SARS病毒M蛋白编码基因真核载体的构建及酵母表达菌株的建立  

Cloning and eukaryotic expression of N-terminal coding gene fragment of SARS-CoV M protein in Pichia pastoris

作　　者：钱超[1] 卢春[2] 余建华[1] 

机构地区：[1]解放军第454医院检验科,南京210002 [2]南京医科大学微生物与免疫学系

出　　处：《陕西医学杂志》2006年第9期1168-1170,1189,共4页Shaanxi Medical Journal

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)

摘　　要：目的：构建SARS冠状病毒M蛋白N端编码基因1～129bp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—M上扩增出M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—M。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M蛋白片段表达。结果：酶切鉴定及核苷酸序列测定表明重组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10000处有重组蛋白的表达。结论：SARS冠状病毒M蛋白N端1～43aa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- 129 base-pair （bp） coding gene of SARS-CoV M protein, and to analyze its expression in yeast GS115. Methods ： M protein coding gene fragments were amplified from plasmid pGEX-6P-1＋ SARS-M and then cloned into eukaryotic expression plasmid pPIC9 to construct recombinant plasmid pPIC9＋SARS-M. The recombinant plasmid, which was confirmed by enzyme digestion and sequence determination and analysis, was transformed to yeast GS115, and expression of recombinant protein was induced by methanol. Results Enzyme 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 10KDa of recombinant protein was visualized on SDS-PAGE when the recombinanl yeast GSn5 was induced by methanol. Conclusion：Secretory expression of N-terminal 1- 43aa of M protein of SARS-CoV was obtained in Pichia pastoris.

关 键 词：冠状病毒属 蛋白 毕赤酵母@真核载体的构建 

分 类 号：R737.110.2[医药卫生—肿瘤]