The Role Of Conservative Sequences Of TNF Superfamily In Their Trimer Formation And Biological Effect

Tumor necrosis factor alpha (TNF-α), is a pleiotropic cytokine with a wide biological activities produced mostly by activated T cells, monocytes/macrophages and NK cells. TNF-αbelongs to tumor necrosis factor superfamily. Although the members of TNF superfamily have the different biological effects, but their form of biological activity requires the formation of trimeric forms. In the sequence 46-58, 119-130, 150-157 of TNF superfamily members there is a very high homology, and they are the highly conserved amino acid residues district. As the molecular structure similarity existing between TNF superfamily is the highly conserved domains, we conjecture that the domains contribute to the trimerization.In this study, mutant TNF-αgenes will be amplified through recombinant ploymerase chain reaction (PCR) from pcDNA3.0-wtTNF-αas a template by site-directed mutation, and try to influence the formation of trimer, which will provide clues for further study of the relationship between the structure and function of TNF-αand give supports to the reconstruction of TNF-αwhich may enhance its biological activities and its clinical application in the future. This experiment have chosen the 58(I58Y) and 124(F124S) of the conserved sequence to be mutated.Then the mutant pIREs-GFP-Xho1-TNF-αrecombinant plasmid were constructed and transfected into 293T cell lines. We can investigate the changes of the cytotoxicity of mutant mTNF-αcompared with wtTNF-α. The major results in the present study are as follows:1. Construction, cloning and identification of mutant pcDNA3.0-TNF-αrecombinant plasmids(1) Construction and cloning of mutant pcDNA3.0-TNF-αrecombinant plasmid: Using the plasmid pcDNA3.0-wtTNF-αas a template, the gene mutant mTNF-αwas amplified with recombinant ploymerase chain reaction (PCR) by site-directed mutation(I58Y and F124S). After digestion with endonuclease(sBamHI and XhoI), the TNF-αfragment was inserted into pcDNA3.0 plasmid by T4 DNA ligase. Then the connected products were transformed into E. coli DH5αand the positive clones were screened by Ampicillin resistance. (2) Identification of positive clones: The positive clones were confirmed firstly by colony PCR, and followed identification with endonucleases digestion, by further DNA sequence analysis also proved that the recombinant contains sequences which sited mutated at positon 58, 124 of TNF-α, suggesting expected mutant pcDNA3.0-TNF-αrecombinant plasmid was successfully constructed.2. Construction, cloning and identification of mutant pIREs-GFP-Xho1-TNF-αrecombinant plasmidsDigest pIREs-GFP-Xho1 plasmid with BglⅡand XhoI sequencely. Digest mutant pIREs-GFP-Xho1-TNF-αrecombinant plasmid with endonucleases BamHI and XhoI. Then join the former`s large fragment and the latter`s small fragment by T4 DNA ligase. Transformed the recombinant plasmid into E. coli DH5αand the positive clones were screened by kanamycin resistance. Then confirm the positive clones by colony PCR.3. The changes of the cytotoxicity of mutant TNF-αcompared with wtTNF-αWe used the 293T cells expressed mutant TNF-αand wtTNF-αrespectively as effector cells and used MCF-7 as target cells, then compared the cytotoxicity of mutant TNF-αand wtTNF-αby MTT. The cytotoxicity results of I58Y and F124S mutants are13% And23% respectively, which indicated that the I58Y,F124Smutants can sharply decrease the cytotoxicity of wtTNF-αby 67%,57%(P<0.01) respectively。In summary, mutant TNF-αhave much lower cytotoxicity on MCF-7 than the wild type of it. The differences of biological effect between mutant TNF-αand wtTNF-αindicate that the mutant site play an essential role in formation of TNF trimer. The conclusions need to beαfurther testing of TNF trimer-forming ability to verify. This study provides a basis and tool for the research of the TNF-αtrimer.