Cloning, Sequence Analysis And Prokaryotic Expression Of Goose Parvorirus VP2 Gene

Goose parvovirus is the causitive agent of Goose plague . which is also named as Derzsy's disease. It is one of highly fatal diseases of goslings and Muscovy ducklings. Goslings and Muscovy ducklings of 4 to 20-day-old are the most susceptible to the virus. The mortality is up to 70/o-100/o. At present,there is no effective medicaments and rapid diagnosis method available. The main prevention methods of the disease depend on classical vaccination and diagnosis that is based mainly on serological and virology methods,but they are all time consuming and laborious. Because of GPV highly contagious,new genetic engineering vaccine and a rapid,special and simple diagnosis method is required to prevent and detect the infection.VP2 is one of viral structural proteins on the surface of provirus. Its neutralizing antigenicity has been proved. In this study. According to the reported complete nucleotide sequence of goose parvovirus in GenBank. with Oligo4.1. a pair of primers were designed and synthesized. By polymerase chain reaction (PCR) the full length of VP2 gene of domestic isolate GPV HI was amplified and sequenced. The result revealed that the full nucleotides sequence of VP2 gene from GPV-H1 isolate was 1764bp and encoded 587 amino acids,which is the same as that of published B strain in GenBank. After comparing the complete nucleotide sequences of VP2 gene between HI isolate and B strain,we discovered that there are 25 nucleotides difference. Among 25nt only lOnt lead to amino acid variation in which 9nt because the first nucleotide of the code mutated. Comparing with GPV-B,GPV-SHM. GPV-YG,MDPV-FRANCE and MDPV-FM isolates show that nucleotide sequence homology are 98.6%. 96.2%. 93.2%,80.3%. 80.0% respectively and deduced amino acid homology are 98.3%,97.5%,96.1%. 88.2%,87.4% separately. According to the phylogenetic tree analysis,they are divided into two groups:GPV-like and MDPV-like. Among them HI isolate is the most relative to B strain. The most variable regions between HI and B strain reside in VP2 and VP3 overlap region (319-470),which are supposedly exposed to the surface of the capsid. It is possible due to the selective pressure from host immune system. The largest divergence between the GPV-H1 and MDPV capsid polypeptides located between the start cordons of VP2 and VP3. It may be related to their host region.By recombinant DNA techniques,the VP2 gene of GPV HI strain was fused in frame with 6His gene of prokaryotic expression vector PPROEX-HTb. The recombinant expressionplasmid of goose parvovirus VP2 gene pPROEX-HTb-VP2 was transformed into E.coli DH5a and induced with IPTG. SDS-PAGE analysis showed an induced expression product band about 72ku,which correspond to the sizes of VP2,reported in the literature. The amount of the goal protein was evaluated by densitometric scanning. It indicated that the product of the VP2 gene was 14% of total bacterial protein of DH5a. Then the product was purified by metal (Ni2-) chelation affinity chromatography and tested by Western-blot. It revealed that 6His-VP2 fusion proteins have a positive reaction with antibody,suggesting that the 6His-tag did not affect the activity of VP2 protein.This study provides the basis evidence for the research of nucleotide sequence evolution relationship between domestic and exterior countries. It also establishes foundation for further research about developing GPV molecular diagnostic reagent and genetic engineering vaccine.Postgraduate:Yun Xia HeMajor:Preventive veterinary scienceAdvisor:Prof. Li Qun Wang. Prof. Jun Wei Wang...