Construction Of Recombinant Attenuated Goatpox Virus Strain Expressing The P1-2A3C Genes Of Asia 1 Foot And Mouth Disease Virus And Its Nonessential Region Selection

Capripox(CP), caused by sheeppoxvirus or goatpoxvirus, is an acute feverish and contagious disease in sheep or goats. It characterized by fever, generalized papules or nodules, vesicles, internal lesions, and death. Foot and mouth disease (FMD), caused by foot and mouth disease virus, is an acute feverish and highly contagious disease. Both FMD and CP are hazardousextremely serious disease, and be classified as group A disease by Office International des Epizooties(OIE)and group first disease in China.In this study, we construct a recombinant attenuated goat pox virus for coexpression FMDV Asia1 P12A3C based on the TK gene and homologous recombination in the cells. Meanwhile, a replication non-essential region was selected by using random cloning method in order to express multigenes in recombinant goatpox virus.(1) TK gene was selected as the extraneous gene cloning site. The TK gene and its flanking fragment was amplified with a pair of specific primers. The PCR product was 2078bp in length and the ORF of TK gene was 534bp encoding 178 amino acids. The identity analysis showed that AV41 shared 95.5%-100.0% identity rates with the reference strains in levels of nucleotides and amino acids. The results showed that TK gene was highly conservative.(2) To express foreign genes in recombinant virus with high efficacy, construction of a eukaryotic gene expression cassette P7.5-EGFP-P was obtained. Then the P7.5 controlled EGFP and the P12A3C gene of FMDV was constructed. Then the segments was ligated into pUC119TK by the kpn I which was chosen as the insertion site for the construction of transfer vector .The transfer vector was named pTK-P7.5-EGFP-P.(3) The transfer vector PTK-P7.5-EGFP-P transfected BHK-21 cells which infected GTPV AV 41. Specific fluorescence could be seen at 24h after transfection. Screened by Green fluorescent, RT-PCR and antigen capture ELISA, the results showed that P12A and 3C genes were expressed successfully. The tilter detection showed that the tilter of the recombinant was 10^5.5TCID50/0.1mL in BHK-21 cells. (4) The goatpoxvirus cultured in Primary lamb testicles was purified by sucrose density gradient centrifugation. Genomic DNA was extracted and digested by HindIII .After cloned into PUC18 , six fragments were obtained: Puc18-A,Puc18-B,Puc18-C,Puc18-D,Puc18-E and Puc18-F . Choosing single restriction enzyme site to insert P7.5-P7.5-LacZ ,the resulting plasmids PUA1, PUB1, PUC1, PUD1, PUE1, PUF1 were acquired and transfected BHK-21 cells with CPV subsequently. After plaques selection, passages and purifications at presence of X-gal, one cloned CPV fragments was identified as replication nonessential regions.To my knowledge, this study is the first time to use goatpoxvirus as live vector to express FMDV gene in the world. It advanced a new way of prevention ruminant's diseases. The construction of goatpoxvirus live vector will lay the foundation for the genetically engineered live vector vaccine of ruminant diseases.