Costruction And Identification Of Recombinant Goatpox Virus Expressing PPRV-H Gene

Peste des petits ruminants(PPR) is an acute and highly contagious viral disease of small ruminants (goats and sheep in particular). It is caused by Peste des petits ruminants virus (PPRV). Has a very high morbidity and mortality. At present, all countries in the world use live vaccine to prevent PPR, the attenuated vaccine play an important role in preventing the disease, but attenuated vaccine can not distinguish between the vaccine strains and wild type infection on serology, that influence the epidemiological and other related studies about the desease. In order to solve the shortcomings of traditional attenuated vaccines, this study constructed PPRV H gene recombination goatpox virus, which supplied a platform for developing genetic engineering vaccine of PPR.1. Identification of back to back prometer 11 and 7.5 fuctionA GPV transfer vector PtkPP, containing the fragments of the non-essentil was constructed TK gene and the back to back promoter of goatpox virus vaccine strain. EGFP gene was inserted into downstream of the promoter 11 or promoter 7.5 to generate two individual transfer vector PtkPegfpP and PtkPPegfp. The two vectors were transfected into LT cells. The consistence expression of EGFP gene in the transfected cells demonstrated that the foreign gene could be expressed downstream either side of the back to back promoter.2. Costruction and identification of recombinant goatpox virus expressing gpt and EGFP geneFusion gene gpt and EGFP were then inserted into PtkPP downstream the late promoter 11, resulted in the generation of transfer plasmid PtkPgpt-egfpP. The recombinant virus was generated by transfection of PtkPgpt-egfpP into LT cells that had been infected with goatpox virus vaccine strain. This study supplied a technique platform for developing goatpox virus live vector vaccine.3. Costruction and identification of recombinant goatpox virus expressing PPRV-H geneBase on the H gene sequences of Peste des petits ruminants virus, design of Peste des petits ruminants virus H gene primers by PCR cloning of the H gene. H gene was inserted into the vector PtkPgpt-egfpP downstream of the promoter 7.5 to generate a recombinant plasmid PtkPgpt-egfpPpprv-H, through restriction enzyme digestion, The transfer vector was transfected into LT cells that had been infected with goatpox virus vaccine strain, through homologous recombination generation of recombinant ruminant ruminants virus H gene goatpox virus, after purifying and the PCR identification, demonstrated that the PPRV H gene had been insertion to goat pox virus.