表达人卵泡抑素的毕赤氏巴斯德酵母工程菌的构建  

Construction of Engineering P.pastoris Which can Express Recombinant Human Follistatin

作　　者：尤汉宁[1] 袁汉英[2] 吕红[2] 高卜渝[2] 陈源文[1] 李育阳[2] 李定国[1] 

机构地区：[1]上海第二医科大学新华医院消化内科,上海200092 [2]复旦大学遗传学研究所

出　　处：《上海第二医科大学学报》2005年第2期107-111,共5页Acta Universitatis Medicinalis Secondae Shanghai

基　　金：国家自然科学基金 (30 1 70 4 1 1 )资助项目

摘　　要：目的在P .pastoris中分泌表达人卵泡抑素基因。方法采用PCR法扩增目的基因FS2 88片段 ,将其亚克隆入pPIC9,构建pPIC9 FS2 88,DNA序列测定验证后 ,将pPIC9 FS2 88采用BamHⅠ和SalⅠ双酶切 ,回收小片段 ,连接pPIC9K ,构建重组分泌型表达载体pPIC9K FS2 88,电穿孔法转化SMD116 8,G4 18筛选多拷贝转化子 ,转化子发酵后 ,取上清液进行SDS PAGE和Westernblot检测重组蛋白表达。结果成功构建了重组分泌型表达载体pPIC9K FS2 88和工程菌株SMD116 8 FS2 88,SDS PGAE显示工程菌发酵上清液在 5 0 0 0 0 ～6 0 0 0 0处有弥散条带 ,并且与Westernblot结果相符。结论工程菌SMD116 8 FS2Objective To express human follistatin gene in P.pastoris. Methods PCR technique was used to amplify FS288 gene fragments encoding mature follistatin polypeptide which were subcloned into pPIC9 for the construction of recombinant vector pPIC9-FS288. Afer DNA sequence analysis, pPIC9-FS288 was completely digested using BamHⅠ and SalⅠ, and then the consequent smaller DNA fragments were isolated and purified for ligation with pPIC9K in order to construct recombinant secretive expression vector, pPIC9K-FS288. The latter was applied to transform competent P.pastoris SMD1168 through electroporation for the construction of engineering P.pastoris SMD1168, multiple copy transformants were screened in selective plates containing G418. After transformant fermentation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot were performed to detect recombinant protein expression in the supernatant. Results The recombinant secretive expression vector pPIC9K-FS288 and engineering P.pastoris strain SMD1168-FS288 were constructed successfully. SDS-PAGE showed that there was a diffuse band between 50 000～60 000 in fermentation supernatant, which was in accord with the results of Western blot hybridization. Conclusion Engineering P.pastoris strain SMD1168-FS288 has the capacity of secretively expressing recombinant human follistatin.

关 键 词：FS 人卵 卵泡抑素 分泌型表达载体 上清液 构建 PCR法 转化子 工程菌 分泌表达 

分 类 号：R346[医药卫生—基础医学]