人CTLA-4胞外区cDNA毕赤酵母表达载体的构建及高拷贝稳定整合菌株的筛选

目的 构建人CTLA－4胞外区蛋白毕赤酵母分泌型表达载体，获得高拷贝稳定整合菌株，为大量获得人CTLA－4胞外区蛋白，进行功能研究及其临床应用奠定基础。方法 从活化人外周血淋巴细胞总RNA中，扩增人CTLA－4基因胞外区cDNA片段，将目的基因插入酵母表达载体pPIC9α分泌信号下游，再插入高拷贝载体pPIC9K，得到pPIC9K-CTLA4,SaL I线性化后电转GS115；G418梯度筛选转化菌。PCR，Suthern blotting鉴定目的基因整合及拷贝数。结果 成功构建了人CTLA－4胞外区cDNA毕赤酵母表达载体，获得了高拷贝稳定整合菌株。结论 为表达人CTLA－4胞外区蛋白及其功能研究和临床应用奠定了基础。

Construction of CTLA4 extracellular domain expression vectors and screening chromosomal integrants in Pichia pastoris

Objective A 400bp cDNA fragment of the CTLA4 extracellular domain coding sequence was amplified by PCR from mRNA of actived human T cells, and then inserted into the P. pastoris expression vector pPIC9 and pPIC9K.Methods Thus the gene's expression was under the alcohol oxidase(AOX1) promoter control and the coding sequence was fused to the alpha mating factor signal.Results Following electroporation of the DNA into the P. pastoris strain GS115(his 4),His+ transformants were recovered and plated on medium containing increasing concentrations of the G418.High copy member of heterogenes was identified by means of PCR and southern blotting to be integrated in chromosome of P pastoris. Conclusion This work offers efficient P pastoris stains for production of CTLA4 extracellular protein for structure/function analyses.