SARS病毒M蛋白编码基因在大肠杆菌中的克隆与表达

Cloning of Protein M Encoding Gene of SARS-Coronavirus(SARS-CoV)and Its Expression in E. coli

目的:克隆SARS冠状病毒膜蛋白M胞外区编码序列,并将其置于大肠杆菌作融合表达。方法:从GenBank获得SARS病毒BJ01株膜蛋白M编码基因序列,人工合成其氨基端129-base-pair(bp)双向单链DNA序列,在5′和3′端分别引入BamH I、Xho I酶切位点,退火后形成双链DNA,常规法将其克隆到原核表达载体pGEX-6p-1中,构建含M蛋白编码基因的重组原核表达质粒。重组质粒经酶切鉴定,核酸序列测定和分析后转化大肠杆菌(E.coli)BL21感受态细胞,以异丙基硫代-β-D-半乳糖苷(IPTG)诱导表达融合蛋白。结果:核酸序列测定与分析的结果表明,克隆的129 bp基因序列与基因数据库中所登记的BJ01毒株序列呈现100％同源性。IPTG诱导后的菌体,经SDS-聚丙烯酰胺凝胶电泳(PAGE),显示有一个大小为30.1 ku的融合表达蛋白产生。结论:M蛋白氨基端胞外区129 bp编码基因在E.coli中获得正确表达。

Objective: To clone extra-celluar region encoding gene of membrane M glycoproteins of SARS-Coronavirus(SARS-CoV) and to evaluate its expression in E. coli. Methods: The amino-terminals 129-base-pair (bp) double-strand DNA of the membrane M glycoprotein encoding gene obtained from GenBank of SARS-CoV (strain BJ01) was synthesized in single strand respectively, and the cut sites of BamH I and Xho I restriction enzymes were engineered on the 5′ends and 3′ends. Then, it was annealed and inserted into plasmid pGEX-6p-1 to construct the recombinant prokaryotic expression vector containing the gene sequence of membrane M glycoprotein. The recombinant plasmid, which had been confirmed by enzymes digestion and sequence determination and analysis, was transformed to E. coli competent cells BL2HDE3) and expression of fusion protein was induced by isopropyl-β-D-thiogalactoside(IPTG). Results: The result of sequence determination, analysis and blasting has demonstrated that a 129bp sequence cloned in this study was the same as the gene sequence of SARS-CoV(BJ01) ,which was previously registered in GenBank. And a 30. 1ku of fusion protein was visualized on 12% of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) when the recombinant bacteria was induced by IPTG. Conclusion: The amino-terminals 129 bp gene of SARS-CoV membrane M glycoproteins was correctly expressed in E. coli BL21 cells.