【摘要】 目的：跨膜型ＴＮＦα（ＴＭＴＮＦα）易在某些蛋白酶的作用下转换为分泌型ＴＮＦα（ＳＴＮＦα）。拟用基因突变技术获得稳定表达的、不能转换成ＳＴＮＦ的ＴＭＴＮＦ突变体（ＴＭＴＮＦｍ）。方法：应用ＲＴＰＣＲ技术，从人外周血单核细胞中扩增出编码ＴＭＴＮＦα的全长ｃＤＮＡ序列并构建ｐＢＳＫＴＮＦα重组体。以此为模板，借助改进的“一次重组ＰＣＲ”定位突变技术，造成ＴＭＴＮＦ转换为ＳＴＮＦ酶切位点的缺失，获得ＴＭＴＮＦ突变重组体（ＴＭＴＮＦｍ）。结果：将ＴＭＴＮＦｍｃＤＮＡ片段克隆至表达载体ｐＧＥＭ３Ｚｆ，利用体外转录翻译系统表达出具有生物学活性的跨膜突变型ＴＮＦα蛋白。经Ｗｅｓｔｅｒｎｂｌｏｔ分析证实，用胶原酶不能将ＴＭＴＮＦ突变体酶解成为ＳＴＮＦ。结论：提示所构建的ＴＭＴＮＦｍ去除了可转换为ＳＴＮＦ的酶解氨基酸序列，为进一步研究跨膜型ＴＮＦ杀瘤作用奠定了基础。更多还原

【Abstract】 Objective: Transmembrane TNF (TMTNF) can readily be converted by certain proteinase into secretary TNF(STNF). Therefore, in the present study, the authors attempt to make use of mutagenesis technique for constructing a kind of stable expressed TMTNF mutant (TMTNFm) which would not be cleaved into STNF. Methods: A full length of TMTNF cDNA was amplified from the human monocytes by RTPCR and cloned into plasmid pBSK to construct recombinant pBSKTMTNF. pBSKTMTNF mutant was then obtained by deletion of enzymatic site for conversion of TMTNF into STNF with the sitedirected mutagenic method of modified "SingleStep PCR". Results: The mutant was subcloned into expression plasmid pGM3Zf and then in vitro transcribed and translated into protein which displayed its biological activities. As shown by Western blot, TMTNF mutant could not be speciafically hydrolyzed into STNF by collagenase. Conclusion: These results suggested that TMTNFm produced was indeed void of the amino acids sequence that could be enzymatically cleaved laying a foundation for the further study on the antitumor activity of TMTNF.更多还原