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目的 构建SARS冠状病毒棘突蛋白部分序列 2 (S2 )的原核表达质粒 ,分析其在大肠杆菌中的表达状况。方法 采用逆转录聚合酶链式反应技术从SARS冠状病毒基因组中扩增出编码S蛋白的第 2 170到 2 814位碱基的基因片段 ,克隆至 pMD18-T载体 ,PCR筛选阳性克隆并测序。用限制性内切酶消化后 ,目的片段亚克隆至表达载体pGEX - 4T - 2 ,转化大肠杆菌JM10 9,PCR和双酶切鉴定转化菌落。将阳性菌落经IPTG诱导 ,SDS -PAGE和免疫印迹分析靶蛋白的表达。结果 RT -PCR扩增出S2特异片段 ,经测序与GenBank比对存在 1处碱基突变。S2基因片段亚克隆至表达载体 pGEX - 4T - 2构建成重组表达质粒 ,并在JM 10 9中表达了S2融合蛋白。结论 成功构建了SARS冠状病毒S2的重组表达质粒 ,并在大肠杆菌中表达了S2融合蛋白。
Objective To construct prokaryotic expression plasmid of partial sequence 2 (S2) of SARS coronavirus and analyze its expression in Escherichia coli. Methods The gene fragment encoding the 2 170th to 2 814th base of S protein was amplified from SARS coronavirus genome by reverse transcription polymerase chain reaction (PCR) and cloned into pMD18-T vector. The positive clones were screened by PCR and sequenced. After restriction endonuclease digestion, the target fragment was subcloned into the expression vector pGEX - 4T - 2 and transformed into E. coli JM109. The transformed colonies were identified by PCR and double enzyme digestion. Positive colonies were induced by IPTG, and the target proteins were analyzed by SDS-PAGE and immunoblotting. Results The specific fragment of S2 was amplified by RT-PCR, and one base mutation was found after sequencing compared with GenBank. The S2 gene fragment was subcloned into the expression vector pGEX - 4T - 2 to construct a recombinant expression plasmid, and the S2 fusion protein was expressed in JM109. Conclusion The recombinant expression plasmid of SARS coronavirus S2 was successfully constructed and the S2 fusion protein was expressed in Escherichia coli.