Research On HFIX Double Screening Site Integration System And Preliminary Exploration Of Two Integrated Related Proteins

Hemophilia B is a kind of hemorrhagic disease caused by the deficiency of human clotting factor Ⅸ(hFⅨ)。Gene therapy is the ultimate therapy method for hereditary disease. However, the immunity [1-4] and safety [1-4] are two main problems should be solved. Previously, long-term expression of clotting factors has been successfully achieved in AAVS1 knock-in mice using rep-RBE gene transfer strategies, and based on this, we constructed a hFⅨ expression plasmid called pCMV-RBE-TK1-N2-EFlα-hFⅨml containing a site-specific integration element RBE and a negative selective gene TK1. AAVS1 site-specific integration hFⅨ stable expression cell lines could be achieved through co-transfecting this plasmid with rep expression plasmid into mammal cell line. Characteristics:a cis-element RBE can mediates rep dependent AAVS1 site-specific integration; cell lines with latent plasmid can be obtained by G418 selection; RBE was located between CMV promoter and TKl gene, non-site-specific gene can be eliminated by ACV; at last AAVS1 site-specific integration hFIX stable expression cell line was successfully achieved. This double plasmids system not only can avoid random mutation and improve the expression efficiency; but also decrease the risk of the potential for insertional mutagenesis and/or insertional activation of proximal genes.Objective:Identify the microRNAs targeting ZNF403 geneMethods:Using microRNA prediction software miRWalk to predict microRNAs targeting ZNF403 gene. luciferase report plasmid of ZNF403 3’UTR was constructed, and dual luciferase activity were detected, preliminary analysis of the possible regulation of ZNF403 gene by miRNAs. At mRNA level, Real-time PCR was used to analysis of the possible regulation of ZNF403 gene by miRNAs.Results:There are 65 possible miRNAs targeting ZNF403 3’UTR predicted by miRNA target gene prediction software miRWalk. According to the prediction results and related papers, we selected four from them and they are miR-23, miR-135, miR-199 and let-7. Dual luciferase assay showed that relative luciferase activity was not affected by miRNA, and no significance difference. Real-time PCR result was consistent with luciferase assay.Conclusion:ZNF403 was not down regulated by miR-23, miR-135, miR-199 and let-7g, ZNF403 3’UTR was lack of these four microRNAs’target sites.The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G1 phase of the cell cycle#。 Activatior Cdc20 binds on key mitosis substrates which would be ubiquitinated by APC/C and then degraded. As important components of PML-NBs, Sp100 takes part in important cell activity for example anti-virus, transcription regulation and apoptosis. Our previous work found that Cdc20 mediated Sp100 degradation in ubiquitin-proteasome pathway, and D-box of Sp100 plays a role in the process of substrate recognition. However, there are typical D-box sequence existing in PML and Daxx, both of them cannot be recognized by Cdc20. While Sp100 D-box residues was weak conserved across all species analyzed. In this study, a set of Sp100 truncation mutants with D-box were established to investigate the necessary sequences for the substrate recognition of Cdc20.We found that the truncation GFP-Sp100(152-182 aa)that contained the D-box cannot be degraded by the APC/C-Cdc20. Further, we found a mutation GFP-Sp 100(151K→G) that damaged the potential ubiquitin binding site was also cannot be degraded by the APC/C-Cdc20. But several potential ubiquitin binding sites far from D-box presented no effect on Sp100 degradation. Our results suggested that a D-box nearby ubiquitin binding site(151K)of Sp100 might be necessary for its Cdc20 mediated substrate degradation.