Construction, Expression And Identification Of Biological Activity Of Domestic Tumor Necrosis Factor (TNF-?) Mutant Gene

Tumor necrosis factor(TNF)-a is a cytokine secreted by activated monocyte-macrophages and is widely involved in biological functions.Thus,TNF-?,is always the hot topic of research since it was found.In view of the TNF-?,two-sided effect in biological activity,there is a lot of limitations in clinical applications of TNF-?,and the means of conquering its limitations are also getting more and more such as mutant TNF-a.Here,we created a mutant TNF-?,In order to conquer its limitations and to increase the activity of antitumor and to decrease the side effect of antitumor,the means are getting more and more such as mutant TNF-?.In this study,we created a mutant TNF-a based on the studies of human TNF-?.In this studies,the full length of coding sequence of pig TNF-a was obtained by reverse transcription PCR,and then a mutant TNF-?(namely PmTNF-a)was created by PCR.In detail,the first 7 amino acids were deleted,and serine at position 8,aspartic acid residues at position 9 and arginine residues at position 156 of pig TNF-a was replaced by lysine residues,leucine and phenylalanine,respectively.Subsequently,the wild type and mutant type TNF-a were used to construct prokaryotic recombinant plasmids to express soluble proteins,and then nickel chelating affinity chromatography was carried out to obtain high purity recombinant proteins.Next,mass spectrometry analysis revealed the molecular weight of mutant TNF-? protein was 16.7 kDa.Compared with wild TNF-?,CCK 8 method and flow cytometry method showed that the tumor cytotoxic activity was significantly increased in L929 cells stimulated by mutant TNF-?,protein,meanwhile,the serum level of lactate dehydrogenase showed that the toxicity was decreased in L02 cells stimulated by mutant TNF-? protein.Finally,the hemolysis activity about these two proteins were also carried out,and which showed that there was no significant difference between these two proteins.In a word,our results indicate that TNF-? mutant(PmTNF-?)protein with higher anti-tumor activity and lower toxicity,which may be used as a potential pig immune reinforcing agent or adjuvant and treatment of disease.