658 results about "Foot-and-mouth disease" patented technology

The invention relates to escherichia coli-derived single-plasmid-tandem soluble coexpression foot-and-mouth disease virus capsid proteins VP0 (which is a VP4 and VP2 fusion gene), VP1 and VP3, and a foot-and-mouth disease virus capsid protein virus-like particle preparation method. Foot-and-mouth disease virus capsid protein virus-like particles can be used for preparation of a foot-and-mouth disease vaccine. According to the method, a plurality of aspects of escherichia coli-derived soluble coexpression foot-and-mouth disease virus capsid protein are studied, by comprehensive use of tandem coexpression and SUMO(suggested upper merged ontology) technology with a tag for soluble coexpression of the foot-and-mouth disease virus capsid proteins VP0 (which is the VP4 and VP2 fusion gene), VP1 and VP3, the ultimate objective protein accounts for about 20% of total bacterial protein, and the foot-and-mouth disease virus capsid proteins obtained by purification can be successfully assembled into the virus like particles.

We have generated novel molecularly marked FMDV A₂₄LL3D_YR and A₂₄LL3B_PVKV3D_YR vaccine candidates. The mutant viruses contain a deletion of the leader coding region (LL) rendering the virus attenuated in vivo and negative antigenic markers introduced in one or both of the viral non-structural 3D^pol and 3B proteins. The vaccine platform includes unique restriction endonuclease sites for easy swapping of capsid proteins for different FMDV subtypes and serotypes. The mutant viruses produced no signs of FMD and no shedding of virulent virus in cattle. No clinical signs of disease or fever were observed and no transmission to in-contact animals was detected in pigs inoculated with live A₂₄LL3D_YR. Cattle immunized with chemically inactivated vaccine candidates showed an efficacy comparable to a polyvalent commercial FMDV vaccine. These vaccine candidates used in conjunction with a cELISA provide a suitable target for DIVA companion tests.

The invention discloses a method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S. A size exclusion high-efficiency liquid-phase chromatographic column in a molecular weight separation range of 2*10<4> to 1*10<7>Da is adopted to carry out the chromatographic separation on a detected sample on a high-efficiency liquid-phase chromatography. The operation pressure of the chromatography is 1.0MPa to 2.5MPa, the flow rate in the chromatographic column is 0.5 to 1.0 ml/min, a flow phase is phosphate buffer (pH 7.0 to 7.5) containing 0.1M sodium sulfate, and the column temperature is 15 to 25 DEG C. An ultraviolet and laser detector is used for detecting an optical signal of effluent at an outlet of the size exclusion high-efficiency liquid-phase chromatographic column, and a peak area of a sample can be analyzed by virtue of a computer software system of the high-efficiency liquid-phase chromatography. A standard curve of the absorption peak area and 146S concentration is established by virtue of a relation between the ultraviolet absorption peak and the concentration of different 146S standard products of different concentrations. Chromatograph is carried out on the detected sample through the size exclusion high-efficiency liquid-phase chromatographic column. The ultraviolet absorption peak area is measured, and the concentration of 146S in the detected sample can be acquired according to the standard curve.

This invention relates to a fusion protein used for preventing aftosa, its preparation method and application. This fusion protein contains O type foot-and-mouth disease virus main cytomembrane protein VP1 epitope, colibacillus thermolability toxin B subunit, thymus derived cell epitope and purification label.

The invention discloses an O type foot and mouth disease virus-like particle and a preparation method of the O type foot and mouth disease virus-like particle and an application. The O type foot and mouth disease virus-like particle is formed by assembling structural protein VP0, VP1 and optimized OP3 structural proteins of O type foot and mouth disease virus, wherein the gene sequence of the VP1 is shown as SEQ ID NO.1; the gene sequence of the VP0 is shown as SEQ ID NO.2; the gene sequence of the optimized VP3 is shown as SEQ ID NO.3. The invention tries to perform artificially missing on a part of section of the structural protein VP3 of O type foot and mouth disease virus; the result shows that the protein expression amount of the VP3 gene after the artificial missing is improved by 20% in comparison to that before mutation; the assembling efficiency of the virus-like particle is also improved by 15%; moreover, the animal test result shows that the immunogenicity thereof is good and free from significant difference with VLPs obtained through unmissed VP3 assembling. The invention provides a new technical manner for the research of the foot and mouth disease vaccine.

The invention relates to a method for expanding the antigen spectrum of a foot-and-mouth disease vaccine strain by reverse genetic operation and a preparation method of a vaccine. The amino acid sequence of the VP3 and VP1 structural proteins of the foot-and-mouth disease virus strain of the invention is represented by the amino acid residues from a position 304 to a position 736 in SEQ ID No.4. Experiments show that the vaccine prepared from the mutant virus strain obtained by the invention can resist porcine epidemic viruses of China O/TL/Taiwan/97 lineage, Pan-Asia O/China/99 lineage and Southeast Asia Myanmar O/GS/2010/98 lineage, has a characteristic of wide antigen spectrum, can immunize pigs and obviously improve the rate of protection against foot-and-mouth disease viruses which are of the same type and have antigenicity difference, achieves an immune effect of cross protection, and is expected to play an important role in the prevention and control of foot-and-mouth disease.

The present invention discloses a method for quantification of 146s content in foot-and-mouth disease antigen by using a liquid chromatography detection system. The method comprises: adopting protamine sulfate to purify virus, adopting PEG6000 to concentrate the virus, carrying out sucrose density gradient ultracentrifugation on the concentrated virus, adopting a liquid chromatography detection system to detect an absorption peak of the centrifugated sample at a wavelength of 259 nm, and calculating 146S content according to the following formula: C=FRS/76Wb. The method can be used for determination of the 146S content in various types of foot-and-mouth disease antigens. With quantification of the 146S content in the foot-and-mouth disease antigen, foot-and-mouth disease vaccine preparation can be guided, vaccine efficacy can be indirectly evaluated, and major guiding significances are provided for enhancing quality of the foot-and-mouth disease vaccine in our country.

The invention provides a method for culturing baby hamster kidney (BHK) 21 cell in a serum-free way, which leads the BHK 21 cell to be inoculated into a cell culture medium for culturing, wherein the cell culture medium comprises a basic culture medium and further comprises 100-200g/100L of soy protein, 100-200g/100L of pea protein, 100-200g/100L of broad bean protein, 0-100g/100L of potato protein, 0-200g/100L of wheat gluten protein and 50-100g/100L of rice protein by taking the volume of solvent of the culture medium as reference. In addition, the invention also provides a vaccine (such asrabies vaccine and foot-and-mouth disease vaccine) preparation method comprising the method for culturing the BHK 21 cell. The BHK 21 cell cultured by the culture medium containing the vegetable protein is high in culture density, beneficial to separating down-stream products of the cells, low in cost, small in batch difference, good in safety and suitable for producing virus host, expression vector and the like of biological products such as vaccine and the like.

The invention provides a method for determining components and estimating the anti-gen content of a foot-and-mouth disease vaccine. The method comprises the following steps: (1) performing emulsion resolving on a foot-and-mouth disease vaccine to separate out an antigen phase; (2) concentrating the antigen phase; (3) using purified foot-and-mouth disease virus Asia 1-type, O-type and A-type 146S antigens to immunize animals and prepare detection serums; (4) diluting the concentrated antigens by different multiples and respectively performing immunodiffusion tests on the detection serums; (5) judging the components of the foot-and-mouth disease vaccine according to precipitation lines, that is, whether the vaccine is an O-type univalent vaccine, an O-type + A-type bivalent vaccine or an O-type + Asia 1-type + A-type trivalent vaccine; (6) estimating the content of the antigen in a detected vaccine sample according to the highest antigen dilution multiple at which the precipitation lines appear. The method can not only identify the components of the foot-and-mouth disease vaccine but also estimate the contents of all the components, and is simple to operate, strong in specificity, good in stability and suitable for basic-level application.

The invention discloses a foot-and-mouth disease genetic engineering mixed epitope vaccine and a preparation method thereof. The vaccine consists of the following four parts: a serial B cell epitope recombinant protein BI consisting of main neutralizing epitops of O-type foot-and-mouth disease viruses in Cathay, Transasia and Mya 98 pedigrees with a gene sequence of SEQ ID NO:1 and an amino acid sequence of SEQ ID NO:2, a T-cell epitope recombinant protein TI consisting of serial connection of universal T-cell epitope and a plurality of foot-and-mouth disease virus specific T-cell epitopes with a gene sequence of SEQ ID NO:3 and an amino acid sequence of SEQ ID NO:4, Toll-like receptor 3 agonist-polyinosinic acid-polycytidysic acid and/or Toll-like receptor7/8 agonist-R848 serving as immunopotentiator, and 201 oil adjuvant. When being used for immunizing a pig, the BI and TI mixed epitope vaccine prepared by utilizing the method can produce a protective immunization effect the same as or better than that of an inactivated influenza virus Vaccines, and has a cross protection effect to viruses of the three pedigrees, so that the vaccine is a novel immune-enhanced O-type foot-and-mouth genetic engineering mixed epitope vaccine.

A method for expressing antigens of foot-and-mouth disease virus in insect in vivo by using re-combinant baculovirus is disclosed, which comprises: cloning different genes combination of foot-and-mouth disease virus into baculovirus carrying vector to construct transfer vector, transfecting baculovirus with the transfer vector to proceed DNA recombination, and thus to get recombinant baculovirus, infecting insect host with the recombinant baculovirus, culturing the infected insect host to express the antigens of foot-and-mouth disease virus, and then collecting and purifying the expressed antigens. The preferred genes, baculovirus carrying vector, baculovirus and insect host are selected from P1-2A3C, ORF and VP1 genes of foot-and-mouth disease virus as shown in SEQ ID NO 1., 3 or 5, pVL1393, Bombyx mori Bm-NPV-ZJ8 and larvae or pupa of Bombyx mori, respectively.

The invention belongs to the field of sanitary examination, and relates to a LAMP kit for testing classical swine fever virus and an establishing method and an application thereof. The kit contains a test system which is composed of the LAMP reaction liquid of six LAMP primers. The tests prove that the kit of the invention has good specificity and sensitivity, fast amplification speed, high efficiency and simple and convenient identification. The test system of the invention can rapidly and conveniently test the classical swine fever virus in high-efficiency, high-specificity and high-sensitivity under the isothermal condition of 65 DEG C without complicate instruments, can better satisfy the spot tests for the classical swine fever virus, provides a novel technical platform for food safety testing, can better meet the urgent requirements for the spot testing of foot and mouth diseases at present, is used for the spot testing of import and export quarantine, food sanitary departments, animal breeding farms, etc, and is easy to be popularized in a wide range.

The invention provides a method for detecting foot-and-mouth disease antigen 146S content with a sucrose density gradient ultraviolet light quantitative method; the method comprises the following steps: sucrose density gradient ultra centrifugation is carried out to virus concentration liquid to be detected, and then OD259nm value of all fractions is continuously detected by an ultraviolet light spectrophotometer; next, peak area of OD value of a sample to be detected is calculated, and the 146S content in the virus liquid is calculated out according to the formula (FR*PA*FSD*1000)/(S*PL*E*W),wherein FR is flow rate of sucrose which passes through a sample cell; PA is peak area; S is speed, FSD is an absorbance unit, PL is optical path length of the flowing sample cell, W is original weight or volume of a sample which is added on the gradient, E is correction coefficient which is optional positive number. The method has better repetitiveness and applicability, and can be used for accurate and quantitative detection of 146S relative content of various foot-and-mouth viruses.

The invention discloses application of a baby hamster kidney(BHK)-21 cell serum-free suspension culture technology in foot-and-mouth disease vaccine production, which comprises the following steps of: 1) performing cell recovery; 2) performing reactor culture and cell amplification culture; and 3) inoculating foot-and-mouth disease virus seed venom and collecting the venom. A process for producing foot-and-mouth disease inactivated vaccines by culturing the BHK-21 cells through serum-free suspension culture and a step-by-step cell amplification method make the production period f the foot-and-mouth disease vaccines shortened and yield increased, and ensure stable quality and obvious benefit. The production process reduces the using amount of a culture medium, and the amount of the collected virus liquid is the culture medium consumption amount, while the culture medium consumption amount in a roller bottle production process is 2 times higher than the amount of the collected virus liquid, and bovine serum which is about 5 percent of the culture medium amount is needed.

The invention discloses a foot-and-mouth disease virus (FMDV) resistant monoclonal antibody and an identified epitope and application thereof, and belongs to the field of prevention and control of the FMDV. The microbial collection number of a hybridoma cell line, which can secrete the neutralizing monoclonal antibody resisting to Asia-1 FMDV, is CGMCC No.2692; and the microbial collection number of the hybridoma cell line, which can secrete the neutralizing monoclonal antibody resisting to O-type FMDV, is CGMCC No.2691. The invention also discloses amino acid sequences of a conformational neutralizing epitope of the Asia-1 FMDV VP1 protein and a linear neutralizing epitope of the O-type FMDV VP1 protein which are identified by the two monoclonal antibodies respectively. In-vitro neutralization tests and in-vivo animal protection tests show that both the two monoclonal antibodies have excellent passive immunity effect, can be applied to emergency prevention of the FMDV and have excellent immunity effect on the passive immunity of the FMDV.

The invention discloses antiviral serotype shared monoclonal antibody of foot-and-mouth disease and a distinguished epitope thereof and the application, belonging to the prevention and control field of foot-and-mouth disease. The invention obtains two hybridoma cell lines which can secrete antiviral serotype shared monoclonal antibody of foot-and-mouth disease (microorganism maintenance numbers thereof are CGMCC No. 3103 and CGMCC No. 3104 respectively), and monoclonal antibodies 4B2 and 3D9 which are secreted by the hybridoma cell lines. ELISA and indirect immunofluorescence assay show that the two hybridoma cell lines both belong to FMDV shared monoclonal antibodies; wherein the 4B2 distinguishes one linear epitope of FMDV, and the 3D9 distinguishes one conformation epitope of FMDV. The4B2 and 3D9 are respectively used as a capture antibody and a detection antibody, a method for capturing ELISA by an antigen is adopted, specificity detection of FMDV can be carried out; the detectionmethod has the characteristics of high sensibility, strong specificity and good repeatability.

The invention discloses an Asia1 type multi-epitope recombinant vaccine of bovine foot-and-mouth disease viruses and a preparation method thereof. The recombinant vaccine comprises the following components: proteins coded by foot-and-mouth disease virus multi-epitope genes and the fusion genes of carrier proteins, and foot-and-mouth disease virus 3D proteins. The preparation method comprises the following steps: diluting the proteins expressed by the foot-and-mouth disease virus multi-epitope genes and the fusion genes of the carrier proteins and the foot-and-mouth disease virus 3D proteins, mixing the diluted proteins uniformly, adding an adjuvant into the mixture to emulsify the mixture. Animal models and animal immune effect experiments show that the bovine Asia1 epitope recombinant vaccine can make comprehensive immune protective response, can induce injected and immunized bovine and guinea pigs to generate high level neutralizing antibodies, and can also induce cell immune response, so the recombinant vaccine can effectively protect animals against the virulent attack of the foot-and-mouth disease viruses.

The invention provides a type O foot-and-mouth disease virus mutant and a preparation method and application thereof, and belongs to the technical field of vaccine candidate strains. According to thetype O foot-and-mouth disease virus mutant disclosed by the invention, an rHN virus strain is used as a female parent virus strain, and the following amino acids in a G-H ring of VP3 protein are mutated: the 173rd aspartic acid is mutated into asparagine, the 174th valine is mutated into glutamic acid and the 179th asparagine is mutated into cysteine. The virus mutant has heredity stability and obtains the capacity of caveolin for performing mediated infestation on CHO-K1cells. The result of detecting the cross neutralization capacity of immune positive serum indicates that compared with a female parent virus strain, the virus mutant has the advantages that the cross protection capacity of the virus mutant for inducing organisms to produce foot-and-mouth disease virus neutralizing antibodies is notably improved, and the virus mutant shows excellent antigen broad spectrum properties. Vaccines prepared through inactivation of the virus mutant can be used for preventing infection with type O foot-and-mouth disease viruses.

The invention discloses a universal primer and probe combination for detecting foot and mouth disease viruses through RPA (recombinase polymerase amplification)-lateral flow assay technology. The forward primer sequence is shown as SEQ ID No.1, the reverse primer sequence is shown as SEQ ID No.2, and the probe sequence is shown as SEQ ID No.3. The invention also discloses a kit for detecting foot and mouth disease viruses. Detection with the primers and probes involved in the invention only needs virus RNA extraction on clinical samples and isothermal amplification after one-step process reverse transcription into cDNA, thermal cycling reaction is not needed, amplification in a PCR instrument is unnecessary, and the result can be clearly displayed on a lateral flow chromatography test strip. The primer and probe combination and the kit provided by the invention have the advantages of high sensitivity, strong specificity, simple reaction procedure, short detection time and the like, and are suitable for rapid, accurate and simple detection of foot and mouth disease viruses on a cattle farm.

The invention discloses antipyretic and toxin removing powder for livestock. The antipyretic and toxin removing powder for livestock is characterized by comprising the following components in parts by weight: 4.2 parts of the root bark of the peony tree, 5 parts of Scutellaria baicalensis, 5 parts of root of common peony, 5 parts of radix scrophulariae, 4 parts of rhizoma anemarrhenae, 20 parts of gypsum, 5 parts of radix rehmanniae, 10 parts of buffalo horn, 4.2 parts of Coptis chinensis, 5 parts of Gardenia jasminoides Ellis, 6 parts of fructus forsythia, 5 parts of Platycodon grandiflorum, 2 parts of liquorice, 3 parts of bamboo leaves, 5 parts of rhizoma cimicifugae, 6 parts of cyrtomium fortune and 4.6 parts of gentian. The antipyretic and toxin removing powder for livestock provided by the invention has the beneficial effects of low cost, obvious curative effect, safety, reliability, low dosage and wide therapeutic range, not only is used for curing heat toxicity disease of animals, purging intense heat for removing toxin, cooling blood and curing heat toxin and spot as well as high heat and coma, but also is used for curing pig foot-and-mouth diseases, and has favorable effects.