| Background: TNF-a, a cytokine with complex bioactivity, is mainly secreted by macrophages and T cells. Besides its tumoricidal activity in vivo and in vitro, TNF-a also has several other activities including antiviral activity, immuno-effective cell activating, such as macrophages and polymorphonuclear leukocytes, major histocompatible complex antigen express upregulating, the function regulation of immune system, and so on. But high level of TNF-a in the body is critically involved in the pathogensis of many diseases such as autoimmune disease, inflammatory disease, dyscrasia and so on. So neutralizing TNF-a in vivo is a useful approach for the treatment of these diseases. At present, anti- TNF-a moncolonal antibodies and soluble TNF-a receptors are clinically used for the treatment of these diseases. But these drugs have some flaws. They need to be used in large amounts. In addition, they are potentially immunogenic compounds that may elicit antibody responses. Vaccines can overcome aboveflaws. So vaccination against TNF-a may be a useful approach for the treatment of these diseases. AIM: To investigate whether or not the phage displayed TNF-a and GST-TNF fusion protein can be used as vaccines in the treatment of diseases with high level of TNF-a. METHODS: In this study,we have cloned human TNF-a gene into phagemid vector pCENTAB5E and displayed it on the N-terminus of phage minor coat protein pill .Then we have cloned human TNF-a gene into fusion expression vector pGEX-4T-l and the recombinant plasmid were transformed into E.coli DH5 a . The transformants were induced by IPTG for expression. GST-TNF fusion protein was purified with affinity chroraatography. The phage displaying TNF-a and GST-TNF were used to immunize mice to investigate the efficacy as vaccines. RESULT: ELISA, dot blot and competition assays showed that human TNF-a displayed succesfully on the phage surface. The phage displayed human TNF-a was found not to possess cytolytic activity by a standard cytolytic assay with L929 cell compared to that of human recombinant TNF-a. The results of Western blot showed that GST-TNF had been expressed successfully in E.coli. After purification by affinity chramotography, the purity of GST-TNF is about 90.4%. GST-TNF possesses cytolytic activity by a standard cytolytic assay with L929 cell. It is neccesary for vaccine development that the cytolytic activity of GST-TNF is inactivated. The sera of mice immunizad with phage displaying TNF-a or purified GST-TNF respectively showed an anti-TNF-a response was detected by ELISA. CONCLUSION:Recombinant phage-TNF has been successfully displayed. GST-TNF fusion protein has been prepared. Displayed phage-TNF can be used as vaccine directly. GST-TNF fusion protein possesses cytolytic activity of TNF-a and need to be inactivated before vaccination. The anti-TNF-a antibodies were elited in mice after immunization with either particles of recombinant phage-TNF or GST-TNFfusion protein. So phage and GST could be used for immunization, and therefore as a tool for vaccine development. |
