Preparation Of Nrf2 Monoclonal Antibody And Its Application In The Study Of EIAV Infection And The Regulation Of Nrf2 Antioxidant Pathway

As the master regulator of cellular defence mechanism against oxidative stress,Nrf2-ARE(transcription factor NF-E2-related factor 2)plays a crucial role in the cytoprotection against the cumulative damaging effects of oxidative stress induced by virus infection.Importantly,cross-talking with other innate immune signals hightlights the role of Nrf2 pathway in regulating the virus replication.Hence it has also become the potential therapeutic targets of oxidative stress-related diseases(include viral diseases,cancer,cardiovascular diseases and inflammation).The equine infectious anemia virus(EIAV)is a member of the genus Lentivirus,family Retroviridae together with human immunodeficiency virus(HIV-1)and simian immunodeficiency virus(SIV).The tipical clinical symptoms induced by lentivirus are repeated fever,persistent infection,swarm inflammation and difficult to produce effective immune protection.Therefore,it is still an urgent problem about how the host overcome the persistent infection,regulating inflammation and producing effective immune control.Equine infectious anemia virus(EIAV)has become a critical model for the study of lentivirus and host immune regulation because 90% infected-horses can acquire the natural immunity from host against dissemination.Thus the unique character of lentiviral EIAV could provide a comprehensive researching platform for oxidative stress pathway.Our preliminary works have revealed the related genes of Nrf2 antioxidant pathway were significantly up-regulated after EIAV infection.How this complicated network regulation remained completely unexplored.Therefore this study attempted to conduct a systematic research on Nrf2-ARE signaling pathways regulated by EIAV infection.In order to implement the study,we firstly obtained horse Nrf2 monoclonal antibody using classical antibody preparation technique.The highly sensitive and specific monoclonal antibody can effectively detect the overexpressed and endogenous Nrf2 protein,and can be used for subsequent mechanism research.Using Nrf2 monoclonal antibody,Nrf2-ARE-luc report system was constructed and used to confirm that EIAV Rev effectively activated Nrf2-ARE pathway combined with the evaluation of Nrf2 phosphorylation and its distribution in cytoplasm and nucleus.The results of Co-IP showed that Rev combined with Keap1,the negative regulator of Nrf2,and the binding domain of Rev and Keap1 is highly similar to that of Nrf2-Keap1.This result s?ggested that these three proteins might be competitive bound with each other.Then,these three expression plasmids were constructed.After expression,purification and biotin labeling,the binding affinity and competitiveness of Rev-Keap1 and Nrf2-Keap1 were tested.The results showed Rev binded to Keap1 to compete the binding of Nrf2 and Keap1.Consequencely,this competitive relationship promotes Nrf2 transportion from the cytoplasme to nucleus.In addition,overexpression of Nrf2 significantly inhibited EIAV replication,indicating that the replication of EIAV could be negatively regulated by Nrf2 activation.In conclusion,we successfully prepared horse Nrf2 monoclonal antibody,and clarified that EIAV Rev competed the combination of Keap1 and Nrf2 by binding with Keap1,resulting in the weak combination of Keap1 to Nrf2,and the entry of Nrf2 into the nucleus and binding with the downstreamrole element ARE.Furthermore,our findings could enhance the comprehension of host national immunity against EIAV dissemination.Therefore,the findings could address the strategy of antiviral treatment via regulation Nrf2-ARE antioxidant pathway as a new potential target.