Cloning And Expressing Of Porcine FC Gamma Receptor Ⅲ And Its Subs -id Iary Protein

The FcRs are cell surface molecules that bind specific the Fc region of immunoglobulins on immune cell surface.Fc receptors can trigger and regulate a variety of immunological effects of the body by ligand binding.Fc receptor is linkages between humoral immunity and cellular immunity.Fc receptors play a very key role in the immune defense.To study the biological function of porcine FcγRIII and FcγRIII-mediated immune response,in this study, cDNA of FcγRIII and its subsidiary proteinβsubunit,γsubunit gene were cloned by RT-PCR from total RNA of porcine alveolar macrophages (PAM) and homology analysis was carried out. The results show that The nucleotide sequence of porcine FcγRIII,βsubunit,γsubunit respectively shared 99.9%,99.5,100% homology with the reference sequence AF372453, AF525403, AF148221.The fragment of gene encoding porcine FcγRIII,FcγRIII extracellular andγsubunit was subcloned into the prokaryotic expression vector pET-32a to construct the recombinant expression plasmid pET-SFR1 (FcγRIII genome), pET-SFR2 (FcγRIII extracellular) and pET-SFRγ(γsubunit) and expressed in E. coli . BL21 (DE3). A size of about 48kDa the SFR1-His (FcγRIII fusion protein), 43kDa SFR2-His (FcγRIII extracellular domain fusion protein), and approximately 29kDa the SFRγ-His (γsubunit fusion protein) were obtained by the IPTG induction. we found SFR1-His and SFR2-His exist in form of inclusion bodies, while most of SFRγ-His present in the supernatant. Then the target protein SFR1-His and SFR2-His were purified by the buffer contained urea and refolded by dialysis method so that the target protein purity can reach 87% and 83%. the fusion protein SFRγ-His was purified by the means of His-bind resin protein purification procedureso that purity of protein can reach 92%. Mices were immunized with recombinated protein SFR1-His, SFR1-His and SFRγ-His to obtain the murine antibodies against SFR1-His ,SFR1–His and SFRγ-His fusion protein ; The antibodies were tested by indirect ELISA and analyzed by Western blot.ELISA result showed obtained antibody titers respectively were 1:16000 and 1:16000,1:8000 and Western blot analysis showed that the recombinant protein has good immunogenicity. Western blotting and ELISA staining demonstrated that the polyclonal antibody was obtained by immunizing mouse with the purified recombinant protein. The preparation of the polyclonal antibody against porcine FcγRIII lays a foundation for the further study on the function of porcine FcγRIII protein.FcγRIII andγsubunit gene were subcloned into eukaryotic expression vector pcDNA3.0 to construct the expressing plasmid pcDNA-SFR1 and pcDNA-SFRγ. Respectively COS-7 cell were transfered with pcDNA-SFR1 and co-transfected with pcDNA-SFR1 and pcDNA-SFR by liposome method,COS-7 cells transfected with empty vector pcDNA3.0 as a negative control, After 48h, transfected COS-7 were detected by flow cytometry and immunoglobulin binding assay . Results show that pocine FcγRIII were not detect on cell membrane of COS-7 transfected with pcDNA-SFR1and COS-7 transfected with pcDNA-SFR1 and pcDNA-SFRγ. Pocine Fc gamma R III expression on cell membrane might need other accessory proteins help.