CAJ | 학술논문

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TFPI cDNA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GS115,利用G418抗性实验筛选含有高拷贝TFPI cDNA的毕赤酵母菌株,采用Western Blot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(1.2 kb),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,Western Blot 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.
목적 구건고고패표체중조인조직인자도경억제인자적필적효모병대단백표체조건진행초보우화,위상관연구전정기출.방법 장PCR확증득도적TFPI cDNA편단여표체질립pPIC9K련접구건중조질립rhTFPI-pPIC9K,채용PCR방법급DNA측서대기진행감정.장중조질립전화효모세포GS115,이용G418항성실험사선함유고고패TFPI cDNA적필적효모균주,채용Western Blot화TFPI검측시제합분석중조단백표체.통과개변유도온도、유도시간급갑순농도대중조단백표체조건진행초보우화.결과 PCR확증rhTFPI-pPIC9K득도예기적확증조대(1.2 kb),경매절도보분석급측서학인성공구건표체질립rhTFPI-pPIC9K.실시정량PCR결과현시,통과G418사선획득료함고고패기인적효모세포,Western Blot 결과현시함기인고패수여단백표체량정정상관.통과대표체조건적우화,명현제고료TFPI중조단백적표체량,중조TFPI표체량고체(9.95+0.78)mg/L,활성체도(3.91+1.37)U/mL.결론 성공구건료고효표체TFPI중조단백적필적효모세포주,위TFPI공능연구급재상관질병방치중적응용제공료견실적실험기출.
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 pPIC9K and the constructed expression vector rhTFPI-pPIC9K was confirmed by restriction endonuclease 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 rhTFPI-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.