| Background To date,there is no effective treatment for autoimmune diseases and malignant tumors,and drugs or methods with fewer side effects are not available.Recent studies showed that in the process of the occurrence and development of diseases,antigen-specific T-cell plays an important role.CTLA4 is one of the main costimulatory signal negative regulatory molecules,which can block the activation of T cells.On the contrary,antibody against CTLA4 can activate T-cells in vitro and in vivo.CTLA4 and its antibody together interfere with immune microenvironment through the regulation of T-cell,opening up a new therapeutic way to these diseases.Objective The purpose of this study was to obtain highly purified cytotoxic T lymphocyte associated molecule 4 extracellular domain(extracellular domain of T-lymphocyte-associated cytotoxic protein,ex CTLA4).The prokaryotic expression vector of p ET-28a-ex CTLA4 was constructed and the recombinant protein was expressed in Transetta(DE3).The recombinant ex CTLA4 protein was successfully purified,providing the basis for studying its function and screening humanized antibody against CTLA4.Methods1.To construct prokaryotic expression plasmid p ET-28a-ex CTLA4.According to the sequence of human CTLA4 gene,extracellular domain of CTLA4 was synthetized by the company.After PCR amplifications,the PCR products were inserted into the p ET-28 a vector within multiple cloning sites.2.To check the expression of ex CTLA4 recombinant protein in small scale.The recombinant ex CTLA4 plasmid was transformed into Escherichia coli Transetta(DE3),and induced with 0.5m M IPTG at 37°C.The engineering bacteria were harvested after4h-culture,and the expression of the recombinant protein was detected.3.To optimize the expression conditions of ex CTLA4 recombinant protein.The induction conditions were optimized in three aspects: IPTG concentration,induction temperature,and induction time.Finally,the most optimal expression conditions were determined by SDS-PAGE.4.To purify ex CTLA4 recombinant protein.The most optimal expression cond itions were used for recombinant protein production.After bacteria harvest and ultras onic disruption,the bacteria inclusion bodies were firstly washed to remove most of th e background of proteins with low concentration of urea(2M),and then were dissolved in high concentration of urea(8M).After that,nickel ion affinity chromatography was employed to purify ex CTLA4.5.The purified recombinant ex CTLA4 protein was refolded using gradient dilution of urea.6.To examine the biological activity of the recombinant protein ex CTLA4.CTLA4 ligand B7-1 and peripheral blood mononuclear cells(PBMCs)were used to detect the biological activity of ex CTLA4 with ELISA kit.Results1.The recombinant expression plasmid p ET-28a-ex CTLA4 was successfully constructed,and the result of the sequencing confirms that the gene of interest is correct.2.The recombinant protein ex CTLA4 was highly expressed and the most optimal condition for ex CTLA4 expression in Transetta(DE3)is 0.75 m M IPTG,25°C,and induction of 6h.However,the recombinant protein was expressed mainly in the form of inclusion body.3.The inclusion body of CTLA4 was successfully refolded and refolded CTLA4 recombinant protein was further purified by Ni-affinity chromatography.The following Western blot analysis identified that the purified protein is ex CTLA4.4.Purified ex CTLA4 recombinant protein has biological activity of CTLA4,as suggested by ELISA test.Conclusion This study investigated the optimal expression conditions of recombinant ex CTLA4 protein in Escherichia coli Transetta(DE3),and we successfully prepared recombinant ex CTLA4 protein after refolding of inclusion body and nickel ion affinity chromatography.Moreover,the purified ex CTLA4 recombinant protein was shwon to have biological activities of CTLA4.Our work will facilitate the application of the protein and provide necessary basis for screening humanized CTLA4 monoclonal antibodies in future. |
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