Isolation、Identification And Complete Genome Sequencing Of Goose Parvovirus And Expression Of Its Structural Proteins

Goose parvovirus (GPV), the pathogen of gosling Derzsy’s disease, has strong pathogenicyto goslings and Muscovy duck with its’ fast spread sped and high mortality. Survival goslingsand Muscovy duck in the disease grow slowly which also get huge economic losses to the goosebreeding industry. The virus has5proteins, three structural proteins VP1, VP2and VP3and twonon structural proteins NS1and NS2.The genes are at length of2199nt,1764nt,1605nt,1884nt and1356nt. The proteins at weight of88ku,65Ku,60Ku and77Ku,50ku. Thegenome of GPV divided into head and "bubble zone", has two terminal ITRs and each is atlength of444nt.Goose parvovirus has existed for a long time in China. China has not carried out systemicmolecular epidemiological survey on the GPV, and even a few isolate stran hasobtained full gene sequence since GPV aseparated by Fang Dingyi in1961. With the rapiddevelopment of goose industry in Jilin province in recent years, gosling plague is popular insome area.Between2012and2013, Changchun, Siping, Shulan Lishu, Baicheng in Jilinoccurred suspected goose plague. In order to diagnose and to know the GPV genotypes,thisresearch collected liver and intestinal contentswere of dead geese from the fivearea,homogenized, centrifuged and inoculated the supernatant to12-15day old gooseembryo. Allantoic fluid was observed under electron microscopy, then identificated by PCR.The sequencing result of PCR products identifies the five strains of GPV.According to the structural characteristics of GPV,5pairs of primers covering the entire GPVgene was designed in this study. GPV DNA was preparated from the goose embryo allantoicfluid containing GPV Changchun strain and Lishu strain by traditional DNA extraction method.The fragments were amplified by PCR and cloned into pMD-18T.The recombinant plasmidswere sequenced.. The genome of GPV CH stran and LS stran both are at length of5050nt.Thegenes of three structural proteins VP1, VP2and VP3are at length of2199nt,1764nt and1605nt and the genes of two non structural proteins NS1and NS2are1884nt and1356nt.However，the length of CH stran and LS stran’s IRT are noly416nt. According to the homology analysisof the relationship, CH strain, LS strain and SHFX1201come from the same stran.VP2and VP3genes, respectively at length of1764bp and1605bp, were amplified fromGPV SP strain pGEX-GPV-VP1plasmid stored in our lab. The PCR products were purifiedand cloned into pMD-18T, then the recombinant plasmid named pMD-18T-VP2and pMD-18T-VP3was transformed into E.coli DH5α. VP2and VP3genes were got from pMD-18T-VP2and pMD-18T-VP3enzyme digesting by the BamH I and Xho I. pGEX-4T-1was cutby the two same enzymes. VP2and VP3genes were cloned into vector pGEX-4T-1.Therecombinant plasmids,named pGEX-GPV-VP2and pGEX-GPV-VP3,were identified byPCR, double enzyme digestion and sequencing.The pGEX-GPV-VP1and the two recombinantplasmids were transformed into E.coli BL21and the three proteins were expressed, thendetected by protein immunoblotting.The expressed VP proteins will be used to preparate formonoclonal antibodies and ELISA detection kits.