| In order to mapping B cell sequencial epitopes and study the antigenicity of viral structural protein of goose parvovirus(GPV), we put the vp gene into 3 fragments with overlapped vp2-vp3 and the middle of vp3. They are 662bp at 5'-end of vp1, 969bp at 5'-end of vp2, and 864bp at 3'-end of vp3. We planed to express these fragments in prokaryotic expression system to study the reactivity of these fusion protein, and tentatively named these expresssed fusion protein as F1, F2, F3, respectively. In previous study , F2 had been expressed and purified. The antiserum against F2 had been prepared by immunizing rabbit, and reactivity of the antiserum had been testified by western blot.In this study, a 662bp fragment at 5'- end of vp\ gene and a 864bp fragment at 3'- end of vp gene of GPV isolate HG5/82 was amplified by PCR. We inserted the 662bp fragment at 5'- end of vp\ gene between BarnH I and Hind III site of prokaryotic expression vector pPROEX?HTb and put the 864bp fragment at 3'- end of vp gene into the same restriction endonuclease site of prokaryotic expression vector pET30a, respectively. After identification of recombinant plasmids by sequencing, the recombinant pPROEX?HTb was transformed into Escherichia coli DH5a and the recombinant pET30a was transformed into Escherichia coli BL21. SDS-PAGE results indicated that transformed Escherichia coli DH5a expressed a 32kDa product and the transformed Escherichia coli BL21 expressed a 34kDa product after inducing with 0.6mmol/L IPTG. Densitometric scanning showed the expressed F1 accounted for as much as 22.8% of total bacterial protein of DH5 α, it indicated that the target gene can be expressed in this prokaryotic expression system in high efficiency. We named the 32kDa product as F1 and the 34kDa product as F3, respectively. As the fusion proteins have 6 x His tag, F1 and F3 were purified with a high purity by ProBond? Resin. By immunizing rabbits the antiserum against F1 and F3 were obtained. Western blot analysis indicated that both of these two kinds of antiserum could recognize the GPV HG5/82 stain's protein, but they identified different viral structural proteins. Antiserum against F1 recognised VP1 and VP2, and antiserum against F1 identified VP1, VP2 and VP3 of GPV HG5/82. The three kinds of structural proteins have identical carboxyl terminal, so the epitope on the VP3 also exists on the VP1 and VP2, while the epitopes on the VP2 are also present on the VP1. Accoding the theory and results of western blot, B cell linear epitopes were mapped in amino acids at 145-198 and amino acids at 220-733 of VP1 sequence of GPV HG5/82 strain. These is no B cell linear epitopes at 198-220 amino acids of VP1 sequence of GPVHG5/82 strain.These data provide more useful information for antigenic epitope prediction of .GPV capsid protein. It established a foundation and prepared experimental material for the future research on the bioactivity of GPV, and also provided a basis for developing GPV recombiant diagnosis reagent and genetic engineering vaccine.
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