摘要
目的 :探讨跨膜型TNF α(TM TNFα)的细胞毒作用及其功能域的关系。方法 :采用重组PCR对TM TNFα基因进行定点突变 ,在微粒体膜的存在下体外转录、翻译突变体 ,并观察这些突变体的胞毒作用。结果 :通过重组PCR分别获得TM TNFα第 - 71、31、35、87、95、14 3与 14 7位氨基酸置换的突变体 ,这 7种突变体对L92 9细胞系的细胞毒作用 ,与野生型TM TNFα相比 :第 - 71、14 3位氨基酸单个置换使TM TNFα胞毒作用下降 4倍以上 ,35、95、14 7位单个置换使TM TNFα胞毒作用下降1 5～ 2 5倍 ;而第 31、87与 133位氨基酸突变则对TM TNFα杀伤作用无影响。结论 :除 - 71位氨基酸外 ,以上 6个位点的氨基酸均参与分泌型TNF α(S TNFα)的胞毒功能 ,提示TM TNFα和S TNFα发挥细胞毒功能的功能域可能不完全相同。
Objective:To determine the region of human transmembrane tumor necrosis factor alpha (TM TNFα),essential for cytotoxic activity against mouse L929 cell.Methods:Single amino acid substituted TM TNFα mutant proteins(muteins) were produced by in vitro transcription linked translation techniques.An expression cDNA for TM TNFα was site directed mutagenized by recombinant PCR.7 single amino acid substituted TM TNFα muteins were generated and assayed for cytotoxic activity.Results:The cytotoxic activities of TM TNFα muteins,eg,TM TNFα -71/Lys was <0.01 of that of parent TM TNFα,and TM TNFα 143/Tyr decreased 4 fold.TM TNFα 35/Gly,95/Cys,147/Phe decreased 1.5～2.5 fold respectively,but that of TM TNFα 31/Gly,87/Phe were not changed obviously.Conclusion:These results indicated that the regions related cytotoxic activity of TM TNFα were different with that of secretion TNF alpha(S TNFα).
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2003年第4期225-227,231,共4页
Chinese Journal of Immunology
基金
国家自然科学基金资助项目 (批准号 :3 9870 713 )
关键词
跨膜型
TNF-α细胞毒作用
功能域
关系
Transmembrane TNF α
Receptor
Site directed mutation
Cytotoxicity
Function region
