Cloning And Expression Of Human FOXP3 CDNA In Escherichia Coli

Chronic HBV infection is one of the major health concerns around the world. The understanding on the mechanism of chronic HBV infection is the foundation for developing a novel effective treatment. The research results indicate that the suppression of specific immune function is a key factor for HBV chronic infection. However, the machanism of this suppressive immune response still remains unclear. The maintainance of immunotolerance is considered to depend on central tolerance and peripherial tolerance, and the regulatory T cell (Treg) has great role in maintaining immune tolerance and stablizing immune response. Treg cell is related to the low specific immune-response in chronic HBV infection patients.FOXP3(the forkhead/winged-helix transcriptional regulators P3) is expressed specifically in regulatory T cells, and exerts great influence on the development and function of Treg cells. Currently, however, very little is known about the mechanism of FOXP3 regulates the development and function of Treg cells. The elucidation on the problems is very important for developing new treatments targetd on Treg for autoimmune diseases, tumors, and chornic infectious diseases including chornic hepatitis B. In this study, human FOXP3 cDNA was successfully cloned, whereafter, prokaryotic expression vector for FOXP3 protein was constructed and successfully expessed in E.coli. It would lay foundation for the purification of FOXP3 protein and the exploration of its immunological function.Methods: Total RNA was extracted from the mononuclear cells which were havested from cord blood. Then, FOXP3 cDNA was amplified by nested RT-PCR. After purifying, PCR products were inserted into pMD18-T vector by T-A cloning, and the plasmids were confirmed by sequencing. Next, recombinant prokaryotic expression vector pRSET-A-FOXP3 was constructed, and then transformed into E.coli. BL-21(DE3) pLysS. 6×His fusion Foxp3 protein was expressed under the induction of IPTG.. Finally, expression products were examined and identified by SDS-PAGE and Western blot.Results and Conclusions: The cDNA of FOXP3 was successfully cloned. The prokaryotic expression vector of Foxp3 protien was constructed and expressed efficiently in E.coli confirmed by SDS-PAGE and western blot.