hITF毕赤酵母表达载体的构建及分泌表达  

Construction of hITF yeast expression vector and secreted expression of hITF in Pichia pastoris

作　　者：孙勇[1] 彭曦[1] 张勇[1] 吕尚军[1] 汪仕良[1] 

机构地区：[1]第三军医大学西南医院全军烧伤研究所创伤烧伤与复合伤国家重点实验室,重庆400038

出　　处：《第三军医大学学报》2006年第6期527-530,共4页Journal of Third Military Medical University

基　　金：国家重点基础研究发展计划项目(2005CB522601);国家自然科学基金资助项目(30200294)~~

摘　　要：目的构建hITF毕赤酵母分泌型表达载体,表达重组hITF,为功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pGAPZαA分泌信号下游,得到重组载体pGAPZαA-hITF。BspHⅠ线性化后氯化锂转化进入X-33,Zeoc in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Tric ine SDS-PAGE分析及W estern b lot检测。结果经测序及PCR证实,hITFcDNA准确插入酵母表达载体pGAPZαA中,氯化锂转化后,重组载体通过同源重组整合进入酵母基因组中。Tric ine SDS-PAGE分析证明hITF的分子量约为10×103,W estern b lot分析表明,表达蛋白具有良好的抗原性和特异性。结论成功构建出酵母表达载体pGAPZαA-hITF,获得重组hITF,为深入研究hITF奠定了基础。Objective To construct Pichia pastoris secreted expression vector of hITF, express recombinant hITF and underlie the base of function analyses. Methods The hlTF gene encoding mature peptide was amplified by polymerase chain reaction, and then inserted into the downstream of the alpha-mating factor signal of the P. pastoris expression vector pGAPZαA. Recombinant plasmid pGAPZαA-hlTF was linearized by BspH Ⅰ and transformed into the P. pastoris strain X-33 with lithium chloride. Zeocin resistant clones were chosen by YPD plates containing 100μg/ml Zeocin and the presence of insert was identified using PCR. The positive transformants were fermented in flask and the proteins in the culture supernatant were deposited with TCA and assayed with Tricine SDS-PAGE and Western blotting. Results It was proved that the fragment amplified was inserted into the P. pastoris expression vector pGAPZαA correctly by PCR and gene sequencing. After lithium chloride transformation, the recombinant plasmid was integrated into the regions of homology within the yeast genome. Tricine SDS-PAGE analysis proved that the molecular weight of hITF was about 10 × 10^3 and Western blotting demonstrated that the expression proteins have good antigenicity and specificity. Conclusion hITF P. pastoris expression vector was successfully constructed and recombinant hITF was expressed. This research lay foundation for the further function studying of hITF.

关 键 词：人肠三叶因子 毕赤巴斯德酵母 分泌表达 

分 类 号：R379[医药卫生—病原生物学] R394-33[医药卫生—基础医学]