人组织因子途径抑制因子在毕赤酵母中高拷贝表达  

High-copy expression of tissue factor pathway inhibitor in pichia pastoris

作　　者：程旭[1] 徐伟杰[1] 宋丽萍[1] 刘兰霞[1] 董霞[1] 张海玲[1] 朱敦皖[1] 冷希岗[1] 

机构地区：[1]中国医学科学院北京协和医学院生物医学工程研究所,天津300192

出　　处：《国际生物医学工程杂志》2011年第3期135-139,共5页International Journal of Biomedical Engineering

基　　金：基金项目：天津市科技攻关培育项目（06YFGPSH03700）

摘　　要：目的构建高拷贝表达重组人组织因子途径抑制因子的毕赤酵母并对蛋白表达条件进行初步优化，为相关研究奠定基础。方法将PCR扩增得到的TFPIcDNA片段与表达质粒pPIC9K连接构建重组质粒rhTFPI—pPIC9K，采用PCR方法及DNA测序对其进行鉴定。将重组质粒转化酵母细胞GSI15，利用G418抗性实验筛选含有高拷贝TFPIcDNA的毕赤酵母菌株，采用WesternBlot和TFPI检测试剂盒分析重组蛋白表达。通过改变诱导温度、诱导时间及甲醇浓度对重组蛋白表达条件进行初步优化。结果PCR扩增rhTFPI-pPIC9K得到预期的扩增条带（1．2kb），经酶切图谱分析及测序确认成功构建表达质粒rhTFPI—pPIC9K。实时定量PCR结果显示，通过G418筛选获得了含高拷贝基因的酵母细胞，WesternBlot结果显示含基因拷贝数与蛋白表达量呈正相关。通过对表达条件的优化，明显提高了TFPI重组蛋白的表达量，重组TFPI表达量高达（9．95±0．78）mg／L，活性达到（3．91±1．37）U／mL。结论成功构建了高效表达TFPI重组蛋白的毕赤酵母细胞株，为TFPI功能研究及在相关疾病防治中的应用提供了坚实的实验基础。Objective To generate recombinant Pichia pastoris for high-copy expression of human tissue factor pathway inhibitor （TFPI）. Methods The cDNA encoding human TFPI was inserted into the expression vector pPICgK and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuelease analysis and DNA sequencing. The recombinant plasmids were subsequently transformed into Pichia pastoris GS115 cells, and the transformants were confirmed by PCR amplification of the genomic DNA. The recombinant Pichia pastoris with high copies of TFPI cDNA was screened by G418 selection. Western blot and TFPI ELISA Kit were employed to analyzing. The temperature, time and concentration of methanol for the induction of recombinant protein were optimized. Results PCR analysis and DNA sequencing confirmed the successful construction of the expression vector rbTFPI-pPIC9K. Real time quantitative PCR and Western blot analysis demonstrated the positive correlation between TFPI expression level and the copy number of TFPI cDNA in Pichia pastoris cells. Optimization of the induction condition significantly elevated the expression level and activity of TFPI （9.95±0.78 mg/L and 3.91±1.37 U/mL）. Conclusion The Pichia pastoris strain with high copy of TFPI expression was successfully constructed, which lays a solid foundation for the further investigation on the function of TFPI and its application in the prevention and therapy of diseases.

关 键 词：组织因子途径抑制因子 毕赤酵母 表达 

分 类 号：Q[生物学]