Construction Of Recombinant Capri Pox Virus Expressing VP2 & VP5 Protein Of BTV And Immunogenicity Evaluation

Bluetongue (BT) is a hemorrhagic disease of ruminants caused by bluetongue virus (BTV). BT is just one of multiple species diseases by the Office International des Epizooties (OIE). For a long time,only two vaccine types for bluetongue are currently commercially available:live attenuated(LA) vaccines and inactivated virus vaccines。 Howerer, they have two common problems.First they are unable to make distinction between infected and vaccinated animals (DIVA). Secondly,the virus exists as 25 distinct serotypes,and protection afforded by vaccines is serotype specific.To address the problems mentioned above,we constructed a recombinant Capri pox virus expressing VP2,VP5 protein of BTV. At the same time,the recombinant Capri pox virus was tested by using PCR identification.And the immunogencity of the recombinant Capri pox virus was tested by animal experiment.This study has established foundation for the production of the new recombinant Capri pox virus vaccine.The new vaccine can overcome the defaut of current vaccine,so the study is of great productive practice significance.Expeiment 1. Construction of recombinant plasmid PTK-BTV-VP5-VP2According to Published VP5,VP2 gene sequence of BTV-12,Primers were designed and synthesized.The complete cDNA clone of VP5 and VP2 gene were amplified by RT-PCR.Next,the two genes were cloned into pBluescript vector and sequenced. Then the fragments of VP5,VP2 gene were subsequently subcloned into the restriction Enzyme cutting sites of vector PTK-GPT-IRES-EGFP:Sall, HindⅢ, XbaI,PstI.naly,we get the recombinant plasmid PTK-BTV-VP5-VP2.Experiment 2 Construction and identification of recombinant Capri pox virus expressing bluetongue virus VP2, VP5 proteinFirst,Capri pox virus genome and recombinant plasmid were respectively digested with restriction endonuclease ApaI.BglI.Then the digested genome and recombinant plasmid were purified.Next,vaccine strain of Capri pox virus (CPV) cultivated and widely used in domestic animals was employed as the Parents virus,and by homologous recombination between the TK gene of Capri pox virus and recombinant plasmid,we constructed recombinant Capri pox virus expressing VP2,VP5 protein of BTV by lipofectin transfection. Finally, inserts of VP2 and VP5 genes into the CPV genomes were confirmed by PCR.Experiment 3 The immunogenicity evaluation of recombinant Capri pox virusFive BALB/C mice were immunized with 106 PFU rCPV-BTV-VP5-VP2 through abdominal cavity injection,and serum was collected after immunization,and was analyzed for the neutralization antibodies.The results show that 21 days after immunization,the neutralization antibodies of CPV and BTV were all positive.In conclusion, this study established the foundation for the development of the recombinant virus against BTV and Capri pox virus at the same time.