SARS冠状病毒N蛋白的原核表达及活性测定

The expression and activity determination of N protein of Sars-CoV

目的克隆编码严重急性呼吸综合征冠状病毒(SARSCoV)N蛋白的DNA,构建原核表达质粒pGEX2T/N,并诱导表达。方法采用RTPCR方法从病毒培养液中获得N基因片段。将N蛋白基因序列定向插入原核表达载体pGEX2T中,在大肠杆菌中表达融合蛋白。用表达产物与抗SARSCoV抗体阳性血清做Westernblot。结果获得N基因片段;GSTN融合蛋白以可溶形式表达;Westernblot检测表明,其与抗SARSCoV抗体阳性血清的反应呈阳性。结论成功地构建原核表达质粒pGEX2T/N,并表达GSTN融合蛋白,为下一步的研究奠定了基础。

Objective To construct an expression vector pGEX-2T/N, and to express the fusion protein consisting of N protein of SARS-CoV in E. coli. Methods The N region gene of SARS-CoV was obtained by RT-PCR. The expression vector PC-EX-2T/N was constructed by DNA recombination. The recombinant plasmid was transformed into E. coli BL21 （DE3）. The expression of the fusion protein was determined by Western blot with anti-SARS-CoV antibody positive blood sera. Results The N region gene of SARS-CoV was obtained. The fusion protein GST-N was soluble. Western blot analysis showed that the reaction of GST-N to anti-SARS-CoV sera was positive. Conclusion The pGEX-2T/N has been constructed and expressed in the form of fusion protein GST-N successfully, and the result lays the foundation for further study of SARS-CoV N protein.