狂犬病毒核蛋白基因的克隆表达及其免疫原性

目的构建狂犬病毒核蛋白(NP)基因的重组杆状病毒表达载体,在昆虫细胞中表达出具有免疫原性的抗原,用于狂犬病的检测及诊断。方法应用RT-PCR方法扩增ERA株狂犬病毒NP基因后克隆到PCR2.1TA载体,转化OneShortTMTOP10感受态细胞构建TA克隆载体,并与pFastBacTM1转座质粒载体分别用KpnΙ和NotΙ双酶切,用T4连接酶连接,构建pFast ERA-NP重组转座质粒,经双酶切、PCR扩增及插入序列方向鉴定后,转化到DH10BacTME.coli感受态细胞,经三抗培养基筛选和PCR扩增鉴定后获得重组Bacmid质粒,将重组Bacmid质粒转染Sf9昆虫细胞制备重组杆状病毒毒液,并继续扩增重组杆状病毒,以第三代毒液感染Sf9昆虫细胞,96h收获细胞,用直接免疫荧光(DFA)、SDS-PAGE和Western blot对表达产物进行检测和分析。结果构建了含有ERA NP基因的重组杆状病毒,并在昆虫细胞中获得表达。结论本研究成功表达出了具有免疫原性的狂犬病毒核蛋白。

Cloning and expression of the nucleoprotein gene of eRA rabies Virus

In order to establish the effective diagnostic reagents,the recombinant baculovirus containing the nucleoprotein(NP) gene of rabies virus were constructed and then the NP was expressed in insect cell.The NP gene was cloned into plasmid PCR2.1TA vector and then ligated into baculovirus donor plasmid pFastBacTM1 after cutting by the restriction enzyme KpnΙ and NotΙ.The pFastBacTM1 were subsequently transferred into the One ShortTMTOP10 competent cells and then into DH10BacTME.coli competent cells,containing the baculovirus shuttle vector(Bacmid) and the helper plasmid to generate a recombinant bacmid.The NP gene was highly expressed in Sf9 insect cell,and the expressed recombinant nucleoprotein obtained from the cell culture was identified by direct immunofluorescence assay.The results of Western blot showed that the protein could be reacted with the sera from the human patient and appeared a specific and predicted band with a molecular weight of 50 kDa.