Canstatin-N基因在Pichia pastoris中表达及产物活性分析  

Expresstion of Canstatin-N in P.pastoris and Its Biology Activity Analysis

作　　者：潘英文[1] 张爱联[1] 屈直[1] 张添元[2] 罗进贤[2] 

机构地区：[1]中国热带农业科学院热带生物技术研究所农业部热带作物生物技术重点开放实验室,海南海口571101 [2]中山大学生命科学学院,广东广州510275

出　　处：《生物技术》2010年第3期25-28,共4页Biotechnology

基　　金：海南省重点科技计划项目(05204);国家自然科学基金项目(30760061);中央级公益性科研院所基本科研业务费专项资金项目(ITBBZX2008-4-2)资助

摘　　要：目的:应用P.pastoris的pAOX1表达系统胞内表达canstatin-N蛋白。方法:通过PCR DNA合成技术以及DNA的酶切和连接技术,除去pPIC9K分泌型表达载体的信号肽,并使其载体的多克隆位点的3′端融合his6纯化标签,获得新的载体pPIC9Ki。以含canstatin N端序列的pET-CTN质粒为模版,PCR法扩增1～89个氨基酸的267bp的canstatin的N端基因片段,将其连接于pPIC9Ki的多克隆位点,获得重组表达质粒pPIC9Ki-CTN-N,用电转法将pPIC9Ki-CTN-N转化P.pastorisGS115,通过G418抗性筛选获得工程菌GS115(pPIC9Ki-CTN-N)。通过摇瓶发酵甲醇诱导表达Canstatin-N蛋白。用蜗牛酶裂解P.pastoris细胞,SDS-PAGE分析蛋白表达情况,在鸡胚中进行活性分析。结果:在pAOX1的调控下,canstatin-N基因能在P.pastoris中经甲醇诱导表达,摇瓶发酵表达量为65mg/L,纯化的目的蛋白具有显著的抑制鸡胚尿囊膜血管生成的作用。结论:实现应用P.pastoris表达Canstatin-N蛋白,为Canstatin-N蛋白的规模化生产和进一步的药用研究奠定了基础。Objective：Using P.pastoris to express of Castatin-N protein.Method：With pET-CTN plasmid which containing the canstatin N end sequence as template,a 267 bp NDA sequence coding for canstatin N-terminal 1-89 amino acids（N domain） was amplified by PCR,then it was inserted into MCS of pPIC9Ki which came from pPIC9K by removing the α-factor secretion signal and fusing the his6 sequence to generate the new expression vector pPIC9Ki-CTN-N.Engineering strain,GS115（pPIC9Ki-CTN-N）,had been obtained from the anti-G418 plate.Under the induction of methanol,Canstatin-N protein was expressed by shaking flask fermentation.The P.pastoris wall was removed by sail enzyme and SDS-PAGE was used to analyze the expressed products.Result：65mg/L Canstatin-N was expressed in the GS115 after fermentation for 3 d.Purified Canstatin-N has obvious inhibition of blood vessel growth on chicken chorioallantoic membrane.Conclusion：Castatin-N gene express successfully in P.pastoris with high activity.This work has established the base for large amount production of Canstatin-N protein with P.pastoris.

关 键 词：canstatin-N P.PASTORIS 基因表达 

分 类 号：Q786[生物学—分子生物学]