Research On RIG-I Transgenic Chicken Model Establishment And Inhibition Of Influenza Virus Activity

RIG-I is a key innate immune pattern-recognition receptor, which can identify intracellular RNA and induce the production of interferon, cytokines and chemokines. The study was set to establish RIG-I transgenic chicken model, through the research of cellular level to reveal in expressing RIG-I of F1 generation CEF cells reestablish the RIG-I signaling pathways, and to inhibit the action of the influenza virus.This study using the equator fenestration, conformed to injections of standard drops restructuring lentiviral through micro needle injected into the fertilized eggs blastoderm inferior vena, to successfully produced express the RIG-I transgenic chickens, using the methods of molecular biology PCR, Southern blot, RT-PCR and Western blot respectively in DNA, RNA and protein level for F0 generation transgenic chickens identification. The resμlts show that the fifteen F0 generation of transgenic chickens have the six with integration and expression in exogenous genes; Using F0 generation positive transgenic rooster hen with varieties of wild type crossbreed F1 generation; Using the above methods of molecular biology for appraisal analysis in F1 generation transgenic chickens, The resμlts show that breeding part of the F1 generation transgenic chickens have the seven with integration and expression in exogenous genes. Proved that F0 generation of transgenic chickens carrying exogenous gene can stable genetic F1 generation.Isolation and culture F1 generation of CEF cells, it can be divided into transgenic group(TG), nontransgenic group(NTG) and wild type group(WT) by the method of molecμlar biology identification. With avian influenza virus infect CEF cells, western blot measure RIG-I protein expression, IFA and TCID50 detection virus replication, q RT-PCR test RIG-I and downstream antiviral molecules(PKR, OASL, MX1), proinflammatory cytokines(IFNβ, IRF7, IFIT5) and influenza genes(PB2) mRNA level model. Western blot and qRT PCR resμlts showed that infected TG group CEF cells the RIG-I mRNA level and RIG-I protein expression quantity are higher than uninfected group; TG group compared with WT group, IFN-β increases 2.4 times, other cytokines IRF7, IFIT5, PKR, OASL and MX1 raised 2.1 times, 1.5 times, 1.9 times, 2 times, 1.9 times respectively; IFA, TCID50 and PB2 gene copy number results show that the TG group CEF cells influenza virus proliferation were lower than that of WT group.This study confirmed the expression of RIG-I transgenic chickens have integration and expression in exogenous gene, which can stably inherited to the F1 generation. verified in expressing RIG-I of F1 generation CEF cells reestablish the RIG-I signaling pathways, and to inhibit the action of the influenza virus. For exploring the RIG-I genes involved in poμltry innate immune mechanism and inhibit influenza virus replication role laying a foundation, and for drug development provides new ideas.