含A(H1N1)流感病毒NP基因病毒样颗粒的构建和表达

Construction and expression of virus-like particles containing NP gene of influenza virus A (H1N1)

目的:构建并表达含有A(H1N1)流感病毒NP基因片段并可抗RNase降解的病毒样颗粒,作为RT-PCR检测的阳性对照。方法:设计带有NcoⅠ、BamHⅠ酶切位点的引物克隆MS2噬菌体的装配蛋白和包膜蛋白基因,化学合成含BamHⅠ和HindⅢ酶切位点的A(H1N1)流感病毒NP基因片段,将这些基因连接到质粒载体pET-28b(+)上,转化到E.coliBL21(DE3)感受态细胞中进行表达,表达产物进行纯化,然后进行RNase攻击试验和稳定性试验。结果:获得含NP基因片段的病毒样颗粒,可抵抗RNase降解,并能在37℃稳定保存30d。结论:该病毒颗粒稳定、安全、可靠,可作为A(H1N1)流感病毒RT-PCR检测、定量分析的有效阳性参考品。

Objective To construct RNase-resistant virus-like particles which can express NP gene of influenza virus A（H1N1）. It can be used as positive control in RT-PCR. Methods The 7 kb assembly protein and envelope protein gene of MS2 phage was cloned by RT-PCR through primers with Nco Ⅰ and Hind Ⅲ recognize sites, and the fragment of influenza NP gene which contained BamHⅠ and HindⅢ recognize sites was synthesized according to its′ sequence from GenBank data. After digestion with restriction enzymes, these DNA fragments were joined into the vector pET-28b（＋）. The recombinant plasmids were transformated into E.coli BL21 （DE3） competent cells and the virus-like particles were obtained after purification. RNase attacking test and stability test were carried out to observe the stability of the particles. Results The RNase-resistant virus-like particles which can express influenza NP gene were successfully constructed and can be stored stable for 30 days at 37℃. Conclusion The virus-like particles with high safety and stability can be used for positive control in influenza clinical diagnosis.