SARS-CoVS2蛋白基因的克隆及其在Vero E6细胞中的表达

Cloning and expression of SARS-CoV S2 protein gene in Vero E6 cells

目的 克隆人SARS病毒S2蛋白分子的编码序列 ,并在VeroE6细胞上获得表达。方法 从人SARS病毒cDNA文库中用PCR技术克隆编码SARS病毒S2蛋白的片段。将它插入质粒载体pVAX1中构建重组S2 pVAX1真核表达载体 ,并对插入片段序列进行测序。采用脂质体法转染VeroE6细胞 ,用Westernblotting检测SARS CoVS2蛋白的表达情况。结果 用PCR方法扩增出一个 1845bp的基因片段 ,并插入pVAX1载体中 ,成功构建出重组S2表达载体 ,经测序证实克隆的S2片段阅读框正确完整。将其转染的VeroE6细胞经WesternBlotting检测获得一条特异性目的蛋白条带。结论 成功克隆基因并获得表达S2分子的VeroE6细胞系。

Objective To clone the DNA sequence encoding SARS CoV S2 protein and to express the protein in Vero E6 cells. Methods SARS coronavirus S2 gene was amplified by PCR from cDNA library of SARS CoV. The fragment of S2 was inserted into the restrictive site of Bam HⅠ and Sal Ⅰ of pVAX1 vector and the successful recombinants were identified by double digestion and sequencing. Following transformation into Vero E6 cells by a liposome transfection reagent (DOTAP), S2 protein expression in Vero E6 cells by the recombinant was detected by Western blotting. Results One DNA fragment with 1 845 bp was amplified by PCR. Double restrictive digestion and sequencing proved that the fragment was cloned into pVAX1 vector correctly with correct ORF and orientation. Recombinant expression vector for S2 was successfully constructed. After transformation into Vero E6 cells, the recombinant could express specific interest protein proven by Western blotting. Conclusion SARS CoV S2 gene has been cloned successfully and the Vero E6 cell line expressing S2 has also been obtained.