狂犬病病毒核蛋白在Bac-To-Bac/AcMNPV杆状病毒系统的表达

Expression of nucleoprotein of Rabies virus in Bac-To-Bac/AcMNPV Baculovirus expression system

为真核表达狂犬病病毒的核蛋白,本研究通过RT-PCR克隆狂犬病病毒ERA株核蛋白基因,将其克隆于杆状病毒转移载体pFastBacHTB中,构建重组质粒pFastBacHTB-NP并将其转化DH10Bac细胞,得到重组穿梭质粒reBacmid-NP;通过转染昆虫细胞sf9包装重组杆状病毒。SDS-PAGE、western blot和间接免疫荧光对表达的蛋白进行鉴定和反应原性分析。分别以重组杆状病毒表达的核蛋白、原核表达核蛋白为包被抗原进行ELISA检测。结果表明,在昆虫细胞中表达的狂犬病病毒重组核蛋白能与鼠抗RV核蛋白单克隆抗体和RV阳性血清特异性结合,其相对分子量约为50.5ku。以重组杆状病毒表达的核蛋白为抗原建立的rNP-ELISA的敏感性、特异性、符合率分别为86.36%,89.83%,90.00%,优于大肠杆菌表达的RV核蛋白。说明杆状病毒系统表达的核蛋白是建立RV核蛋白ELISA抗体检测方法的理想抗原。

The nucleoprotein （NP） gene of rabies virus ERA strain was amplified by RT-PCR, cloned into the baculovirus vector pFastBacHTB and transformed into competent cells of E.coli DH10Bac. The recombinant shuttle plasmid was then transfected into sf9 insect cells. Expression of the recombinant NP （rNP） was detected by immunofluorescent assay, SDS-PAGE and western blot. The rNP had a molecular weight of 50.5 ku, and was successfully used as a coating antigen in ELISA and used the anti-NP monoclonal antibody and anti-RV positive serum as primary antibodies. Comparing with the American SYNBIOTICS kit, the rNP-ELISA assay had 90.48 % sensitivity, 89.83 % specificity, and 90.00 % coincidence, and was superior to the E.coh expressed rNP antigen.