摘要
目的：跨膜型ＴＮＦα（ＴＭＴＮＦα）易在某些蛋白酶的作用下转换为分泌型ＴＮＦα（ＳＴＮＦα）。拟用基因突变技术获得稳定表达的、不能转换成ＳＴＮＦ的ＴＭＴＮＦ突变体（ＴＭＴＮＦｍ）。方法：应用ＲＴＰＣＲ技术，从人外周血单核细胞中扩增出编码ＴＭＴＮＦα的全长ｃＤＮＡ序列并构建ｐＢＳＫＴＮＦα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ＴＭＴＮＦ转换为ＳＴＮＦ酶切位点的缺失，获得ＴＭＴＮＦ突变重组体（ＴＭＴＮＦｍ）。结果：将ＴＭＴＮＦｍｃＤＮＡ片段克隆至表达载体ｐＧＥＭ３Ｚｆ，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ＴＮＦα蛋白。经Ｗｅｓｔｅｒｎｂｌｏｔ分析证实，用胶原酶不能将ＴＭＴＮＦ突变体酶解成为ＳＴＮＦ。结论：提示所构建的ＴＭＴＮＦｍ去除了可转换为ＳＴＮＦ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。
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.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
1999年第6期254-256,共3页
Chinese Journal of Immunology
基金
国家自然科学基金
关键词
跨膜型TNF-Α
免疫
TM-TNFm-α
体外转录翻译
Transmembrane TNF(TMTNF)Transmembrane TNF mutant (TMTNFm) Sitedirected mutagenesis
by singlestep PCRIn vitro translation
