Red Panda Ifn-Γ And IL-17Prokaryotic Experssion And Anti-viral Activity Of IFN-Γ

IFN-γ (Interferon-gamma) has a variety of biological activity and secreted by activatedTh1cells, CT8cells and NK cells. Promoting the activation of macrophages, fibroblasts andepithelial cells synthesize moleces and interfere with viral proliferation of involved in theclearance of pathogenic microorganisms. Interleukin-17(IL-17) is mainly composed of Th17cells secrete a cytokine with mtiple functions, and its main functions include target cells tosynthesis and secretion proinflammatory cytokines and chemokines involved in the bodyimmune clearance of pathogens. Red pandas in captivity were prone to infecting malignantdiseases distemper, parvovirus and a variety of parasitic diseases. Hence the urgent need for apanda for having anti-virus, anti-parasitic drugs and enhance immunity.In this study, red panda peripheral blood lymphocytes stimated by ConA to obtain totalRNA, specific primers were designed based on gaint panda gene sequences and cDNAsynthesized by reverse transcription PCR (RT-PCR), amplified and cloned IFN-γ and IL-17gene inserted into a cloning vocter pGEM-T and experssion vocter pET-32a (+). Therecombinant expression vectors transformed into prokaryotic expression strain BL21(DE3),and fusion protein red panda IFN-γ (RpIFN-γ) and red panda IL-17(RpIL-17) induced byIPTG and expression conditions detected by SDS-PAGE. Rabbits were immuned by RpIFN-γpurified for polyclonal antibody. F81and MDCK cells were detected RpIFN-γ inhibitedreplication of CAV and CPV respectively. The rests obtained are as follows:1. Cloned panda IFN-γ and IL-17gene was501bp and462bp, and the similarity of thepanda was95.4%and93.7%, respectively,167and152encode the amino acid residue.2. Constructed the recombinant expression vector (pET-32a-IFN-γ and pET-32a-IL-17),the optimum express condition that IPTG concentration0.5mmol/L, temperature andinduction time was16℃38h, and25℃20h.3. Purified by nickel column RpIFN-γ expression product and RpIL-17, west-blotidentified a molecar weight of approximately38ku and35.5ku.4. Immunofluorescence test and real-time PCR test proved that the role RpIFN-γ at2-fold dilution of the most obvious effect has not been seen after64diluted.The above rests show that the study successfly cloned red panda IFN-γ and IL-17gene,reconstructed prokaryotic expression vector; find out the optimal expression conditions of pET-32a-IFN-γ and pET-32a-IL-17, obtained purified RpIFN-γ and RpIL-17recombinantprotein; proved RpIFN-γ has good antiviral activity. The rests for improve clinical red pandaimmune function and enhance their resistance to disease provides a new basis...