SARS病毒S蛋白N端片段真核载体的构建及酵母表达菌株的建立

目的：构建SARS冠状病毒S蛋白N端1～501bp的真核表达载体,并分析其在毕赤酵母GS115中的表达情况.方法： 用PCR方法从质粒pGEX-6P-1＋SARS-S上扩增出S编码基因片段,克隆至真核表达载体pPIC9上,构建重组质粒pPIC9＋SARS-S.重组质粒经酶切鉴定和核苷酸测序鉴定后,转化毕赤酵母GS115,用甲醇诱导重组S蛋白片段表达.结果： 酶切鉴定及核苷酸序列测定表明重组真核表达质粒的构建完全正确.对甲醇诱导后重组毕赤酵母培养上清行SDS-PAGE电泳分析,在相对分子量约为41 000处有重组蛋白的表达.结论： SARS冠状病毒S蛋白N端1～167aa在毕赤酵母中获得了高效、分泌性表达.

Cloning and Eukaryotic Expression of N-terminal Gene Fragment of SARS-CoV S Protein in Pichia Pastoris

Objective: To construct eukaryotic expression vector containing N-terminal 1-501 base-pair(bp) coding gene of SARS-CoV S protein, and to analyze its expression in yeast GS115.Methods: S protein coding gene fragments were amplified from plasmid pGEX-6P-1+SARS-S and then cloned into eukaryotic expression plasmid pPIC9 to construct recombinant plasmid pPIC9+SARS-S.The recombinant plasmid,which was confirmed by enzymes digestion and sequence determination and analysis,was transformed to yeast GS115,and expression of recombinant protein was induced by methanol.Results: Enzymes digestion and sequence determination and analysis confirmed that the construction of recombinant plasmid was correct.SDS-PAGE was performed to analyze the cultural supernatant of recombinant yeast GS115,and a 41KDa of recombinant protein was visualized on SDS-PAGE when the recombinant yeast GS115 was induced by methanol.Conclusion: Secretory expression of N-terminal of S protein of SARS-CoV was obtained in Pichia Pastoris.