The Role of Conservative Sequences of TNF Superfamily in Their Trimer Formation and Biological Effect
|School||Huazhong University of Science and Technology|
|Keywords||TNF-α Trimer Site-directed mutagenesis Cytotoxicity|
Tumor necrosis factor (tumor necrosis factor, TNF-α) by activated T cells, monocytes / macrophages, NK cells produced having a variety of biological functions of cytokines. It belongs to the tumor necrosis factor superfamily members, members of the family, although the biological function of the same, but the amino acid sequence in the primary structure of protein molecules 46-58,119-130,150-157 area highly homologous (height conserved structural domain); addition, they must be in order to the homotrimer form of their corresponding receptors binding can be mediated biological effects. Therefore, we speculate that the TNF superfamily member trimer formation and their amino acid sequence is highly conserved sequence-related. The subject by overlapping PCR method conserved sequences of TNF-α in the first 58 isoleucine mutation of tyrosine (I58Y) 124 phenylalanine mutation of serine (F124S), and the two TNF -α mutants were subcloned into plasmids of pIREs-GFP-Xho1, then the two mutant recombinant plasmids were transfected into 293T cells, MCF-7 cells using the MTT assay TNF-α mutant cytotoxicity, as provide clues depth study of the molecular structure and function of the relationship between TNF-α has important theoretical significance and practical value of basic research and its clinical application of TNF-α. The results are as follows:. PcDNA3.0-TNF-α mutation Body recombinant plasmid, cloning and identification of recombinant build and clone: ??pcDNA3.0-wtTNF-α plasmid as a template the overlapping PCR legal point mutant 58 (I58Y), 124 (F124S), the target gene and pcDNA3.0 vector with BamHI and XhoI double digestion, and ligated with T4 DNA ligase, and the ligation product is transformed DH5α was selected by their ampicillin-resistant positive cloning. 2 Identification of positive clones: colony PCR and the restriction enzyme double digestion preliminary identification of positive clones, further DNA sequencing analysis confirmed point mutation succeed, only in the expected site mutation, the rest nucleoside acid sequence is the same as with the wild-type TNF-α sequence. Second. PIREs-GFP-Xho1-TNF-α mutant body recombinant plasmid, cloning and identification will the fluorescent carrier pIREs-GFP-Xho1 plasmid sequence was digested with Bgl Ⅱ and XhoI, In addition to the two mutant recombinant plasmid (I58Y F124S) and pcDNA3.0-wtTNF,,-alpha, double digested with BamHI and XhoI, taking the former digested large fragment of the latter small fragments connected with T4 ligase, and then transformed into DH5α was selected by kanamycin resistance positive clones. Colony PCR positive clones were screened. MTT assay of wild-type and mutant TNF-α cytotoxic effect of the transient transfection of mutant TNF-α and the wild-TNF-α in 293T cells as effector cells, MCF-7 target cells by MTT The method compare their cytotoxic effects. The results showed that the cytotoxic effect of I58Y, F124S two mutants were 13% and 23%, compared to its cytotoxic effect with the wild-type TNF-α decreased significantly reduced by 67% and 57%, respectively. Conclusion 58,124 amino acids: I58Y, F124S mutants can weaken mTNF-α cytotoxicity, suggesting that TNF-α may be associated with the formation of TNF-α trimer conclusions need further testing the forming capacity of the TNF trimer to verify . This study provide the basis for the TNF-α trimer and tools.