1.Study On The Interaction And Their Interaction Mechanism Between Keap1 And P65 2.Prokaryotic Expression And Purification Of Different Truncated Protein Of Mayven

Our preliminary study that yeast two hybrid system (Y2H) was used to screen adult liver cDNA library found the interaction between Keap1 and p65. This research aims to comfirm the authenticity of interaction between Keap1 and p65 and to explore their interaction mechanism.CHAPTERⅠAUTHENTICITY OF THE INTERACTION BETWEEN KEAP1 AND P65Objective: To confirm the authenticity of the interaction between Keap1 and p65.Methods: Yeast retransforming, Co-immunoprecipitation and fluore- scence co-localization methods were performed.Results: The interaction between Keap1 and p65 has been proved by yeast retransforming and Co-immunoprecipitation methods. It was found that Keap1 co-localize in the cytoplasm with p65. Moreover, it was indicated that the interaction between Keap1 and p65 needs the full length of Keap1.Conclusion: The interaction between Keap1 and p65 is authentic and special.CHAPTERⅡIMPACT OF THE INTERACTION BETWEEN KEAP1 AND P65 ON NF-κB SIGNALING PATHWAYObjective: To explore whether the interaction between Keap1 and p65 could affect the NF-κB signaling pathway.Methods: The effect of exogenous Keap1 on p65 transcriptional activation was studied by Dual-Luciferase Reporter Assay System. In addition, the effect of exogenous Keap1 on p65 protein level was analyzed by Western Blot.Results: Our findings suggested that Keap1 have no effect on the transcriptional activity of p65 both in the resting state and in the stimulating state. Moreover, Keap1 have no effect on the protein level of p65.Conclusion: The interaction between Keap1 and p65 has no significant effect on the transcriptional activity and protein level of p65. CHAPTERⅢIMPACT OF THE INTERACTION BETWEEN KEAP1 AND P65 ON KEAP1-NRF2-ARE SIGNALING PATHWAYObjective: To explore whether the interaction between Keap1 and p65 could affect Keap1-Nrf2-ARE signaling pathway.Methods: The effect of p65 on Nrf2-mediated transcription was investigated by Dual-Luciferase Reporter Assay System.Results: We found that p65 inhibited the transcriptional activation of Nrf2 and enhanced the inhibitory effect of Keap1 on Nrf2. Moreover, p65, Keap1 and the complex of p65 and Keap1 all can inhibit the Nrf2-mediated transactivation of NOQ1 promoter.Conclusion: The interaction between Keap1 and p65 enhances the inhibitory effect of Keap1 on the transcription of Nrf2.CHAPTERⅣMECHANISM OF P65 ENHANCING THE INHIBTORY EFFECT OF KEAP1 ON NRF2Objective: To explore the mechanism of p65 enhancing the inhibitory effect of Keap1 on the transcription of Nrf2.Methods: The effect of p65 on the translocation, the transcription level and the protein stability of Keap1 was examined each by indirect immunofluorescence , semiquantitative RT-PCR and protein stability method.Results: Both in resting state and in TNFαstimulating state, p65 couldn't affect the translocation of Keap1. p65 have no effect on the transcription level of Keap1. p65 could increase the protein stability ofKeap1.Conclusion: P65 enhancing the inhibitory effect of Keap1 on Nrf2 maight be attribute to p65 augmenting the protein stability of Keap1.PARTⅡPROKARYOTIC EXPRESSION AND PURIFICATION OF DIFFERENT TRUNCATED PROTEIN OF MAYVENObjective: To understand the function of Mayven and explore the pathogenesis of multiple sclerosis.Methods: Several prokaryotic vector which expressed different truncated fragments of Mayven were constructed.And these protein were expressed in prokaryotic system.Results: Recombinant express vectors of different truncated GST-Mayven were successfully constructed and were expressed in soluble form protein induced by IPTG. The fusion proteins have good reactivity to GST antibody.Conclusion: The construction of recombinant express vectors of different truncated GST-Mayven lay a basis for further function study on Mayven.