人肠三叶因子毕赤酵母表达载体的构建及稳定整合菌珠的筛选

Construction of human intestinal trefoil factor yeast expression vector and screening chromosomal integrants in Pichia pastoris

目的构建hITF毕赤酵母分泌型表达载体,获得稳定整合酵母菌珠,为大量获得重组hITF、进行功能研究奠定基础。方法通过PCR获得hITFcDNA片段,将目的基因插入酵母表达载体pPICZαA分泌信号下游,得到重组载体pPICZαA-hITF。SacⅠ线性化后氯化锂转化进入X-33,Zeocin筛选转化酵母菌,PCR鉴定目的基因,MD、MM鉴定基因型。结果经测序证实PCR扩增的hITFcDNA与基因文库中的完全一致并准确插入酵母表达载体pPICZαA中,氯化锂转化后,PCR鉴定证明重组载体整合进入酵母基因组中,基因型鉴定表明获得的酵母菌株均为Mut+。结论成功构建出酵母表达载体pPICZαA-hITF,获得稳定整合hITFcDNA的酵母菌株,为hITF的大量表达及其功能研究奠定了基础。

Objective To construct Pichia pastoris（P, pastoris） expression vector of hITF,screen stable chromosomal integrants in Pichia pastoris,and underlie the base of large scale production and functional analysis. Methods The hITF 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 pPICZαA. Recombinant plasmid pPICZαA-hITF was linearized by Sac 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. Finally, the presence of insert was identified using PCR and the Mut phenotype was confirmed by testing on MD and MM plates. Results The fragment amplified by PCR was the same as the original sequences of hITF gene from GenBank and inserted into the P. pastoris expression vector pPICZαA correctly. After lithium chloride transformation, the recombinant plasmid was integrated into the regions of homology within the yeast genome and all the Pichia transformants were Mut^＋ by screening Mut phenotype. Conclusion hITF P. pastoris expression vector was successfully constructed and stable chromosomal integrants in P. pastoris were screened. This research lays foundations for hITF expression and its further function studying.