马西尼罗热病毒E蛋白部分基因在大肠杆菌中的表达

E.coli Expression of Equine West Nile Virus Partly E Protein

参考GenBank发表的西尼罗病毒(west nile virus,WNV)的E蛋白基因序列,自行设计合成一对引物,利用RT-PCR扩增出了WMV E基因318bp片段,将其克隆入pMD18-T-Vector载体中,阳性克隆命名pMD-E,并进行序列分析。进一步亚克隆入表达载体pET-32a(+)。重组质粒pET32a-E转化BL21(DE3)感受态细胞中表达,表达产物经SDS-PAGE可检测到分子量约为32kD的目的蛋白带,经薄层扫描分析,目的蛋白占菌体总蛋白的33．1%。表达产物纯化后,Wester-blotting分析证明表达产物能被WNV的阳性血清所识别,为下一步建立以表达产物为包被抗原建立检测马的WNV的ELISA方法打下了基础。

According to the GenBank published sequence of equine west nile virus （WNV） E protein gene, a pair of primer was designed in ordex to amplify equine WNV partly E gene by RT-PCR. The fragment was 318bp in length and was cloned into pMD18-T-Vector. The positive clone was named pMD-E and was sequenced. Then it was sub-cloned into pET-32a （ ＋ ） . The recombinant pET32a-E plasmid, which includes the gene fragment of the equine WNV E protein, was transformed into E. coli BL21, and expressed about 33. 1% . The expressed product was about 32kD molecular weight by SDS-PAGE. The purified product could be recognized by positive serum of WNV by Wester-blotting. It was laid a foundation for developing an ELISA to detect equine WNV used the purified production as coated antigen.