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Dvl1及其结构缺失突变体ΔDIX(dsh and axin)和ΔDEP(dsh,Egl-10 and pleckstrin)腺病毒载体的构建并验证其在MSCs(mesenchymal stem cells)中的表达情况。将目的基因定向克隆至pAdTrack-CMV穿梭载体上,并经PmeⅠ线性化后在BJ5183细菌中与pAdEasy-1骨架质粒同源重组,获得重组腺病毒载体,在QBI-293A细胞中包装及扩增,实时荧光定量PCR和Western bolt验证Dvl1在MSCs中的表达情况。经PCR、PacⅠ单酶切鉴定及测序分析,成功构建Ad-Dvl1、Ad-ΔDIX和Ad-ΔDEP腺病毒载体,目的基因序列与GenBank报道一致,并选出150 MOI为最适感染MSCs的感染复数,成功构建了Ad-Dvl1、Ad-ΔDIX和Ad-ΔDEP腺病毒载体,并获得高滴度的病毒子,qPCR和Western blot证实Dvl1在MSCs中高表达并增加了β-catenin在细胞中的累积,为进一步研究Dvl1在MSCs迁移过程的作用奠定了基础。
Dvl1 and its structural deletion mutants ΔDIX (dsh and axin) and ΔDEP (dsh, Egl-10 and pleckstrin) adenovirus vector and verified its expression in MSCs (mesenchymal stem cells). The target gene was cloned into the pAdTrack-CMV shuttle vector and linearized with PmeI. The recombinant plasmid was homologously recombined with the pAdEasy-1 backbone plasmid in BJ5183 bacteria to obtain a recombinant adenovirus vector, which was then packaged and amplified in QBI-293A cells. Real-time quantitative PCR and Western bolt verified the expression of Dvl1 in MSCs. Ad-Dvl1, Ad-ΔDIX and Ad-ΔDEP adenovirus vectors were successfully constructed by restriction enzyme digestion with restriction endonucleases (PCR) and PacⅠ restriction enzyme digestion and sequencing. The sequence of the target gene was reported in GenBank and 150 MOIs , Successfully constructed Ad-Dvl1, Ad-ΔDIX and Ad-ΔDEP adenovirus vector and get high titers of virus. QPCR and Western blot confirmed that Dvl1 is highly expressed in MSCs and increases the accumulation of β-catenin in cells, Which laid the foundation for the further study on the role of Dvl1 in MSCs migration process.