钙网蛋白-N58在毕赤酵母中的表达及活性分析

Expression of Calreticulin-N58 in Pichia pastoris and Its Activity Analysis

目的:利用毕赤酵母表达系统表达Calreticulin-N58蛋白。方法:利用重叠延伸PCR方法合成Calreticulin-N58基因,构建成分泌型的重组表达载体pPIC9K-CRT-N58。该重组表达载体经SacⅠ线性化后,电激转化入毕赤酵母GS115,经MD板和不同浓度G418筛选得到多拷贝重组菌株。经甲醇诱导表达目的蛋白,SDS-PAGE分析重组蛋白的表达情况,在鸡胚中进行活性分析。结果:重组质粒pPIC9K-CRT-N58在毕赤酵母中经甲醇诱导能分泌表达CRT-N58蛋白,摇瓶表达量大约为60mg/L,纯化的目的蛋白有显著的血管生成抑制作用。结论:实验成功地在毕赤酵母表达系统中实现了Calreticulin-N58的分泌表达。为Cal-reticulin-N58蛋白的进一步药用研究奠定了基础。

Objective： To express protein Calreticulin- N58 in Pichia pastor/s. Method： Calreticulin- N58 gene was obtained by overlap extension PCR to constructed secreting recombinant expression plasmid named pPIC9K- CRT- N58. Then pPIC9K- CRT- N58 was lined by Sac and then transformed into Pichia pastor/s cell GS115 by electroporation. Multi - copy recombinant strains were screened by MD plate culture and G418 screening. The expression of recombinant protein was induced by methanol, SDS - PAGE was used to analyze products expressed. Bioactivity of CRT- N58 was measured by Chorioallantoic membrane（CAM） assays. Result： recombinant expression plasmid pPIC9K- CRT- N58 secreted target protein CRT - N58 in Pichia pastoris under the induction of methanol. The expression level was about 60mg/L in shaking flask. Purified CRT- N58 have obvious anti-vein biological activity. Conclusion： CRT- N58 secretion expression was successfully achieved in Pichia pastor/s expression system, which laid a solid foundation for the further research and development of CRT- N58.