Ⅰ型鸭肝炎病毒vp3基因的截短表达及鉴定

PROKARYOTIC EXPRESSION AND CHARACTERIZATION OF TRUNCATED VP3 OF DUCK HEPATITIS VIRUS TYPE 1

根据I型鸭肝炎病毒(Duck hepatitis virus type 1,DHV-1)A66株vp3基因序列,设计了一对扩增vp3截短基因(-468bp)的特异性引物,将vp3截短基因与表达载体pET-32a(+)连接,构建了vp3截短基因的重组表达质粒pET-VP468。pET-VP468转入表达菌E.Coli Rosetta-gami(DE3)pLysS中,使用IPTG诱导表达融合蛋白Trx-VP468。Trx-VP468经过Ni-NTA亲和层析柱和DEAE-Sepharose阴离子柱纯化,SDS-PAGE电泳结果表明,pET-VP468在大肠杆菌中表达了一个相对分子量约为36kDa的蛋白,免疫印迹试验表明Trx-VP468得到了正确表达。该结果为VP3蛋白的免疫学功能研究奠定了基础。

In this study, a pair of primers was designed to amplify truncated vp3 gene （1-468 bp） of vp3 of Duck hepatitis virus 1 （DHV- l） A66 strain. The truncated vp3 gene was cloned into vector pET-32a（~） to construct recombinant plasmid pET-VP468, which was transformed into E. coli Rosetta-gami（DE3） pLysS. The fusion protein Trx-VP468 was expressed with induction of IPTG and purified with Ni-NTA affinity chromatography and DEAE-Sepharose anion exchange column. The presence of Trx-VP468 with molecular mass of approx. 36 kDa was determined in SDS-PAGE and Western blotting using DHV-1 specific antibody. These results lay a foundation for further studies of immunological function of VP3 of DHV- 1,