SARS-CoV S1蛋白在甲醇营养型酵母中表达及鉴定

Cloning,expression and identification of SARS-CoV S1 gene in yeast p.methanolica

目的获得保持生物学活性、易于纯化、高产量的严重急性呼吸道综合征冠状病毒(severe acute resp iratorysyndrom coronavirus,SARS-CoV)S1蛋白,以便进一步研究S1蛋白及其抗体的功能。方法将SARS-CoV S1基因重组入酵母表达载体pMETαA,经过酶切和测序鉴定后,通过电穿孔法将重组质粒S1-pMETαA转化入宿主酵母菌株PMAD11,使其在甲醇的诱导下表达目的蛋白。最后用覆盖分析法对重组转化子初步分析,SDS-PAGE和W esntern b lotting法鉴定目的蛋白的表达和特异性。结果酶切鉴定和测序结果证实了S1基因成功地克隆到pMETαA载体中;覆盖分析、SDS-PAGE和免疫印迹法证实S1基因得到正确的表达。结论利用酵母表达系统,成功地克隆和表达了SARS-CoV S1蛋白,为进一步纯化该蛋白和研究其功能奠定了基础。

Objective To obtain high-yield and easy-purification severe acute respiratory syndrome coronavirus （SARS-CoV） S1 protein with biological activity and to study the activity of S1 protein and its antibody further. Methods SARS-CoV S1 gene was inserted into yeast expression vector pMETαA by ligation reaction. The recombinant plasmid was verified by enzyme digestion and sequencing, followed by being transformed into yeast host strain PMAD11 with electroporation. After induced with methanol, the S1 gene expression was verified with overlay assay and Western blotting. Results The positive clones of S1 gene into pMETαA were approved by restriction enzyme digestion and sequencing. The expression of S1 protein was confirmed subsequently by overlay assay and Western blotting. Conclusion SARS-CoV S1 gene has been cloned and expressed in yeast p. methanolica, which can provide experimental data for next study on the activity of this protein and its antibody during SARS-CoV infection.