Construction Of Recombinant Attenuated Goatpox Virus Strain Expressing The P1-2A-3C Genes Of Type O Foot And Mouth Disease Virus

Foot and mouth disease(FMD), casued by foot and mouth disease virus, is an acute feverish and highly contagious disease.Capripox(CP), casued by sheeppoxvirus or goatpoxvirus, is an acute feverish and contagious disease in sheep or goat.FMD and CP both is the hazardousextremely serious disease, they are classified as group A disease by Office International des Epizooties(OIE)and group first disease in China.Fowl pox viruses vector were one of the earliest study and most successful the virus vectors. FPV possessed the wide range of hosts,the proliferation of high-titer,good stability,large genome capacity and contained a number of non-essential areas.It is easy to carry out genetic engineering operations and more than suitable for insertion of foreign genes, stability of the antigenicity and duration of immunity, can also induce humoral immune and cellular immune at the same time, relatively safe. The experiment made use of our country extensive Goatpoxvirus vaccine Attenuated strains (AV41), to TK gene for the breakthrough point.Useing the virus in the homologous recombination in cells, Construction of Recombinant Attenuated Goatpox Virus Strain Expressing the P1-2A-3C Genes of Type O Foot and Mouth Disease Virus.vector live vaccine was Constructed successfully that can also prevent foot and mouth disease and Capripox virus.(1)The P1-2A gene and the 3C gene of O/China 99 strain foot-and-mouth disease virus were amplified by RT-PCR.The P1-2A gene fragment was inserted into pUC119 vector and the 3C gene was inserted into pMD18-T vector,then the recombinant plasmid pUC119-P1-2A and pMD18-T-3C were constructed.the recombinant plasmid pUC119-P1-2A was digested by Hindâ…¢and BamHI and the recombinant plasmid pMD18-T-3C was digested by BamHI and NheI, then could achieve the ligated of P1-2A gene and 3C gene .Due to the two gene fragments P1-2A,3C have the same BamHI enzyme site ,and constructed the transfer vector pMD18-T-P1-2A-3C.At last the two directions poxvirus p7.5 gene controlled Enhanced Green fluorescent protein gene and P1-2A-3C gene was ligated.The vector was named p-EGFP-N1-P7.5-P1-2A-3C.The positive vector was analyzed by restriction enzyme, PCR and nucleic acid sequencing.The results showed that the gene expresstion cassette pEGFP-N1-P7.5-P1-2A-3C was constructed successfully.the P7.5 promoter controlled P1-2A-3C gene in one direction and in the opposite oriention controlled Enhanced Green fluorescent protein..Then construction of the plasmid has provided an element of gene expression cassette for conveniently construction of related recombinant GPV.Then the segments was ligated into pUC119-TK by the kpnâ… was chosen as the insertion site for the construction of transfer vector .The transfer vector was named pUC119-TK -EGFP-N1- -P7.5-P1-2A-3C.(2)Based on the construction of a recombinant goatpox Virus expressing the P1-2A,3C gene of FMDV and the EGFP gene previously, Transfection in BHK21 cells infected with goatpox virus by lipofectin method, recombinant GPV was got by propagation finally. That P1-2A-3C gene inserted in the genome was identified by RT-PCR, and the expression protein of which was detected by ELISA test.The result indicated that it was obtained successful correct expression of the recombinant protein goatpox virus strain TK + /EGFP + / FMDV P1-2A-3C + / GTPV.This study used the Attenuated sheeppox virus strains for expressed FMDV P1-2A-3C genes genetic engineering of live vector research .Created a new way of thinking.for the prevention of foot-and-mouth and capripox epidemic. Construction of live capripox virus vector, it provide technical and resource support for research of ruminant animal disease live vector genetic engineering vaccine.