猪繁殖与呼吸综合征病毒N基因的克隆及高效表达

Cloning and Expression of Nucleocapsid Protein Gene of Porcine reproductive and respiratory syndrome virus in E.coli

本试验参照GenBank公布的Porcine reproductive and respiratory syndrome virus(PRRSV)VR2332株的核苷酸序列,设计并合成了一对引物,应用RT-PCR方法扩增出了PRRSV的核衣壳蛋白基因(N基因)。在对N基因及pET32a载体双酶切后进行连接,构建了高效原核表达载体pETN。将pETN重组质粒转化BL21(DE3)宿主菌后,对培养条件及诱导表达条件(IPTG最佳浓度、作用时间)等影响表达的因素进行优化,实现了PRRSV核衣壳蛋白基因的高效表达。

The gene of nucleocapsid protein of Porcine reproductive and respiratory syndrome virus (PRRSV) VR2332 strain was amplified by RT-PCR. The PCR product was purified and digested with BamH Ⅰ and Xho Ⅰ, then directly cloned into the prokaryotic vector pET32a. Consequently the recombinant plasmid was constructed, designated pETN. PETN was transformed into the host cell BL21 (DE3) and the expression procedure was optimized including cultivated temperature, optional induction concentration and time of IPTG. The result indicated that the nucleocapsid protein can be expressed efficiently with 0.8mmol/L IPTG and 4-hour induction.