猪繁殖与呼吸综合征病毒核衣壳蛋白基因的克隆与原核表达

摘要

[目的] 克隆猪繁殖与呼吸综合征病毒核衣壳蛋白基因并在原核表达系统中表达.[方法] 根据GenBank上登录的猪繁殖与呼吸综合征病毒(PRRSV)核酸序列设计1对引物,用RT-PCR方法扩增出PRRSV的核衣壳蛋白(N)基因,并将其克隆到原核表达载体pGEX-4T-1上,得到重组质粒pGEX-4T-1-N.将重组质粒转化大肠杆菌BL31(DE3)感受态细胞,经TPTG诱导,SDS-PAGE电泳检测.[结果] PRRSV肺组织所提取的RNA,经RT-PCR扩增,得到一段392 bp的片段,与预期结果一致.SDS-PAGE电泳分析,所得重组蛋白的分子量约39.866 Da,与预期结果相同.重组菌体超声裂解后,经10%的SDS-PAGE电泳分析,结果显示该重组蛋白为可溶性蛋白.[结论] 该研究为重组蛋白的纯化奠定了基础.%[Objective] The aim was to clone nucleocapsid protein gene of porcine reproductive and respiratory syndrome virus (PRRSV) and express it in procaryotic expression system. [Method] According to the nucleic acid sequence of PRRSV addressed in GenBank, a pair of primers was designed for amplifying the nucleoprotein (N) gene with RT-PCR. The N gene was cloned into the procaryotic expression vector pGEX-4T-1 and the recombinant plasmid pGEX-4T-1-N was obtained, then the recombinant plasmid was transformed into competent cell of Escherichia coli BL31 (DE3), after TPTG induction, it was detected by SDS-PAGE electrophoresis. [Result] A fragment of 392 bp was amplified from PRRSV RNA extracted from lung by RT-PCR, which was consistent with anticipated result. The analysis on SDS-PAGE electrophoresis showed that the molecular weight of the obtained recombinant protein was about 39.866 Da, which was consistent with anticipated result. After ultrasonic degradation, the recombinant was analyzed by 10% SDS-PAGE electrophoresis and the result showed that the recombinant protein was soluble. [Conclusion] The research laid the foundation for the purification of the recombinant protein.