A组人轮状病毒NSP3基因的克隆及其在大肠杆菌中的表达

目的:获得轮状病毒NSP3基因的表达产物及其抗血清。方法:将TB-Chen株轮状病毒NSP3基因插入质粒pETL,构建重组表达质粒pET-NSP3,并转化大肠杆菌BL21(DE3)表达重组蛋白NSP3;用凝胶分离回收的方法纯化该蛋白,免疫豚鼠制备该蛋白的抗血清。结果:构建了重组表达质粒pET-NSP3,并在大肠杆菌中高效表达了重组蛋白NSP3,目的蛋白表达量占菌体总蛋白量的28.6%;有效地纯化了目的蛋白并制备了该蛋白的抗血清,Western印迹表明该抗血清能与重组蛋白NSP3发生特异性免疫反应。结论:通过质粒pETL能高效表达轮状病毒NSP3蛋白,该重组蛋白具有较好的免疫反应性,为进一步研究其结构、功能及免疫学性质奠定了基础。

Cloning and Expression of NSP3 Gene from a Group A Human Rotavirus in E.coli

Objective:To obtain recombinant protein NSP3 of rotavirus and its antiserum.Method:The gene of rotavirus NSP3 cloned from rotavirus strain TB-Chen was inserted into the expression vector pETL,and the recombinant plasmid pET-NSP3 was constructed.The recombinant NSP3 was expressed in E.coli BL21(DE3)cells and recovered after SDS-PAGE.The purified recombinant NSP3 was inoculated into guinea pigs to produce antiserum.Results:The recombinant NSP3 was highly expressed(28.6% of total protein)in E.coli BL21(DE3)cells.It was shown by Western blot that the recombinant NSP3 can react with its antiserum specifically.Conclusion:The recombinant NSP3 can be highly expressed by plasmid pETL and has remarkable immunoreactivity.These results lay the foundation for further study on the structure,function and immunological properties of the NSP3.