Cloning And Prokaryotic Expression Of Goose Parvovirus SRW Strain VP2 Gene

Goose plague (GP) was firstly found at Yang Zhou China by Fang Ding-Yi in 1956. The virus was isolated from infected gosling ultilizing embryonated goose eggs in 1961. Hungarian scholar Derzsy denominated this virus as Goose parvovirus (GPV). GPV is member of Parvovirus in Parvoviridae. GPV genome has 5106bp and two ORFs, the left ORF(LORF) encodes non-structral protein NS1 and NS2, the right ORF(RORF) encodes structral protein VP, including three kinds of protein VP1, VP2 and VP3. Some research indicated that VP2, the protective antigen, could induce gosling neutralizing antibody against GPV infection. In order to prepare the recomninated GPV vaccine, VP2 gene from strain SRW of GPV was cloned and expressed in the E.coli.Refered to the nuclease sequence of the VP2 gene of GPV B strain reported in GenBank (register number U25749), a pair of primer which has BamH I and Sal I restriction enzyme site was designed to amplify the VP2 gene from GPV SRW strain genome by PCR. The PCR products of the VP2 gene were recombinated into pUCm-T vetor by restriction enzyme terminerts. The results of sequence of the recombinats have 1758 base pairs of VP2 gene and 98.46% homology with GPV B strain VP2 gene. The deduced amino acid sequence homology between VP2 gene of GPV SRW strain and 9 other GPV strains' VP2 gene reached to 94.18%. It was not significance in the antigenicity index and hydrophilicity site distribution beteen the protein deduced from the GPV VP2 recombinats from SRW strain and GPV B strain.The pE-VP2 recombinats was sub-cloned into the expression vector pET-21a and identified by digestion of BamH I and Sal I restriction enzymes, it showed the ORF of VP2 gene and been transformed into E.clio BL21(DE3). The recombinant bacteria E.coli BL21(GVP2) was selected and cultured to expression inducing by 1 micromol of IPTG at 30°C. The cultures was treated by supersonic wave and centrifuged. The pellets of the bacteria were resuspentioned and denaturated using 6 Mol of guanidine hydrochloride buffer. The esprssioned VP2 protien was purified by N²⁺ affinity column and has 74 kiloDalton of molecular weight by SDS-PAGE. The specific band was fonded in the Western-Blot of the esprssioned VP2 protien with the antibody against GPV SRW strain. The results indicated that the E.coli BL21(GVP2) expresssed the VP2 protien of GPV SRW strain and have the value in developing GPV genetically engineering vaccine and ELISA kit to detect the tire of the serum in gosling aginst the GPV infection.