Dvl1及其突变体腺病毒载体的构建与在MSCs中表达鉴定

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。

Construction of Adenovirus Vectors Dvl1 or Its Depletion Variants and Analysis Their Expression in MSCs

To construct recombinant adenovirus vector carrying Dvl1 or variants without the two conserved regions（ΔDIX, ΔDEP） and identify their＇s expression in MSCs. Dvl1, ΔDIX, and ΔDEP cDNA were inserted into pAdTrack-CMV transfer vector, linearized with PmeⅠdigestion and pAdEasy-1 backbone vectors were further cotransformed into the bacteria BJ5183 competent cells for homologous recombination adenoviruses of Ad-Dvl1, Ad-ΔDIX and Ad-ΔDEP. Then they were linearized with PacⅠ digestion and transfected into the human embryonic kidney 293（QBI-293A） cells by LipofectamineTM 2000. The expressions of Dvl1 were examined by western blot and qPCR. Using PCR, PacⅠdigestion and gene sequencing, the genes were verified to be correctly cloned in adenovirus vector. The high expression of green fluorescence protein in QBI 293A cell line wes found under fluorescent microscope. The optimal virus concentration for MSCs was 150 MOI. And the successful infection and expression of Dvl1, ΔDIX, and ΔDEP in MSCs was determined by using qPCR and western blot. The high titer recombinant virus was obtained, and infection and expression of Dvl1, ΔDIX, and ΔDEP in MSCs, which can serve as a tool for the further research of the mechanism of Dvl1 in MSCs migration.