| Retinoic acid inducible gene-1(RIG-1) is a protein that acts as an intracellular RNA receptor andsenses double stranded virus RNA. It is composed of two N-terminal caspase activation and recruitmentdomains, a C-terminal repressor domain and a helicase domain in the middle. RIG-1was firstdiscovered to bind dsRNA and trigger the expression of typeⅠIFN by Yoneyama et al. in2004, andthen key findings have identified that ssRNA containing a5’tri-phosohate motif was required forrecognition by RIG-1. It is reported by Barber et al. that RIG-1plays a role in clearing an influenzainfection and it may be absent in chickens. This study was set to search the antiviral activity of RIG-1and to get the RIG-1Monoclonal antibodies (mAb) with the purpose of doing some preparations to thegeneration and detection of RIG-1transgenic chickens.To investigate the RIG-1gene expression in different tissues and its antiviral activity in vitro, theduck RIG-1gene was amplified by RT-PCR and cloned into pcDNA3.1to construct pcDNA-RIG. Thesequencing result showed that the duck RIG-1gene was2,802bp encoding934amino acids. In addition,the expression of RIG-1in different tissues was qualified by SYBR Green-based real time RT-PCR; theresults showed that mRNA of RIG-1expressed in a high level in the kidney and spleen, and midumlevel in liver, but low level in the heart, lung and muscle. Furthermore, the replication of avian influenzavirus (AIV) was significantly inhibited in pcDNA-RIG-1transfected DF-1cells detected by standardtitration method, indirect immunofluorescence assay and real time RT-PCR. Evidently, the results alsodisplayed that the expressions of IFN-βand Mx were significantly increased in pcDNA-RIG-1transfected DF-1cells, resulting in inhibition of the AIV replication.The mAb plays an important role in immunological detection. To prepare the mAb against duckRIG-1protein and identify the characteristics of the mAb, RIG-1segment were cloned into pET-30a toconstruct pET30a-a, pET30a-b, pET30a-c and pET30a-d recombinant plasmidand then they weretransformed into E. coli BL21(DE3) and induced to express with IPTG. The fusion proteins werepurified by Ni-NTA resin and then they were used to immunize mice to obtain the specific antibody. Theidentification and analysis of mAb was carried in the method of ELISA, Western blot and indirectimmunofluorescence assay. The results show that recombinant expression plasmid was correctlyconstructed and the fusion proteins were expressed and purified;29hybridoma cell lines were obtainedin which28lines have satisfactory antigen-antibody reactivity with prokaryotic expressed RIG-1inELISA detection and9lines have satisfactory antigen-antibody reactivity with eukaryotic expressedRIG-1in Western blot and IFA detection. Evidently, RIG-1protein mAb was prepared and theantibodies have high titers and hydro-affinity.In order to determine indexes of evaluation experiment on anti-AIV transgenic chickens, this studywas set to establish the Highland white chicken model infected with GD1/96AIV by searching themedian lethal dose (LD50) and pathogenicity of H5N1AIV on Highland White Chickens. The resultsshow that LD50of GD1/96AIV on Highland white chickens is104.5EID50, and GD1/96AIV has highpathogenicity to Highland white chicken. Furthermore, high titers of AIV were detected in dyingchickens’lungs and swabs, and the expression of IFN-αand TNF-αwere increased in lungs. According tothe results above, time of death, virus titers in pharynx swabs, rectal swabs and lung tissue and relativeexpression levels of IFN-alpha, TNF-alpha can be used as an index of evaluation experiment foranti-AIV transgenic chickens infected AIV.In this study, we cloned duck RIG-1gene and obtained the RIG-1protein mAb. The results also demonstrated that duck RIG-1has a significant antiviral activity in vitro. This study will lend somereferences to avian RIG-1associated innate immune research and generation of RIG-1genetic chickenand its antivirus research. |
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