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  

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

作　　者：汤巧[1] 钱超[1] 夏永祥[2] 卢春[1] 

机构地区：[1]南京医科大学微生物学与免疫学系,江苏南京210029 [2]南京医科大学附属南京第一医院检验科,江苏南京210006

出　　处：《江苏大学学报（医学版）》2005年第5期377-379,383,共4页Journal of Jiangsu University：Medicine Edition

基　　金：江苏省卫生厅非典快速启动项目(H200308);江苏省教育厅SARS专项研究课题(JH03-054);南京市科技局SARS专项研究课题

摘　　要：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.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.

关 键 词：SARS冠状病毒 S蛋白 真核表达 毕赤酵母 

分 类 号：R373.21[医药卫生—病原生物学]