人抑瘤素M在毕赤酵母中的高效分泌表达

目的：在毕赤酵母中高效分泌表达重组人抑瘤素M（rhOSM）。方法：以人胚胎组织DNA为模板通过PCR技术获得hOSM基因，构建毕赤酵母真核表达载体pPICZαC-hOSM，电转化毕赤酵母菌株X-33，PCR法筛选阳性克隆，SDS-PAGE和Western blotting方法筛选能够稳定、高效分泌表达rhOSM的酵母工程菌。结果：经PCR法获得了hOSM基因，培养液上清经SDS-PAGE和Western blotting证实获得了相对分子质量约为28 000的rhOSM，表达量为45　mg?L^-1。结论：毕赤酵母表达系统能够稳定、高效分泌表达rhOSM，摇瓶规模表达量为45 mg?L^-1。

High level secretory expression of human oncostatin M in Pichia pastoris

Objective To explore the method of secretory expression of recombinant human oncostatin M (rhOSM) in Pichia pastoris. Methods The human embryonic genomic DNA was used as templet to obtain the sequence of mature hOSM gene by PCR,the expression vector of Pichia pastoris of pPICZαC-hOSM was constructed. The recombinant plasmid was transformed into Pichia pastoris X-33 via electroporation.The transformed positive strains were screened by PCR. The supernatant were analyzed by SDS-PAGE and Western blotting to screen Pichia pastoris engineer bacteria with highly effective expression of rhOSM. Results The mature hOSM DNA was obtained by PCR. SDS-PAGE and Western blotting analysis showed there was rhOSM in the culture supernatant induced by methanol with 28 000 of molecular weight.Conclusion Pichia pastoris can express rhOSM efficiently,the expression level of rhOSM reaches at 45 mg·L-1 in flask scale.