Cloning Of VP7 Gene From A Group Porcine Rotavirus Of Strain OSU And Its Expression In Baculovirus

Porcine Rotavirus (PRV) belongs to the family reovindae. It is one of the major pathogensthat cause life threatening diarrhea in piglets. This kind of diadrrhea was the easiest infected byyoung animal. Its infectious ratio is over 80%, and its mortality in pigS 1 to 4 weeks is 20%.Rotavirus diarrhea's typically clinical manifestation is seriously diarrhea. Partial infected pigletSwere deaded due to severe Ioss of water, imbalance of acid and alkaIi and secondary infection.Accurate diagnosis and effectiv Veccination are main means for the prevention of Porcinediarrha ReSearh showed tha the outer caPsid glycoprotein VP7 of PRV particle is majorprotective anigen. It can make body generating the antibody to neutralize rotavirus andeldriinate infechon. So gaining VP7 gene and itS exPressing product is key to the preparaion ofthe new tyPe vaccine and sPeCific diagnOsis anigen.In this StLIdy, G5 serotyPe A gIDuP POreine rotavha stanha strain OSU was propagatedon MA-l04 monolayer cell. A Pair of prirners was designed to amplify VP7 gene by RT-PCRaccording to the published Sequence of PRVs VP7 gene with Oligo4. l software. The product ofPCR named VP7 is aPprotriate l.0kb in length. The VP7 gene was cloned into the vectorPMD-l8T and the recombinan was named PMD-l8T-VP7. Identifications of restrictionempme, PCR and sequencing inditaled tha the VP7 gene has been cloned successfullyPMD-18T-VP7 was analpe in comParison with the other fOur G serotyPe PRV strain wngenes, Gene sequence comparison indibed that OSU shad 99.4%, 77.9%, 71 .7%, 75.3% and87.7% identities with OSU-1 (G5), ICB2l85 (G9), P343 (G10), Gottiffied (G4) and CI34 (G2)resPectively The deduced arnino acid Sequences were 99.4%, 83. l %, 87. l%, 84.7% and 96.3%cormsPOndingly The results affinned that VP7 gene of PRV strain OSU was not conservative.mp was insertd in Bgl IIand HindIII multiple cloning sites of the vector PblueBAChisCusing Another pair ofpriIners designed accoIding to the sequence of VP7. PblueBAChisC-VP7was identified and analped by compnding restriction endonuclease, PCR and nested PCRon the basis of the genetic siteS of PblueBAChisC, which was idenhfied as VP7 gene of PRVhafied PblueBAChisC-VP7 and BarmidN-blue DNA were cbeinfected insect cell sroand harvested Mmbinan beculovirus 5d later Identification with restriction empme andPCR showed VP7 gene has been arembwt comehy with baculovirus DNA and namdA-l. A-l was infected on monolayer sffi ther the third plaque purification to reproduce inquantity and named it A-2. After A-2 was infeCtal on sffi monolayer cell 4d harvested the CPEcell. The analysis resultS with SDS-PAGE and Dot-ELISA showed that PRV VP7 gene was-- o --Abstractexpressed in baculovirus.the eukaryotic expression of PRV VP7 gene provided not only the important basis for further research of the structure and function of the PRV VP7 protein and the preparation of vaccine, but an new viral diagnosis method.Postgraduate: XU Hongjun Major: Preventive Veterinary Science Supervisor: Prof. WANG Junwei Prof. LI Yijing...