中文摘要：

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。

英文摘要：

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.