蝙蝠SARS样冠状病毒S蛋白受体结合域在昆虫细胞中的表达和纯化

Expression and purification of receptor binding domain of bat SARS-like coronavirus spike proteins in insect cells

目的:在昆虫细胞表达系统中表达蝙蝠SARS样冠状病毒Spike蛋白的受体结合域,并探讨其表达动力学和纯化条件。方法:以PCR扩增蝙蝠SARS样冠状病毒Spike基因的受体结合域片段,产物连接到pMD-T载体,再亚克隆至供体质粒pFAST-HTB,经序列测定确认基因正确克隆,进一步将其转座入Bacmid中,在昆虫细胞Sf9中进行表达,采用SDS-PAGE和Western blotting对表达产物进行分析,并通过镍离子螯合树脂纯化重组蛋白。结果:在昆虫细胞表达系统中表达出蝙蝠SARS样冠状病毒Spike蛋白的受体结合域片段,当感染复数(multiplicity of infectiin,MOI)为2时,重组病毒感染细胞56 h后,重组蛋白的表达量达到峰值。结论:蝙蝠SARS样冠状病毒Spike蛋白的受体结合域片段在昆虫细胞表达系统中得到了有效表达,并可通过镍离子螯合树脂纯化重组蛋白。

Objective ：To express the receptor binding domain （RBD） of bat SARS-like coronavirus spike proteins in the expression system of insect cells, and to explore its dynamics of expression and purification conditions. Methods Gene amplification of RBD located in the spike gene of bat SARS-like coronavirus was completed with PCR. The PCR products were connected to the pMD-T vector, which was then subcloned into pFastBacHTB donor plasmid. After confirmed by DNA sequencing, the recombinant plasmid was further transferred into Bacmid and expressed in insect Sf9 cell line. The proteins products of recombinant gene expression were analyzed by using SDS-PAGE and Western blotting, then purified with the nickel chelating resins. Results. The receptor binding domain of bat SARS-like coronavirus spike protein was expressed in the expression system of insect cells, at 56 hours after infection with recombinant virus at a multiplicity of infection 2, the peak expression of the recombinant protein was obtained. Conclusion：The receptor binding domain of bat SARS-like eoronavirus spike protein may could be effectively expressed in insect cell expression system, and the recombinant proteins may be purified using nickel chelating resins.