严重急性呼吸综合征冠状病毒刺突蛋白在酿酒酵母中的表达和纯化

Expression and purification of spike protein of severe acute respiratory syndrome coronavirus in Saccharomyces cerevisiae

目的构建重组表达质粒pYES6-S,诱导SARS冠状病毒刺突蛋白(spike protein)在酿酒酵母中的表达并纯化。方法反转录获得SARS冠状病毒DNA片段,PCR法获得刺突蛋白基因互相重叠的四部分片段,酶切连接成全长,在大肠埃希菌E.coli JM109中构建重组表达克隆pYES6- S,在酿酒酵母中诱导表达并纯化。结果重组克隆pYES6-S经酶切、测序鉴定确定,插入片段大小、方向、碱基匹配与预期一致,获得1个完整的刺突蛋白基因,表达产物纯化后,电泳鉴定,相对分子质量大小近110 000。结论成功克隆SARS冠状病毒刺突蛋白基因全长,并在酿酒酵母中诱导表达成功,有助于抗SARS分子疫苗的研究。

Objective To construct the recombinant plasmid pYES6-S and express and purify spike protein of severe acute respiratory syndrome（SARS） coronavirus in Saccharomyces cerevisiae. Methods DNA fragments of SARS coronavirus were obtained by reverse transcription. Four overlapped fragments of spike protein genes were amplified by polymerase chain reaction（PCR） and ligated into an integral spike protein gene by restriction enzyme digestion. The spike protein gene recombined with pYES6 and cloned into E. coll. The recombinant plasmid pYES6-S was induced and expressed in Saccharomyces cerevisiae（INVScl）by galactose. Results The recombinant plasmid pYES6-S was confirmed that inserted fragment was right in length, direction and base matching by restriction enzyme digestion and sequencing. The purified protein encoded by the whole spike protein gene was about Mr 110× 10^3 identified by electrophoresis. Conclusion The whole spike protein gene of SARS coronavirus is cloned into E. coli and the protein is expressed in Saccharomyces cerevisiae successfully, which can be helpful in SARS vaccine research.