Preparation And Research For Immunogenicity Of Low Activity Human Tumor Necrosis Factor

As a pleiotropic cytokine mediated of inflammatory reaction and immune regulating,Tumor Necrosis Factor alpha(TNF-α)was closely related to the generation and development of many diseases,such as rheumatoid arthritis and inflammatory enteritis.In recent years,there were five alpha monoclonal which had been marketed and made a success.It means that drugs which are targeting the TNF-α will have a broad prospect.This research aimed at preparing the point mutation of human TNF-α protein which had low bioactivity for vaccine and researching its immunological properties with the TNF-α as targets.On the basis of open literature as well as our experience,we replaced specific amino acid on 8 important amino acid site of human TNF-α,built the plasmid and mutant proteins which were purified by Ni affinity chromatography,and then detected bioactivity of human TNF-α and its mutant proteins through L929 cell survival experiment.We immunized mouse with TNF-α and its mutant proteins,used ELISA to detect the production of antibody in mouse serum,and detected the combination of mutation proteins with antibody which was anti-human TNF-α through ELISA.The results showed that the mutant proteins’ bioactivity of Y119 W,R31T,R32 Y,S95F and Y115 F were respectively reduced to 28.6 %,7.53 %,7.56 %,7.53 % and 7.56 %.The bioactivity of S86 F was down to 0.37 % when it compared to human TNF-α,A145 R had bioactivity lower than S86 F.While all the mutant proteins kept the protein immunogenicity and produced antibodies which could combine with human TNF-α specifically.At the same time,there existed the ability for all mutant proteins to combine with the anti-human TNF-α.