H7N9亚型禽流感病毒核蛋白NP原核表达载体的构建与表达

Cloning and prokaryotic expression of nucleoprotein of influenza A virus subtype H7N9

目的构建甲型H7N9禽流感病毒NP基因的原核表达载体,并表达其编码重组蛋白。方法 RT-PCR法扩增H7N9病毒NP基因,克隆至原核表达载体PET28a(+)中,构建表达载体PET28a(+)-NP质粒;经PCR、双酶切、测序鉴定后,将PET28a(+)-NP质粒转化表达菌BL21(DE3),用IPTG诱导表达NP重组蛋白。应用western blot法鉴定NP蛋白的表达。结果成功构建NP基因的原核表达载体,在大肠杆菌中表达出分子量为56kD的NP重组蛋白。结论实现了禽流感病毒H7N9NP重组蛋白在原核系统中的高效表达,为禽流感诊断试剂及单抗的研发工作奠定了基础。

Objective To construct the prokaryotic expression vector of nucleoprotein （NP）of influenza A virus subtype H7N9 and express the recombinant protein in E. Coll. Methods NP gene was amplified by RT-PCR and cloned into PET28a （＋） vector to construct PET28a（＋）-NP plasmid ,which was validated by PCR, double digestion and sequencing analysis and subjected to transformation into E. Coli BL21（DE3）. The expression of recombinant NP was induced by IPTG, and confirmed by Western Blot. Results The prokaryotic expression plasmid PET28a（＋）-NP was successfully constructed. A 56kD NP re-combinant protein was expressed in E. Coll. Conclusion NP recombinant protein of influenza A virus subtype HTN9 was ex-pressed successfully with high efficiency in prokaryotic system, which should lay a foundation for research and development of novel diagnostic kit and monoclonal antibody of AIV.