40 results about "Orf virus" patented technology

The invention relates to a preparation method of a permanent cell line for multiplying an orf virus. The aim that the virus can be stably multiplied in a large amount is achieved by inoculating a calf testicular cell line to the orf virus and a stable cell environment and a standardized culture system for developing and producing an attenuated vaccine of the orf virus are provided. The preparation method comprises the following steps of: firstly, acquiring, separating and culturing a calf testicular cell; secondly, permanently establishing the calf testicular cell; and thirdly, carrying out multiplication culture on the orf virus in the cell line.

The invention discloses a cell line used for separation culture and multiplication of an Orf virus (ORFV) as well as a preparation method and application of the cell line. The cell line disclosed by the invention is a newly born bovine testis supported cell line obtained by separating and purifying newly born bovine testis primary culture cells; research results show that the cell line is more sensitive to inoculation of contagious ovine ecthyma virus, virus titer multiplied by utilizing the cell line is about 1 virus titer higher than that of newly born bovine testis primary culture cells and 1.9 virus titer higher than that of a madin-darby bovine kidney (MDBK) cell line. ORFV is separated by utilizing the cell line, uniformity and stability of virus can be guaranteed, and a risk that the separated viruses carry other pathogens as the primary culture cells are utilized for multiple times can be prevented; besides, the passage cell line is utilized for preparing a vaccine, a production process of the vaccine can be simplified, a production cycle is shortened, production cost is reduced, and stable quality of the prepared vaccine is also guaranteed, so that the cell line has a certain practical significance on large-scale production of the vaccine.

The invention discloses a goatpox, sheeppox and contagious ecthyma triple cell attenuated vaccine and a preparation method and application thereof. The effective components of the vaccine are separated by an inventor automatically, and after attenuation culture is conducted, goatpox virus, sheeppox virus and Orf virus cell passage attenuated viruses or passage viruses thereof are obtained, wherein a goatpox virus is a Goatpox Virus HN-XY2010F43 strain, a sheeppox virus is a Sheeppox Virus GS-WW 2010 F44 strain, and an Orf virus is an Orf Virus HB-TS09F65 strain. It is shown through attacking protection test results that the prepared goatpox, sheeppox and contagious ecthyma triple cell attenuated vaccine is safe and capable of producing an effective immune protection force, and important practical significance in developing safe and efficient goatpox, sheeppox and contagious ecthyma triple cell attenuated vaccine and preventing and controlling goatpoxes and ecthyma is achieved.

The invention discloses an orf virus (ORFV) protein ORFV059 monoclonal antibody hybridoma G3 and a monoclonal antibody. The ORFV059 monoclonal antibody hybridoma G3 was collected in China General Microbiological Culture Collection Center (CGMCC) on November 4, 2011, wherein the collection number is CGMCC No.5425. The recombinant ORFV059 protein coats an enzyme-linked immuno sorbent assay (ELISA) plate; activity of a purified antibody is detected by an ELISA method; and the purified ORFV059 protein is subjected to Western-blot by the purified antibody, which proves that the antibody can identify the ORFV059 protein. The purified antibody is used for performing immunofluorescent staining on the ORFV059 and immunohistochemical staining on the orf tissue, which proves that the purified antibody can identify the natural ORFV059 protein. The monoclonal antibody secreted by the hybridoma cell lays basis for further development of ORFV059 function and a diagnosis reagent for developing the ORFV059 and provides a powerful tool for clinical diagnosis on the orf and confirmation of a therapeutic target.

The invention discloses a real-time fluorescent quantitative PCR (polymerase chain reaction) detecting reagent kit for an orf virus and belongs to the technical field of viral molecular biology detection. The real-time fluorescent quantitative PCR detecting reagent kit for the orf virus comprises a pair of primers of nucleotide sequences shown in SEQ ID NO:1-2 and a TaqMan probe of a nucleotide sequence shown in SEQ ID NO:3. The detecting reagent kit can quickly and accurately detect the orf virus from samples and is high in detecting sensitivity and simple and convenient to sample, detecting efficiency is greatly improved, pollution can be effectively prevented, and the detecting reagent kit is of practical significance in the field of inspection and quarantine of import and export animals.

The invention provides a goatpox and sheep pox bivalent cell attenuated vaccine. The goatpox and sheep pox bivalent cell attenuated vaccine comprises effective ingredients, namely, a goatpox virus cell passage attenuation virus and a sheep pox virus cell passage attenuation virus, wherein the goatpox virus cell passage attenuation virus is a goatpox virus cell passage attenuation vaccine strain Goatpox Virus HN-XY2010F43 strain CCTCC V201503, and the sheep pox virus cell passage attenuation virus is a sheeppox virus cell passage attenuation vaccine strain Sheeppox Virus GS-WW2010F44 strain CCTCC V201504. The goatpox and orf virus bivalent cell attenuated vaccine is safe, can have the effective immune protection force and has the important practical significance in development of safe and efficient attenuated vaccines as well as prevention and control on goatpox and sheeppox through large-scale production of goatpox and sheep pox virus bivalent cell attenuated vaccines by the aid of the attenuation virus strains.

The invention discloses a double PCR detection primer, a double PCR detection reagent kit and a double PCR detection method for identification of orf virus and capripoxvirus. The method provides a rapid detection method for diagnosis of the orf virus and capripoxvirus. Particularly, the PCR primer for detection of the capripoxvirus relates to all the members of capripoxvirus, including goat capripoxvirus, sheep capripoxvirus, and lumpy skin disease virus, so that the detection method has the characteristics of highlight integration and high-throughput performance, etc., and can provide technical support and help for diagnosis of the orf virus and capripoxvirus, and investigation, prevention and treatment of epidemic virus. The method has the advantages of high specificity, high sensitivity and time saving and labor saving.

The invention discloses ORFV (Orf virus) oncolytic virus and a preparation method and application thereof. The recombinant contagious ecthyma oncolytic virus preparation method disclosed by the invention comprises the following steps: (1) overlap PCR amplifying gene p53 of protein shown in claim 7; (2) connecting to a pSPV-EGFP carrier, inserting into the p53 gene to obtain recombinant plasmid p53-pSPV-EGFP and then inserting into the left side and the right side of ORFV132 gene to construct shuttle plasmid ORFV132LF-p53-pSPV-EGFP-ORFV132RF; (3) converting host bacteria TOP10 to obtain positive recombinant bacteria; (4) performing transfection on OFTu cells and performing fluorescent identification on fusion expression of the p53 and the EGFP to prepare the purified recombinant contagiousecthyma oncolytic virus. The prepared recombinant contagious ecthyma oncolytic virus can be duplicated and multiplicated in OFTu and varieties of tumor cells and can effectively inhibit growth of varieties of tumor cell. The recombinant contagious ecthyma oncolytic virus disclosed by the invention can be observed in a microscope; thus, easiness in operation is achieved, convenience and quickness are achieved, and screening time is greatly shortened.

The invention discloses a Taqman fluorescence quantitative PCR kit and method for simultaneously detecting eight common sheep viruses and bacteria, and relates to the technical field of molecular biology. The eight common sheep viruses and bacteria include peste des petits ruminants virus (PPRV), orf virus (ORFV), goatpox virus (GTPV), chlamydophila abortus (C.a), staphylococcus aureus (S.a), escherichia coli (E.coli), streptococcus (Stre.) and salmonella (Salm.). PCR reaction liquid comprises eight groups of primers and eight groups of Taqman probes. The kit can rapidly and effectively detect8 sheep pathogens simultaneously, the detection method is accurate, the specificity and the sensibility are high, the stability is good, and it can be achieved that to-be-detected pathogens is rapidly diagnosed and effectively detected.

The invention provides a detection method for detecting the orf virus of sheep. Real-time fluorescent quantitative PCR (polymerase chain reaction) detection is performed on a DNA (deoxyribonucleic acid) template of a sample by designing a pair of primers and a probe. The invention further provides a kit for detecting the orf virus of the sheep by utilizing the detection method. The real-time quantitative method which is established against H2L genes in a conserved region of the ORFV (orf virus) is high in specificity and good in stability. The kit for detecting the orf virus of the sheep, provided by the invention, is simple and convenient to operate, a user only needs to add DNA of the sample to be detected into a reaction tube, detection results can be obtained according to an amplification curve, the operation is time-saving and labor-saving, the cost is lower, and the time is shortened from original 3-4 hours to 1-2 hours; and furthermore, the sensitivity is higher than that of the ordinary PCR, and the detection method and the kit can be used for detecting the low content of the orf virus of the sheep, can be suitable for all levels of prevention and control units, basic-level veterinary stations, large and medium-sized breeding farms and the like in China, and has better application prospects.

The invention discloses a sheep pox and aphtha bigeminy cell attenuated vaccine and a preparation method and application thereof. The effective ingredient of the sheep pox and aphtha bigeminy cell attenuated vaccine is sheep pox virus and contagious pustular dermatitis virus cell passage attenuate virus or passage virus thereof which are separated through the inventor and obtained after passage attenuate culture, wherein the sheep pox virus cell passage attenuate virus is Sheeppox Virus GS-WW 2010 F44 strain, the preservation number of CCTCC NO: V201504, the sheep pox virus (contagious pustular dermatitis virus) cell passage attenuate virus is Orf Virus HB-TS09F65 strain, and the preservation number is CCTCC NO:V201406. Toxin attacking protection test results show that the sheep pox and aphtha bigeminy cell attenuated vaccine is safe and capable of producing effective immune protection force. Accordingly, the sheep pox and aphtha bigeminy cell attenuated vaccine is of an important practical significance in researching safe and efficient attenuated vaccine and preventing and controlling sheep pox and aphtha bigeminy.

The invention discloses an orf F1L vaccine based on ferritin nanoparticles and a preparation method of the orf F1L vaccine. According to the invention, a fusion protein is characterized in that an orfF1L protein is externally connected to the surface of ferritin through a genetic engineering method to form nanoparticles, namely the orf F1L/Fe fusion protein. The target protein provided by the invention has good immunogenicity and can be used for preventing infection of the orf virus.

The invention provides a preparation method for a goat testis fibroblast membrane system yeast cDNA library. The preparation method comprises the following steps: preparation of goat testis fibroblasts; extraction of RNAs of the goat testis fibroblasts; synthesis of cDNAS and homogenization of a cDNA library; linkage of the cDNAS with a pPR3-N vector, transformation of the host cell DH5alphja witha linkage product so as to obtain the goat testis fibroblast membrane system yeast cDNA library, and cloning and verification; screening of the goat testis fibroblast membrane system yeast cDNA library; etc. The goat testis fibroblast membrane system yeast cDNA library prepared in the invention has a cDNA storage capacity of 7.5*10<5>; fragments have sizes in a range of 200 to 2000 bp and an average length of 1000 bp; and the recombination rate of the library is 100%. The screening of the goat testis fibroblast membrane system yeast cDNA library with the membrane protein of Orf viruses provesthat the library is a high-quality library.

The invention relates to a LAMP visual detection kit for an orf virus. A detection primer in the kit can specifically detect the orf virus aiming at a conservative VIR sequence of the orf virus; a detection means of constant-temperature reaction is adopted, so that the whole reaction can be realized only by heating, and the dependence of a traditional nucleic acid detection technology on a PCR instrument is eliminated; the LAMP detection kit can realize rapid on-site detection of the orf virus, and a detection result can be directly observed by visual inspection, so that the LAMP detection kitis suitable for on-site detection; and the method is high in detection sensitivity, high in specificity, good in repeatability and high in detection speed, and can be used as the effective basic detection means for the orf virus.

The invention provides an orf virus attenuated strain and an application thereof. The attenuated strain is an orf virus attenuated strain QFnm2015, the preservation institution is the China Center forType Culture Collection, and the preservation number is CCTCC NO: V201903. According to the invention, the isolated orf virus is proliferated and cultured on lamb testicular cells, and the orf virusattenuated strain is cultured through continuous passage. The isolated strain of the orf virus provides a high-quality material basis and selection space for developing an effective vaccine of endemicORFV, and has important significance for prevention and treatment of orf.

The invention provides a recombinant DNA vaccine and a recombinant plasmid thereof, the DNA vaccine recombinant plasmid comprises an antigen coding sequence and a plasmid vector, the antigen coding sequence comprises an orf antigen coding sequence and a sheep pox antigen coding sequence which are connected in series, and an orf antigen and a sheep pox antigen are expressed as independent antigen proteins. The invention provides construction of a double-gene recombinant DNA (Deoxyribose Nucleic Acid) vaccine of an orf virus gene and a sheep pox virus gene and analysis of a mouse immune effect. According to the invention, an orf virus B2L (011) gene and a sheep pox virus P32 gene are selected, the two genes are connected in series by using a self-cleaving peptide P2A, and the self-cleaving peptide P2A generates self-cleaving in eukaryotic cells, so that two antigen proteins are independently expressed on an eukaryotic expression vector pcDNA3.1 (+). The recombinant DNA vaccine prepared by the invention also has a better immune effect after being used for immunizing a mouse, and can be used as a reference of a candidate vaccine.