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Vasostatin在毕赤酵母中的表达及抗血管生成性质研究
作者:林玉丽 华子春 
单位:南京大学生命科学学院
关键词:毕赤酵母 表达纯化 抑制血管生成 抗肿瘤  
分类号:
出版年·卷·期(页码):2011·30·第五期(692-695)
摘要:

Vasostatin是一种有应用前景的抗血管生成蛋白质,它在大肠杆菌中表达时主要呈无活性的包涵体形式存在,从而影响到其进一步的研究和应用。本文中为了表达具有生物活性的Vasostatin,我们将Vasostatin基因克隆入酵母诱导型表达载体pPIC9K中,经过Sac I线性化、电转化毕赤酵母蛋白酶缺陷菌株Pichia pastoris KM17、筛选阳性克隆、PCR鉴定和DNA序列分析,获得了Vasostatin基因重组到酵母染色体中的阳性重组子。阳性重组子经甲醇诱导和分离纯化,获得了重组Vasostatin蛋白质,产量达12 mg•L-1。CAM实验表明重组Vasostatin能够显著抑制新生血管生成。有生物活性的重组Vasostatin的表达为进一步研究其结构与功能关系及其潜在应用奠定了良好的基础。

Vasostatin is one of angiogenesis inhibitors which have been studied intensively because of its clinical application potential. When vasostatin protein was produced in E.coli, it expressed in insoluble and inactive inclusion bodies form. For efficient production of soluble and biologically active recombinant vasostatin, vasostatin gene was cloned into expression vector pPIC9K, then linearized by Sac I enzyme, transformed into yeast Pichia pastoris KM17 by electroporation. The transformants with vasostatin gene integration were selected by G418+ selection, then confirmed PCR and DNA sequencing. The recombinant proteins were induced to express by methanol and purified with a yield of 12mg•L-1. CAM assay demonstrated that the yeast-expressed recombinant vasostatin obviously inhibited the angiogenesis. Production of biologically active recombinant vasostatin lays a solid foundation for further structure-function relationship study of vasostatin and its potential application.

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