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目的拟借助微粒体膜和体外转录翻译系统来研究分泌型ＴＮＦα（Ｓ－ＴＮＦ－α）的产生机制及Ｓ－ＴＮＦ－α和跨膜型ＴＮＦα（ＴＭ－ＴＮＦ－α）的关系。方法首先将分子量为１７×１０３Ｓ－ＴＮＦ（不含编码ＴＮＦ信号肽的基因）、２６×１０３ＴＭ－ＴＮＦ（含信号肽基因）和１７×１０３Ｓ－ＴＮＦ突变体（Ｓ－ＴＮＦｍ，用ＩＬ－２信号肽密码子置换ＴＮＦ信号肽序列）的全长ｃＤＮＡ片段分别亚克隆于含Ｔ７启动子的ｐＧＥＭ－３Ｚｆ载体，然后利用体外转录翻译系统，在微粒体膜存在和不存在的情况下，体外翻译合成Ｓ－ＴＮＦ、ＴＭ－ＴＮＦ及其Ｓ－ＴＮＦｍ。结果经Ｗｅｓｔｅｒｎｂｌｏｔ分析结果表明：微粒体的存在并不改变２６×１０３ＴＭ－ＴＮＦ的分子量，但微粒体的存在能将Ｓ－ＴＮＦｍ上的ＩＬ－２信号肽切除，证实ＴＮＦ引导序列与一般分泌性蛋白的“信号肽”不同，在粗面内质网翻译过程中不被切除。进一步酶切分析表明ＴＭ－ＴＮＦ是通过某些金属蛋白酶酶解作用而被转换成Ｓ－ＴＮＦ的。结论实验结果提示１７×１０３Ｓ－ＴＮＦ产生机制可能是：ＴＮＦ产生细胞经ＬＰＳ等激活后，导致ＴＮＦ基因转录翻译增加，首先形成２６×１０３ＴＮＦ，并借助其信号肽疏水氨基酸部分将之“锚定”?
Objective To investigate the mechanism of secreting TNFα (S-TNF-α) and the relationship between S-TNF-α and transmembrane-type TNFα (TM-TNF-α) by means of microsomal membrane and in vitro transcriptional translation system. Methods First, the 17x103S-TNF (excluding the gene encoding the TNF signal peptide), the 26x103TM-TNF (containing the signal peptide gene) and the 17x103S-TNF mutant Peptide codon replacement TNF signal peptide sequence) were subcloned into the pGEM-3Zf vector containing the T7 promoter, respectively, and then translated in vitro using the in vitro transcriptional translation system in the presence and absence of microsomal membranes S-TNF, TM-TNF and S-TNFm. Results Western blot analysis showed that the presence of microsomes did not change the molecular weight of 26 × 103TM-TNF, but the presence of microsomes cleaved the IL-2 signal peptide of S-TNFm and confirmed that the interaction between TNF-targeting sequences and general secreted proteins Of the “signal peptide” is different from the rough endoplasmic reticulum translation process is not removed. Further digestion analysis revealed that TM-TNF is converted to S-TNF by enzymatic hydrolysis of certain metalloproteases. Conclusion The experimental results suggest that the mechanism of 17 × 103S-TNF production may be as follows: After TNF-α-producing cells are activated by LPS and other factors, the transcript of TNF gene is increased. First, 26 × 103TNF is formed and “anchored” by the hydrophobic amino acid part of its signal peptide ?