58 results about "Goatpox virus" patented technology

The invention relates to a method for screening non-essential regions for replication of a goat pox virus and universal transfer vectors for same. The method comprises the steps of amplifying two-end gene segments of any two regions of a goat pox virus gene by using a PCR (Polymerase Chain Reaction) method; then, inserting an enhanced green fluorescent protein (EGFP) gene and a xanthine-guanine phosphoribosyl transferase (gpt) gene expression cassette into the segments; establishing two universal transfer vectors of the goat pox virus; and acquiring a recombinant virus expressing an exogenous gene stably from the transfer vectors, thereby determining the selected regions to be non-essential regions for replication of the goat pox virus, wherein each universal transfer vector contains one unique restriction enzyme cutting site Sal I and allows gene expression cassettes of other items to insert in. The recombinant virus obtained by means of the two universal transfer vectors provided by the invention not only has a growth performance similar to a parent virus, but also has better safety because a plurality of toxicity related genes in a genome are knocked out in an orientation way, and has the potential to be developed into an attenuated vaccine strain for gene engineering.

The invention discloses a goatpox virus-orf virus combined cell attenuated vaccine and its preparation method and use. The goatpox virus-orf virus combined cell attenuated vaccine contains an active component which comprises goatpox and contagious pustular dermatitis virus cell passage-attenuated viruses or passaged viruses separated by the inventor and subjected to passage attenuation culture, wherein the goatpox virus cell passage-attenuated virus is a Goatpox Virus HN-XY2010F43 strain and has a preservation number of CCTCC NO: V201503 and the contagious pustular dermatitis virus cell passage-attenuated virus is an Orf Virus HB-TS09F65 strain and has a preservation number of CCTCC NO: V201406. A toxin counteracting protection test result shows that the goatpox virus-orf virus combined cell attenuated vaccine is safe and can produce an effective immunization protection function. The goatpox virus-orf virus combined cell attenuated vaccine and its preparation method have important practical meanings for development of safe and efficient attenuated vaccines and large-scale production of the goatpox virus-orf virus combined cell attenuated vaccine for preventing and controlling goatpox and orf from the disclosed attenuated strain.

The invention discloses a capripoxvirus (CPV) Taqman-MGB (minor groove binder) probe multiple real-time fluorescence quantitative PCR (polymerase chain reaction) detection primer, a kit and a detection method. The multiple Taqman-MGB real-time fluorescence quantitative PCR detection method can be used for rapid detection of Lumpy Skin Disease Virus (LSDV), Goatpox Virus (GTPV) and Sheeppox Virus (SPPV). According to the method, on the basis of conserved domains of target sequences of the LSDV, the GTPV and the SPPV, a pair of primer and three Taqman-MGB probes are designed. The method only needs two step amplification method and simple reaction conditions for fast, efficient, specific and highly-sensitive detection of an objective target sequence, is simple in operation, does not need expensive equipment and reagents, has no technical requirement on operators, is low in detection cost and short in testing time, and can avoid cross contamination caused by agarose electrophoresis so as to improve the detection accuracy and reliability.

The invention discloses a colloidal gold test strip for detecting goatpox virus and a preparation method thereof. The colloidal gold test strip for detecting goatpox virus comprises a sample pad, a colloidal-gold conjugate pad, a nitrocellulose membrane, an absorbent pad and a backing, wherein the sample pad, the colloidal-gold conjugate pad, the nitrocellulose membrane and the absorbent pad are successively adhered to a nonabsorbent support sheet; the colloidal-gold conjugate pad is a fiberglass membrane bonded to gold-labeled anti-goatpox virus P32 protein monoclonal antibody; and the nitrocellulose membrane has a detection line prepared by spraying of rabbit anti-P32 protein IgG and a quality control line prepared by spraying of goat anti-mouse polyclonal antibody. According to the invnetion, composition of a confining liquid used for preparation of the colloidal-gold conjugate pad and composition of a coating buffer solution used for preparation of the detection line and the quality control line are further optimized. It shows through specificity and sensitivity test results that the colloidal gold test strip for detecting goatpox virus has high specificity and high sensitivity.

The invention belongs to the technical field of biology, and discloses a kit for detecting bovine nodular skin disease virus by eliminating goatpox virus as well as a preparation method and an application thereof. The kit comprises a specific primer and a probe, wherein the specific primer comprise an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is as shown in SEQ ID NO.1, the nucleotide sequence of the downstream primer is as shown in SEQ ID NO.2, and the nucleotide sequence of the probe is as shown in SEQ ID NO.3. The kit provided by the invention has the advantages of strong specificity and high sensitivity.

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 a natural passage cell line for lamb testis sertoli cells and application of the natural passage cell line to separate culture and proliferation of goatpox viruses. The cell line disclosed by the invention is a lamb testis sertoli natural passage cell line which is obtained by separating and purifying primary cells of a lamb testis to obtain the lamb testis sertoli cells and performing natural passage on the lamb testis sertoli cells. A research shows that the cell line is relatively sensitive to inoculation of the goatpox viruses; the virus titer proliferated by the cell line is greater than that of the primary cells, Vero cells and BHK21 cells of the testis of a newborn lamb by about 1.5, 2.5 and 2.3 respectively. The cell line for separating the goatpox viruses can guarantee the uniformity and the stability of the viruses and prevent the risk that the separated viruses carry other pathogenies due to the fact that the primary cells are used for multiple times. Furthermore, by the use of the passage cell line for preparing vaccines, the production process is simplified, the production period is shortened, and the production cost is reduced; meanwhile, the quality of the vaccines are kept stable; the cell line has certain practical significance for large-scale production of the vaccines.

The invention relates to the field of vaccine preparation, and particularly relates to a cell strain for producing a goatpox vaccine. The cell strain is an African green monkey kidney cell Vero B10 and is collected at China General Microbiological Culture Collection Center with the collection number of CGMCC No. 9705. Compared with a primary cell production method, a preparation method of the cell strain has the advantages that a goatpox virus strain AV41 is cultivated by using a VeroB10 cell, good in virus adaptability and low in strong recurrence risk; a passage cell vaccine is superior to a primary cell vaccine in terms of virus yield, uniformity, pureness and the like; the cost is low, and animals are not needed; and the safety of the passage cell vaccine is superior to that of the primary cell vaccine, and the immunity efficiency of the passage cell vaccine is not lower than that of the primary cell vaccine.

The invention provides primers and a probe for detecting the fragment S of a rift valley fever virus, wherein, a forward primer is 5'-GGATTACTTTCCTGTGATATCTGTTG-3'; a reverse primer is 5'-GTATCCTGGGAGGRCCATCWC-3', R refers to A or G and w refers to T or A); and a probe is 5'-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3', F1 is a fluorescent reporter and Q1 is a fluorescent quencher. The invention also provides a real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection method for the fragment S of the rift valley fever virus. In the method, pseudovirus grains of the rift valley fever virus are taken as a template, and the primers and the probe are used to carry out real-time fluorescent RT-PCR, thus judging whether the detected virus is the rift valley fever virus according to the detected result. The probe provided by the invention has high sensitivity and good specificity, and has no cross reaction with all of Africa swine fever virus, irides virus and goatpox virus.

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 kit for identifying and detecting sheep pox viruses and goat pox viruses. The kit comprises three groups of primers: SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11 and SEQ ID NO. 12. The kit can quickly identify the sheep pox viruses and the goat pox viruses and can be applied to epidemiologic research on sheep and goat pot viruses.

Through homologous recombination and by innovative utilization of plaque and reverse plaque screening technologies, the selection marker-free recombinant virus, especially the rGTPV-F1L recombinant goat-pox virus is finally obtained. The method is simple and effective, is easy to operate, provides the selection marker-free recombinant virus for the development of a recombinant virus vaccine, provides new ideas and measures for the researches on a novel ORF vaccine, and has broad application prospects.

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 relates to a virus detection kit and a use method thereof, in particular to a kit for detecting and identifying capripoxvirus (goatpox virus, sheeppox virus and lumpy skin disease virus)and a use method for non-disease diagnosis. The kit for detecting and identifying the capripoxvirus (goatpox virus, sheeppox virus and lumpy skin disease virus) comprises specific primers of which the sequences are SEQ ID No. 1 and SEQ ID No. 2. The kit has high specificity and high sensitivity. The kit is high in cost performance, can screen unknown mutations on any amplicon by only needing a pair of specific primers, and is greatly reduced in cost and simple and convenient to operate compared with a Taq-man probe method.

The invention provides a recombination transfer vector used for constructing a recombinant goatpox virus. The transfer vector mainly comprises the left arm and the right arm of a TK gene, two reversep7.5K promoters, an orf virus B2L gene and F1L gene independently regulated and controlled by the two reverse p7.5K promoters, a p11K promoter, an enhanced green fluorescence protein (eGFP) gene and two forward Loxp sequences, wherein the transfer vector and the goatpox virus generate homologous recombination in goat testicular cells so as to obtain the recombinant goatpox virus capable of expressing the eGFP, under the function of Cre enzyme, the eGFP gene and the p11K prompter are specifically knocked out, and therefore, the potential recombinant goatpox virus capable of carrying out the coexpression on the orf virus B2L and F1L protein is obtained.

The invention provides primers and a probe for detecting the fragment S of a rift valley fever virus, wherein, a forward primer is 5'-GGATTACTTTCCTGTGATATCTGTTG-3'; a reverse primer is 5'-GTATCCTGGGAGGRCCATCWC-3', R refers to A or G and w refers to T or A); and a probe is 5'-F1-ACTCCACTGACACAACACGACGACCACT-Q1-3', F1 is a fluorescent reporter and Q1 is a fluorescent quencher. The invention also provides a real-time fluorescence RT-PCR (reverse transcription-polymerase chain reaction) detection method for the fragment S of the rift valley fever virus. In the method, pseudovirus grains of the rift valley fever virus are taken as a template, and the primers and the probe are used to carry out real-time fluorescent RT-PCR, thus judging whether the detected virus is the rift valley fever virus according to the detected result. The probe provided by the invention has high sensitivity and good specificity, and has no cross reaction with all of Africa swine fever virus, irides virus and goatpox virus.

The invention provides a TK gene deletion type goat pox virus and a preparation method thereof. The preparation method comprises the following steps: firstly, constructing two kinds of goat pox virus shuttle vectors pGVTK and pGVTKalpha, wherein core elements contained in a plasmid pGVTK are a TK gene homologous left arm, a pox virus general early and late promoter PE / L, a fluorescent marker gene, a transcription terminator and a TK gene homologous right arm sequentially, and core elements contained in a plasmid pGVTKalpha are a TK gene homologous left arm, an insignificant sequence and a TK gene homologous right arm sequentially; and then specifically knocking down a TK gene by utilizing a secondary homologous recombination technology, wherein a selection marker can be knocked down together. The toxicity of the constructed TK gene deletion type goat pox virus is reduced to some extent, but virus proliferation is not influenced, the growth characteristics of a parent strain are still kept, and the TK gene deletion type goat pox virus also has good genetic stability, is applicable to preparation of a goat pox vaccine and has good safety.

The present invention relates to a Peste des petits ruminants-goat pox bivalent live vaccine and a production method thereof. According to the present invention, a Peste des petits ruminants virus PPRV 75 / 1-Clone 9 strain (CGMCC No.13388) and a goat pox virus temperature sensitive attenuated strain (CVCC No.AV41) are used as vaccine production strains and are respectively cultured in Vero cells and goat kidney primary (secondary) cells to harvest virus liquids, the virus liquids are mixed, a freeze drying protection agent is added to the mixed virus liquid, and vacuum freeze drying is performed to prepare the bivalent live vaccine for effectively preventing Peste des petits ruminants and goat pox.

The invention discloses a visual rapid nucleic acid detection method of bovine nodular skin disease virus (LSDV), the method is characterized in that an RPA primer is designed for orf068 gene of bovine nodular skin disease virus, amplification is carried out, then Cas12a protein is added into an amplification product, enzyme digestion reaction is carried out, the reaction system comprises the Cas12a protein, sgRNA and a nucleic acid probe, and the signal emitted by the nucleic acid probe can be detected. The sensitivity for detecting orf068 plasmids is 5 copies / [mu] L, the sensitivity for detecting LSDV and goat pox virus (GTPV) is 0.1 TCID50 / [mu] L, the diagnostic sensitivity for detecting clinical samples is 96.3% (95% CI: 81.0%, 99.9%), the specificity is high, the changes in color are observed by eyes for result judgement, and the method has the advantages of simplicity, convenience, rapidness, suitability for field detection and the like.

The invention discloses a kit for detecting goat pox virus. The kit disclosed by the invention at least comprises four primers of SEQ (sequence) No. 1, SEQ No. 2, SEQ No.3 and SEQ No. 4, and the kit further comprises dNTP (deoxy-ribonucleoside triphosphate), Tris-HC, KCl, (NH4)2SO4, MgSO4, Tween 20 and a Bst (bacillus stearothermophilus) enzyme, as well as template DNA (deoxyribonucleic acid). The primers in the kit disclosed by the invention have specificity and can not amplify the nucleic acid of the goat pox virus under same conditions; and simultaneously, the kit is higher in detection sensitivity and also has the advantages that the detection operation is relatively simple and fast.

The invention relates to a combined inactivate vaccine for sheep pox and contagious caprine pleuropneumonia (CCPP), and a production method of the combined inactivate vaccine. The method comprises thesteps of inoculating suspension cultured BHK-21 cells and a proper culture medium with a capripox virus AV41 strain and a mycoplasma capricolum pneumonia subspecies C87001 strain respectively for culturing, harvesting the culture, concentrating and purifying; inactivating with BEI and thimerosal respectively, adding a suitable adjuvant according to a certain proportion, mixing and emulsifying. The combined inactivate vaccine is used for preventing the sheep pox and the CCPP. The combined inactivate vaccine, provided by the invention, for the sheep pox and the CCPP is obtained internationallyfor the first time, and can achieve the aim of preventing the two diseases by means of one injection; furthermore, a live vaccine for the sheep pox is produced by means of the suspension cultured BHK-21 cells; compared with the original inactivate vaccine prepared from ovine testicular cells, the combined inactivate vaccine provided by the invention has obvious advantages, and can solve the problems that in the production process of the live vaccine for the sheep pox, the sheet testis is difficult to obtain, lamb death and ewe abortion are easily caused, subcutaneous immunization needs to be performed when the vaccine is used, an operation is difficult during clinical application, and the like.

The invention discloses a construction method of a rabies virus G protein-goatpox virus recombinant vaccine. The construction method comprises the following steps of: 1, design of a recombinant virusvaccine construction model; 2, amplification of each gene and fusion amplification of each combined gene; 3, construction of various combined recombinant transfer vectors; 4, recombination of rabies virus G protein and goatpox virus and virus purification; 5, rabies virus G protein expression detection; and step 6, detection of the immune effect of a rabies virus G protein-goatpox recombinant virus vaccine strain. The vaccine constructed by the method can effectively prevent rabies of goat, has the characteristics of high production efficiency, simplicity, convenience and practicability, and is suitable for popularization and application.

The invention discloses a preparation method of a goat pox virus recombinant protein antigen and application thereof. The invention provides a product having the function of 1) or 2): 1) preventing, diagnosing and / or treating animal goat pox, and 2) inhibiting or neutralizing a goat pox virus. The product is any of the following products: protein ORF056, protein ORF112 and protein ORF117; or a composition containing the protein ORF056, protein ORF112 and protein ORF117; or a fusion protein containing the protein ORF056, protein ORF112 and protein ORF117. The experiment of the invention provesthat a mouse immunized by a subunit vaccine prepared by using IMV ORF056, ORF112-encoded protein and EEVORF117-encoded protein can produce a neutralizing antibody for the goat pox virus more than thatproduced by a mouse immunized by a vaccine prepared by single ORF056, ORF112 or ORF117 protein, so that the goat pox virus recombinant protein antigen is an ideal vaccine with the potential goat poxprevention effect.

A multiplex-PCR (polymerase chain reaction) kit for detecting six viruses of sheep and goats simultaneously comprises six pairs of specific primers, wherein the primers used for detection of the bluetongue virus are BTV-F and BTV-R respectively; the primers for detection of the foot and mouth disease virus are FMDV-F and FMDV-R respectively; the primers for detection of the peste des petits ruminants virus are PPRV-F and PPRV-R respectively; the primers for detection of the sheep pox virus are SPPV-F and SPPV-R respectively; the primers for detection of the goat pox virus are GTPV-F and GTPV-R respectively; the primers for detection of the sore mouth disease virus are ORFV-F and ORFV-R respectively. The sequences of the primers are nucleotide sequences shown in SEQ ID NO: 1-12. The multiplex-PCR kit can be used for detecting nucleic acid of the six viruses including the bluetongue virus, the foot and mouth disease virus, the peste des petits ruminants virus, the sheep pox virus, the goat pox virus and the sore mouth disease virus in sheep and goat clinical samples and has the characteristics of high sensitivity, high specificity, simple operation and reduction of detection time.

The invention discloses a goat pox virus recombination system free of bacteriophage plaque cloning and screening labels and dual-expression PPRV (peste des petits ruminants virus) H / F protein vaccine construction. The recombination system is not only capable of constructing single-expression and dual-expression recombinant viruses or recombinant vaccines and even capable of constructing recombinant vaccines or recombinant viruses which expressing more than three proteins, the recombinant viruses obtained by the recombination system are free of any screening labels, and accordingly potential adverse effects caused by the screening labels in vaccine use can be avoided. In addition, the invention discloses a peste des petits ruminants virus H and F protein dual-expressed screening-label-free recombinant goat pox virus prepared by the recombination system, wherein the recombinant goat pox virus. According to researches, the recombinant is capable of inducing cell and humoral immunity more comprehensive than single antigen expression and simultaneously preventing two important diseases including PPR and goat pox, and an induced high-enough neutralizing antibody is beneficial to immunoserology monitoring, so that the system has a promising application prospect in prevention and treatment of peste des petits ruminants and goat pox.

The invention discloses a colloidal gold test strip for detecting goatpox virus and a preparation method thereof. The colloidal gold test strip for detecting goatpox virus comprises a sample pad, a colloidal-gold conjugate pad, a nitrocellulose membrane, an absorbent pad and a backing, wherein the sample pad, the colloidal-gold conjugate pad, the nitrocellulose membrane and the absorbent pad are successively adhered to a nonabsorbent support sheet; the colloidal-gold conjugate pad is a fiberglass membrane bonded to gold-labeled anti-goatpox virus P32 protein monoclonal antibody; and the nitrocellulose membrane has a detection line prepared by spraying of rabbit anti-P32 protein IgG and a quality control line prepared by spraying of goat anti-mouse polyclonal antibody. According to the invnetion, composition of a confining liquid used for preparation of the colloidal-gold conjugate pad and composition of a coating buffer solution used for preparation of the detection line and the quality control line are further optimized. It shows through specificity and sensitivity test results that the colloidal gold test strip for detecting goatpox virus has high specificity and high sensitivity.

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 provides a recombinant transfer vector used for constructing a recombinant goatpox virus. The transfer carrier mainly comprises the left arm and the right arm of a TK gene, two reverse p7.5K promoters, a peste des petits ruminants virus H gene and F gene independently regulated and controlled by the two reverse p7.5K promoters, a p11K promoter, an enhanced green fluorescence protein(eGFP) gene and two forward Loxp sequences, wherein the transfer vector and the goatpox virus generate homologous recombination in goat testicular cells so as to obtain the recombinant goatpox virus capable of expressing the eGFP, under the function of Cre enzyme, the eGFP gene is specifically knocked out, and therefore, the potential recombinant goatpox virus capable of carrying out coexpressionon peste des petits ruminants virus H and F protein is obtained.

The invention discloses a natural passage cell line for lamb testis sertoli cells and application of the natural passage cell line to separate culture and proliferation of goatpox viruses. The cell line disclosed by the invention is a lamb testis sertoli natural passage cell line which is obtained by separating and purifying primary cells of a lamb testis to obtain the lamb testis sertoli cells and performing natural passage on the lamb testis sertoli cells. A research shows that the cell line is relatively sensitive to inoculation of the goatpox viruses; the virus titer proliferated by the cell line is greater than that of the primary cells, Vero cells and BHK21 cells of the testis of a newborn lamb by about 1.5, 2.5 and 2.3 respectively. The cell line for separating the goatpox viruses can guarantee the uniformity and the stability of the viruses and prevent the risk that the separated viruses carry other pathogenies due to the fact that the primary cells are used for multiple times. Furthermore, by the use of the passage cell line for preparing vaccines, the production process is simplified, the production period is shortened, and the production cost is reduced; meanwhile, the quality of the vaccines are kept stable; the cell line has certain practical significance for large-scale production of the vaccines.