The Preliminary Study Of The Impact Of Various Proteins For TNFα Immunogenicity

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.But these drugs have some flaws.They need to be used in large amounts, require repeated used for a long time. They increase the risk of infection such as tuberculosis, and bacteria. Meanwhile, these expensive price also make the kinds of drugs are not widely used. According to statistics, the average annual cost of clinical patients receiving TNF monoclonal antibody therapy more than $ 20,000, such expensive medical bills prohibitive to many patients.At the same time, these drugs expensive price and make the kinds of drugs can not be widely used.According to statistics, TNF monoclonal antibody drug therapy clinical patients spend more than $20000 per year on average, such expensive medical bills for many patients.Vaccines can overcome above flaws.So vaccination against TNF-αmay be a useful approach for the treatment of these diseases.TNF-Kinoid vaccine is a kind of protein chemical conjugate vaccine.It based on a vaccine comprised of a keyhole limpet hemocyanin-h TNFα heterocomplex immunogen(h TNFα kinoid)adjuvanted in incomplete Freund’s adjuvant,and treated with formaldehyde to inactivate TNF biological activity.After 10 years of research and development, it can produce a good immune response in mice, and have good effect of neutralizing antibodies.But the Phase II clinical results did not achieve good results in 2014.Analyze the reasons may be because larger vaccine menace process changes its molecular conformation, destroyed the vaccine antigen, it did not produce for the TNF neutralizing antibodies.Due to its biological activity, if directly used in vaccines, TNF will produce some side effects to human body. So after our previous point mutation screening, we greatly reduced its activity named TNFY119N(abbreviated to TNFt). We connect different carrier protein and TNF by chemical coupling methods to study the effect of different carriers on the TNF protein immunogenic.In this study, TNF-α as a target was fused to the C-terminal and N-terminal of CTB using homologous recombination.TNF-CTB and CTB-TNF was purified by affinity chromatography method after expression in E. coli TG1. We connect different carrier protein and TNF by chemical coupling methods. Immunized Balb / C mice with recombinant proteins and protein conjugates, used ELISA to detect the production of antibody in mouse serum and the titer of antibodies. Results show that,all proteins are able to produce antibodies and antibody titer greater than 1 × 105.Meanwhile, the antibody titer of CTB-TNF kinoid and CTB-TNF coupling itself protein are greater than TNF coupling itself. Other groups antibody titer are lower than the control group.So we can preliminary assessment cholera toxin B subunit(CTB) can be used as a better carrier protein to enhance the immunogenicity of TNF in the body.