猪流感H1N1亚型NP基因杆状病毒表达载体构建及其在昆虫细胞中的表达

目的获得研发A型猪流感病毒ELISA检测试剂盒和制备NP蛋白单克隆抗体的抗原物质。方法根据已报道的猪流感病毒H1N1亚型NP基因序列(GenBank登录号:GQ422386)人工合成NP基因,通过XbaⅠ和EcoRⅠ特异性酶切,将NP基因克隆于昆虫杆状病毒表达载体pFastBacHTB,经PCR、酶切、测序鉴定后,获得携带NP基因的重组质粒pFast-BacHTB-NP。该重组质粒转化含有杆状病毒穿梭载体的DH10Bac感受态细胞,经抗生素、PCR筛选,获得转座的杆粒Bac-mid-NP。在脂质体介导下转染sf9昆虫细胞,获得重组杆状病毒,再感染细胞,收获目的蛋白。结果通过SDS-PAGE和Westernblotting分析表明该蛋白得到表达,且具有良好的生物活性,大小约为57KD。结论实验结果表明已成功构建了携带NP基因的重组质粒pFastBacHTB-NP和转座的杆粒Bacmid-NP,转染后在sf9昆虫细胞上成功的表达。

Construction of baculovirus transfer vector NP gene of swine influenza virus (H1N1) and its expression in insect cells

In order to gain antigenic properties that was used to construct an ELISA kit to test SIV antibody and to prepare monoclonal antibody,NP gene of A/Swine/Influenza Virus/2009（H1N1）was syntheticed according to the reported SIV virus NP gene（GenBank accession number：GQ422386）.The NP gene fragment was cloned into the vector pFastBacHTB.The recombinant plasmid pFastBacHTB-NP was constructed and identified by PCR and enzyme digestion and sequenced.Then the plasmid pFastHTB-NP was transformed into DH10Bac complement cells and identified by antibiotics and PCR.It is showed the baculovirus expression vector to NP gene was constructed.On this basis,transposition bacmid DNA was extracted to transfect sf9 insect cells.After 96 hours,baculovirus was harvested.Then the SDS-PAGE and western blotting showed that the NP protein was expressed in insect cells.It has a good biological activity and is approximate 57kDa.It is a base to construct an ELISA kit to test SIV antibody and to prepare monoclonal antibody.The result showed that the recombinant plasmid pFastBacHTB-NP and baculovirus expression vector to NP gene were constructed,and the NP gene was expressed in insect cells.