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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,SaLⅠ线性化后电转GS115 ;G418梯度筛选转化菌。PCR、Southernblotting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
OBJECTIVE: To construct a secreted recombinant expression vector of Pichia pastoris in human extracellular domain of CTLA 4 and to obtain a high copy stable and stable strain. This study will lay the foundation for the functional study and clinical application of recombinant human CTLA 4 extracellular domain. Methods The human extracellular domain of human CTLA 4 gene was amplified from the total RNA of human peripheral blood lymphocytes. The target gene was inserted into the downstream of pPIC9α secretion signal of yeast expression vector and inserted into pPIC9K vector to obtain pPIC9K CTLA4 and SaL Ⅰ linearization After electroporation GS115; G418 gradient screening transformed bacteria. PCR, Southern blotting to identify the target gene integration and copy number. Results The human CTLA 4 extracellular domain of Pichia pastoris expression vector was successfully constructed and a stable, high-copy stable strain was obtained. Conclusions provide a basis for the study of the extracellular domain of human CTLA 4 and its function and clinical application