摘要
目的：构建肠三叶因子（ITF）的真核表达载体,并在酵母菌GS115中表达,为进一步研究ITF的生理和药理功能奠定基础.方法：通过RT-PCR获得ITF cDNA片断,将目的基因插入pPICZαA酵母表达载体,得到重组载体pPICZαA-ITF.经BspH I线性化后氯化锂转化进入GS115,Zeocin筛选转化酵母菌,PCR鉴定目的基因.阳性转化子经摇瓶表达,取上清TCA沉淀后做Tricine SDS-PAGE分析及Western blot检测表达蛋白.结果：经测序及PCR证实ITF cDNA准确插入酵母表达载体pPICZαA中.Tricine SDS-PAGE分析证明ITF的分子量约为14×10^3,表达蛋白具有良好的抗原性和特异性.结论：成功构建了真核表达载体pPICZαA-ITF,在酵母菌GS115中表达,获得重组ITF蛋白.
Objective： To construct eukaryodc expression vector of FIT and express the recombinant ITF in Pichia pastoris GSll5. The research establish the base for further research of physiological and pharmaceutical functions of ITF. Methods： ITF DNA fragment was amplified using polylnerase chain reaction （PCR）, the gene was cloned into the yeast expression vector pPICZαA, and the recombinant expression vector pPICZαA-ITF gained. The recombinant plasmid pPICZαA-ITF was linearized by BspH I and transformed into the yeast strain GSll5 with lithium chloride, Zeocin resistant transformed yeast strains were cho- sen, and the presence of insert was identified by PCR. The posidve transformants were expressed and the proteins in the culture supernatant were deposited in flask with TCA and analyzed with Tricine SDS-PAGE and Western blotting. Result： PCR result and gene sequencing proved that the fragment amplified was inserted into the yeast expression vector pPICZαA correctly. It was proved that the molecular weight of expressed proteins was about 14 × 10^3 by Tricine SDS-PAGE analysis and Western blotting showed the proteins have good andgenicity and specificity. Conclusions： pPICZαA-ITF eukaryodc expression vector was constructed successfiully, recombinant ITF was expressed in yeast fungus GSll5 and recombinant ITF protein was gained.
出处
《现代生物医学进展》
CAS
2007年第8期1124-1126,1141,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金(30300294)
国家重点基础研究发展计划项目(2005CB522601)
关键词
肠三叶因子(ITF)
酵母
表达
Intestinal trefoil factor （ITF）
Yeast
Eukaryodc expression
