摘要
用限制酶将pSK TTR质粒上TTR基因切下,分别插入分泌型载体pPIC9和pPIC9K.将重组的pPIC9K TTR用BglⅡ酶切后,转化PichiapastorisGS115菌株,筛选获得G418高抗性转化子.转化子经摇瓶发酵,上清液用SDS PAGE检测,有明显的rTTR表达.经DEAE sepharoseF.F.离子交换柱和Superdex75分子筛层析,得到蛋白纯度大于95%的rTTR.经Westernblotting,分子质量和等电点测定等证明,毕赤氏酵母表达的rTTR与人血浆提取的TTR极为相似.
TTR gene was cut off from the plasmid pSKTTR by restriction enzymes, and was inserted into secretion vectors pPIC9 and pPIC9K. The recombined pPIC9KTTR was digested by BglⅡ and transformed into Pichia pastoris strain GS115, then the transformants with high G418 resistance were selected. After the transformants grew in the shaking flask, the SDSPAGE analysis of supernatant showed that they could express rTTR in a high level. rTTR was isolated and purified by DEAESepharose F. F. and Superdex75 gel permeation chromatography with more than 95% purity. The results obtained through experiments such as Western blotting and Mr and PI determination indicated that purified rTTR protein expressed by Pichia pastoris had very similar characteristics compared with those of natural TTR protein purified from human serum.
出处
《复旦学报（自然科学版）》
CAS
CSCD
北大核心
2002年第6期646-649,共4页
Journal of Fudan University：Natural Science
基金
国家自然科学基金资助项目(39970019)
