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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 FS2 88能分泌表达重组人卵泡抑素
Objective To secret the human follistatin gene in P. pastoris. Methods The fragment of target gene FS2 88 was amplified by PCR and subcloned into pPIC9 to construct pPIC9 FS2 88. After DNA sequence analysis and validation, pPIC9 FS2 88 was digested with BamH Ⅰ and Sal Ⅰ to recover the small fragment and connect with pPIC9K Recombinant secretory expression vector pPIC9K FS2 88, electroporation transformation SMD116 8, G4 18 screening multicopy transformants, after transformant fermentation, the supernatant was taken for SDS PAGE and Western blot detection of recombinant protein expression. Results Recombinant secretory expression vector pPIC9K FS2 88 and engineering strain SMD116 8 FS2 88 were successfully constructed. The SDS PGAE showed that the supernatant of the engineered bacteria had a diffusion band between 50000 and 60000, The result is consistent. Conclusion The engineered strain SMD116 8 FS2 88 can express recombinant human follistatin