Structure And Bioactivities Analysis Of Human Tumor Necrosis Factor Alpha Mutated In Position 30 And 42 Amino Acid

Human tumor necrosis factor alpha(hTNFα),as a pleiotropic cytokine capable of killing tumor cell in vitro and in vivo, has been thought a potential anti-tumor agent, but its side effects limiting its using in clinic. In order to reduce systemic toxicity in human, while still exerting their direct anti-tumor activities, we had developed four hTNFα derivatives with genetic engineering techniques. The structure-function relationships of hTNFα molecules were discussed.The anti-tumor activities of hTNFα were mediated through the specific receptor, which exhibited on some tumor cells surface. When hTNFα receptors were activated by binding of its ligand, they activate a serials signaling cascades and induce the target cells apoptosis. Based on the further analysis of the hTNFα structure, we had developed four hTNFα derivatives by using PCR techniques, they were hTNFα ml: deletion at the N-terminus for eight amino acids; hTNFαm2: ml+Asn30Asp; hTNFαm3: ml+Asn30His; hTNFαM4: ml+Glu42Gly. These mutants had been expressed at high level in E.coli. They were produced in E.coli as soluble protein except hTNFαm4, which produced in cytoplasm as inclusion bodies. After purification, characters of the different mutants were analysised.Western blot and immunoprecipitation tests were used to investigate the abilities of the mutants binding to the wild type hTNFα antibodies. Four hTNFα mutants were recognized by polyclonal antibodies specifically, these results indicated, after we mutated the hTNFα's primary structure, they still keep their basic space conformation. The different autoradiogram bands were displayed when the wild type hTNFα monoclonal antibody bound with the same quantities of radiolabelled mutants, it indicated that the relative binding affinities of the hTNFα mutants to wild type hTNFα monoclonal antibody were different. We had compared...