摘要
目的构建大鼠促衰变因子(DAF)的酵母真核表达载体,诱导其表达并对生物学特性进行鉴定。方法采用RT-PCR技术从大鼠肝脏组织中扩增出DAF基因并克隆到毕赤酵母表达载体pPIC9K质粒中。经酶切鉴定后,将pPIC9K-DAF转化至毕赤酵母菌GS115,通过G418-YPD平板筛选高拷贝转化子。转化子经甲醇诱导表达后,对表达的DAF蛋白进行SDS-PAGE及Western blot鉴定并检测重组蛋白的生物活性。结果成功克隆大鼠DAF基因,并筛选出pPIC9K载体中的DAF阳性克隆,证实所获得相对分子质量约为70×103大小的重组蛋白具有大鼠DAF蛋白的抗原性和生物学活性。结论采用分子克隆的方法成功构建大鼠DAF蛋白的毕赤酵母真核表达载体,能为补体及免疫研究提供高效的大鼠DAF蛋白来源。
Objecitve To construct yeast eukaryotic expression vector carrying the gene of rat decay- accelerating factor （DAF）, and to induce the expression of recombinant protein. Methods The cDNA of rat DAF was amplified by RT-PCR from the fresh hepatic tissue of rat. The PCR product was inserted into the Pichia pastoris vector pPICgK. Then the recombinant plasmid pPICgK-DAF was transformed into yeast GSl15 through electroporation. The positive high copy number transformants were rapidly selected by using G418-YPD and were induced by methanol. The induced product was analyzed by DNA sequencing, SDS-PAGE and Western blot. Finally, the bio-activity of recombinant rat DAF was examined. Results The rat DAF cDNA was successfully cloned in pPICgK. The positive clone in pPICgK expression vector （pPICgK-DAF） was sieved, and the pPICgK- DAF showed the same seqencing result as reported in GenBank. And the recombination protein of rat DAF （relative molecular mass is 70×10^3） has natural rat immunogenicity and bio-activity. Conclusion Successfully cloning and high-level expression of rat DAF recombination protein in Pichia pastoris laid a foundation for further research on immunity and complement system.
出处
《四川大学学报（医学版）》
CAS
CSCD
北大核心
2013年第3期333-336,共4页
Journal of Sichuan University(Medical Sciences)
基金
国家自然科学基金(No.81101261)资助
关键词
毕赤酵母
真核表达
PPIC9K
促衰变因子
Pichia pastoris Eukaryotie expression pPIC9K Decay-accelerating factor
