犬圆环病毒Cap蛋白的生物信息学分析及原核表达

犬圆环病毒(CanineCV)是近年被发现的圆环病毒属新成员,获取CaineCV Cap蛋白,为研究Cap蛋白的功能以及抗原表位奠定了基础。本研究以CaineCV GX2017株基因组作为模板,使用PCR方法扩增出Cap蛋白的基因序列,对其进行生物信息学分析,并克隆至pET-28a原,进行原核表达。结果表明:GX2017的N端2~26位氨基酸存在一个核定位信号,同时含有3个B细胞表位(aa7~aa14;aa150~aa174;aa233~aa253);第134位氨基酸为N-糖基化位点,第169和第238位氨基酸为O-型糖基化位点;进化分析表明,本研究的CanineCV Cap蛋白基因序列与欧美株同源性较低,且处在不同的分支;SDS-PAGE结果显示,重组Cap蛋白在E.coli BL21(DE3)中不能正确表达,切除了NLS的d(1-26)Cap蛋白能在E.coli BL21(DE3)中大量表达;Western blot 分析表明,该重组蛋白能与Anti-His 标签抗体发生特异性反应。本研究成功构建了pET-d(1-26)Cap重组原核表达质粒,并在大肠杆菌中获得高水平表达,为进一步制备Cap蛋白的抗体奠定了基础。

BIOINFORMATICS ANALYSIS AND PROKARYOTIC EXPRESSION OF CANINE CIRCOVIRUS CAP PROTEIN

Canine circovirus(CanineCV)is a newly discovered circovirus in recent years.To further investigate function and epitopes of Cap protein,polyclonal antibodies to Cap protein were prepared in the present study.The gene sequence of Cap protein was amplified by using the CaCV GX2017 strain genome as template,then the gene were cloned into pET-28a vector for prokaryotic expression.Bioinformatics analysis of Cap protein showed that the N-terminal amino acids 2 to 26 acted as nuclear localization signals and contained three B-cell epitopes(aa7-aa14,aa150-aa174 and aa233-aa253.The amino acid at position 134 was N-glycosylation site and the amino acids at positions 169 and 238 were O-glycosylation site.Phylogenetic analysis showed that GX2017 had low homology with the European and American reference strains and was located in different branches.SDS-PAGE analysis revealed that the recombinant Cap protein was not correctly expressed in E.coli BL21(DE3)cell.However,the d(1-26)Cap protein,which excised the nuclear localization signal,could be expressed in E.coli BL21(DE3)cell.Additionally,the recombinant Cap protein specifi cally reacted with anti-His tag antibody in Western blot.The results from the present study laid the foundation for the preparation of Cap antibodies and subunit vaccines.