A组人轮状病毒NSP3基因的克隆及其在大肠杆菌中的表达

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

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。

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 SDSPAGE. 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.