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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 of Spike protein of SARS-like coronavirus in bat cells and to investigate its expression kinetics and purification conditions. Methods: The fragment of receptor binding domain of spike gene of SARS-like coronavirus was amplified by PCR. The product was ligated to pMD-T vector and subcloned into donor plasmid pFAST-HTB. The gene was confirmed by sequencing and further cloned. Transposing into Bacmid, expressing in insect cell Sf9, analyzing the expressed product by SDS-PAGE and Western blotting, and purifying recombinant protein by nickel ion chelating resin. Results: The receptor binding domain of spike protein of SARS - like coronavirus was expressed in insect cell expression system. When the multiplicity of infectiin (MOI) was 2, the expression of recombinant protein The amount reached its peak. CONCLUSION: The receptor-binding domain of spike protein of SARS-like coronavirus of bats is efficiently expressed in insect cell expression system, and the recombinant protein can be purified by nickel ion chelating resin.