人血管内皮抑制素基因在毕赤甲醇酵母中的表达

Successful Expression of Human Endostatin Gene in Pichia pastoris

对发生突变的人血管内皮抑制素基因进行了PCR修正,并将其连接到pAO815载体上,然后克隆进大肠杆菌TOP10F’中,提取质粒测序,证实序列正确。再用电激法转化毕赤甲醇酵母KM71,利用RDB平板和PCR技术筛选出阳性克隆菌落后,甲醇诱导表达,SDS-PAGE电泳检测显示重组的人血管内皮抑制素蛋白在毕赤甲醇酵母KM71中获得了有效表达。

The mutant human endostatin modified by PCR, is ligated into pAO815 vector and cloned into E.coli TOP10F＇.The sequence of this modified gene is proved to be correct by measuring the sequence of the collected particles. Then the isolated plasmid pAO815-endostatin is trans- formed into Pichia pastoris KM71 by electroporation. After the positive recombinants are screened out by using RDB plate and PCR technique and induced with methanol, the SDS-PAGE results show that human endostatin protein is effectively expressed in Pichia pastoris KM71.