无血凝性兔出血症病毒VP60蛋白分子特性的分析及其在大肠杆菌内的高效表达

Analysis of molecular characteristics of VP60 protein of a strain of nonhaemagglutinating RHDV and high-level expression of the VP60 protein in Escherichia coli

分离出1株在4、25、37℃条件下均不能凝集人"O"型红细胞的兔出血症病毒(RHDV),命名为SQ-1株;采用RT-PCR扩增获得病毒主要结构蛋白VP60蛋白基因,并对目的基因进行测序及分析;将目的基因定向克隆入原核表达载体pET-30a的多克隆位点,在大肠杆菌Rosetta(DE3)中诱导表达,纯化重组蛋白,用Western-blot检测其反应原性。测序与同源性分析结果显示,该毒株属于RHDV抗原遗传变异株(RHDVa),所扩增的VP60基因的ORF为1 740bp,编码579个氨基酸残基,与世界上其他RHDV毒株的核苷酸序列的同源性为89.9%～98.0%,与已报道的无血凝特性的病毒株whn-1株及低血凝性的Rainham株的核苷酸序列的同源性分别为95.2%及90.8%。重组质粒经IPTG诱导后目的基因获得表达,重组蛋白与预期大小一致,分子质量约为60ku,主要以包涵体形式存在。Western-blot检测证实,纯化蛋白能够与RHDV阳性血清发生良好的杂交反应,说明该重组蛋白具有良好的反应原性。

A variant SQ-1 strain of rabbit hemorrhagic disease virus lacking haemagglutinating activity at 4 ℃,25 ℃ and 37 ℃,respectively,was isolated.The capsid protein VP60 gene was cloned by RT-PCR and sequenced.The VP60 gene was then sub-cloned into the multiple cloning sites of pET-30a,the positive recombinant plasmids were screened through restriction endonuclease digestion,and transformed into Rosetta（DE3） competent cells for protein expression.The secretory proteins expressed in Escherichia coli were purified to detect the reactogenicity with the antiserum.In results,the VP60 gene was 1 740 nt in length,which encoded 579 amino acids.Alignment results indicated that the SQ-1 strain belonged to the RHDVa subtype.The homology of VP60 gene with that of other 44 strains were from 89.9% to 98.0%,and only 95.2% and 90.8% respectively with the non-haemagglutinating whn-1 strain and the low-haemagglutinating Rainham strain.The VP60 protein was efficiently expressed in inclusion bodies and soluble proteins,and the molecular weight of the recombinant protein was 64ku according to the SDS-PAGE.The Western-blot analysis indicated that the soluble proteins could hybridize with antiserum specifically.Therefore,the VP60protein can not only be efficiently expressed in Rosetta（DE3） cells,but also possess reactionogenicity with antiserum.