Molecular Cloning And Characterization Analysis Of Carp (Cyprinus Carpio L.) FcRγ

Fc receptors present as membrane receptors or as soluble molecules which are named for binding of immunoglobulin Fc(Fragment, crystallizable) domains, they are classified according to the antibodies that they recognize: FcγR associate with IgG, FcαR associate with IgA, FcεR associate with IgE, FcμR associate with IgM associate with FcδR bind IgD. It is well-known that macrophages start to ingest and digest a pathogen coverd with IgG by phagocytosis due to FcγR binding IgG at Fc section. FcγRIIIA (CD16) on the surface of natural killer (NK) cells associate with human IgG1 and IgG3 mostly, this process to kill the antibody coated target cell is named antibody-dependent cell-mediated cytotoxicity (ADCC). FcεRI also have a function to project the granulocytes from parasitic infections. In mammals, FcR includes oneαchain,oneβchain andγhomodimer, Most activating receptors have a short cytoplasmic tail, which associates with the combine receptor to activate intracellular signals. As we known the FcRγplay significant roles in immune reaction through meditating intracellular signals transmiting from variety of cell surface receptors. FcRγwas known to cross-linking of the osteoclast-associated receptor on dendritic cells and monocytes, revealing FcRγmay associate with osteoclast function. Since the FcRγpossesses so many function described above, we must notice that unusual signals transmiting can lead to various diseases.Common carp (Cyprinus carpio L.) was bought from the mart. Total RNAs were extracted from the kidney and other various tissues of carp using Trizol reagent following the user's manual. The quality and concentration of total RNAs were separated by agarose gel electrophoresis。FcRγgene expresses in a wide range of tissues, therefore a RT-PCR methed was performed to detect tissue distribution of FcRγmRA from buccal epithelium, liver, kidney, spleen, intestine, branchia, skin, heart and muscle. Expression of FcRγwas measured in previously non-exposed fish, 1, 2, 3 and 4 weeks after experimental Vibrio anguillarum challenge to determine if they were associated with host response to V. anguillarum infection by real-time quantitative PCR.The first strand of cDNA was served as the template to amplify part of the gene FcRγ. The product of amplification was treated by double endonuclease digestion (BamHⅠand EcoRⅠ) and linked to pcDNA3.1/Myc-His(+) vector. The recombinant expression vector pcDNA3.1/Myc-His(+)/FcRγwas then transformed into E.coli DH5α, and the recombinant expression vector was subjected to Restriction Endonuclease Assay and DNA sequencing. The double-endonuclease-treated recombinant vector broke down into two fragments, the size of which corresponded to pcDNA3.1/Myc-His(+) expression vector and PCR amplification product respectively. Moreover, DNA sequencing demonstrated a sequence completely identical to the gene in GenBank. Plasmids were purified with the EndoFree Plasmid Mega purification kit according to the manufacturer's instructions, aliquoted at 1 mg/mL in sterile endotoxin-free phosphate-buffered saline (PBS). The recombinant vector was adjusted as the DNA vaccines. Antiserum was gained from animal immunization, then double agar diffusion experiments and Western blotting were conducted afterwards. And the titer of the antiserum was measured as 1:4 according to the results of double agar diffusion experiments.In brief, the full coding region of FcRγwas gained by the method of RT-PCR; FcRγplay an important role in innate and adaptive immunity of common carp with Vibrio anguillarum challenge by RT-PCR and real-time PCR; The recombinant vector was adjusted as the DNA vaccines. Antiserum was gained from animal immunization, and the titer of the antiserum gained was 1:4. Immunohistochemistry results indicated that FcRγexisted in a wide range of tissues of common carp.