蒙古绵羊内源性绵羊肺腺瘤病毒全基因组序列测定与囊膜蛋白的表达分析

Determination of complete genome of endogenous jaagsiekte sheep retrovirus and expression of Env protein in Mongolian sheep

为了克隆内源性绵羊肺腺瘤病毒（en JSRV）全基因组序列,针对env基因分析其结构并构建原核表达质粒,并诱导囊膜蛋白表达。根据Gen Bank中提供的en JSRV DNA序列（AF152615）设计引物,采用PCR方法进行序列扩增并克隆,并对克隆结果进行测序和生物信息学分析。然后将添加了Bam HⅠ和HindⅢ酶切位点的env基因片段克隆至p ET-32a载体构建表达质粒,并将质粒转化至大肠杆菌（E.coli）BL21（DE3）中诱导囊膜蛋白表达,最后利用SDS-PAGE电泳及Western-blot检测、鉴定囊膜蛋白。结果显示,en JSRV序列长7 382 bp,其中env基因片段长1 836 bp,en JSRV与en JSRV-23的同源性为97.3%。PSORTⅡ及DNAStar等软件预测分析en JSRV囊膜蛋白主要定位于细胞质、线粒体内膜等位置,且存在多个抗原表位。在37℃条件下,0.5 mmol/L IPTG诱导转入p ET32-Env的E.coli BL21（DE3）,8 h后可获得分子质量约为89 ku的以包涵体形式存在的囊膜蛋白。本试验测定了en JSRV全基因序列,并成功构建了表达囊膜蛋白的p ET32a-Env载体,为今后制备en JSRV-Env抗体奠定了试验基础,同时为囊膜蛋白生物学功能的深入探讨提供了理论参考。

The objective in this study is to clone the complete genome sequence of endogenous jaagsiekte sheep retrovirus（en JSRV）,and to construct prokaryotic expression plasmid of env gene,then induce the expression of envelope protein.The primers were designed on the based on endogenous jaagsiekte sheep virus DNA sequence（AF152615）available in Gen Bank.The sequence was amplified by PCR. And the sequence was cloned.Then the cloned plasmids were sequenced and bioinformaticly analyszed.The env gene fragment with Bam H I and Hind Ⅲ restriction sites was cloned into p ET32 a vector to construct expression plasmid,and the expression plasmid was transformed into E.coli BL21（DE3）to obtain envelop protein.ally,the envelop protein were detected and identified by SDS-PAGE electrophoresis and Weste rn-blot.The length of en JSRV sequence was 7 382 bp,the env gene fragment was 1 836 bp,and the homology between en JSRV and en JSRV-23 was 97.3%.Using PSORT Ⅱ and DNAStar software it was predicted that en JSRV envolope protein located in the cytoplasm,mitochondrial inner membrane and other locations and the presence of multiple epitopes.The E.coli BL21（DE3）transformed into p ET-32 a-Env was induced by 0.5 mmol/L IPTG at 37 ℃ for 8 h,the envelop protein（about 89 ku）was obtained as a inclusion bodies.In this study,the en JSRV gene sequence was determined and the p ET-32 a-Env expression vector was successfully constructed to express the envelop protein.In conclusion,this study laid the foundation for the preparation of en JSRV-Env antibodies in the future,and provided a theoretical reference for the further exploration of the biological function of envelop protein.