleic acids coding an improved influenza virus HA, as well as to vectors. The IVPs can be used in developing the influenza vaccines or for the treatment of existing vaccines.
EFFECT: presented group of inventions can be used in medicine.
18 cl, 44 dwg, 1 ex
FIELD: biotechnology, veterinary medicine.
SUBSTANCE: invention relates to the development of biological preparation for prophylaxis and treatment of colibacillosis (escherichiosis) and for control of carriage of escherichious infections pathogens in animals and poultries also. Also, invention can be used in producing curative fodders and ecologically pure human foodstuffs. Biopreparation for prophylaxis and treatment of escherichiosis in animals and poultries comprises strains of bacteriophages Phagum Escherichia coli Ec022-DEP and/or Phagum Escherichia coli Ec021-DEP, and/or Phagum Escherichia coli Ex0782-DEP, and/or Phagum Escherichia coli Ec0781-DEP, and/or Phagum Escherichia coli EPZ-1-DEP, and/or Phagum Escherichia coli EPZ-2-DEP, and/or Phagum Escherichia coli EG-5-DEP, and/or Phagum Escherichia coli BC-1-DEP, and/or Phagum Escherichia coli M78-DEP, and/or Phagum Escherichia coli Sheksna 2k-DEP taken in the effective amount. The biopreparation comprises also antiseptic, for example, quinosol and a stabilizing agent. Protein (for example, soybean protein), vegetable meal, organic polymer, milk, serum, albumin can be used as a stabilizing agent. Among organic polymers can be used: dextran, polyglucin, starch, polyvinylpyrrolidone. The biopreparation can be dried by lyophilization, granulated and placed in polymeric matrix. The biopreparation has no toxic properties on animals, it shows good hygroscopicity and can be good dispersed in water. The biopreparation can be used in liquid and dry prescription formulations and in different methods of its administrations: both by subcutaneous, intraperitoneal, intramuscular injections and as an aerosol, by administration of phage particles into lung compartments including applying as curative fodder and supplement to fodder, and by applying on surface of cutaneous integuments. Invention provides enhancing the effectiveness of treatment of animals and poultries with gastroenteric infections due to reducing treatment period, expanding spectrum of lytic effect of the biopreparation, resistance to effect of digestive tract enzymes and convenience in using.
EFFECT: valuable veterinary properties of biopreparation.
9 cl, 5 tbl, 7 ex