β-catenin和突变体ΔN90腺病毒载体的构建及其在MSCs中的表达  

Construction of Adenovirus Vector Containing Rat β-catenin or ΔN90Gene and Its Expression in MSCs

作　　者：崔恒进 王春燕[1] 胡君[1] 谢桂琴[1] 徐晓静[1] 张焕相[1] 

机构地区：[1]苏州大学基础医学与生物科学学院江苏省干细胞研究重点实验室,苏州215123

出　　处：《生物技术通报》2013年第11期153-158,共6页Biotechnology Bulletin

基　　金：国家自然科学基金资助项目(30870642;31071220)

摘　　要：旨在研究β-catenin对MSCs迁移的作用,构建β-catenin和突变体ΔN90的重组腺病毒载体,在MSCs中验证其表达情况。将目的基因定向克隆到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细胞中包装和扩增。结果显示,酶切和测序证实Ad-β-catenin和Ad-ΔN90质粒构建正确,荧光显微镜可观察到GFP的表达,并选出150 MOI为最适感染MSCs的感染复数,Western blot检测到Ad-β-catenin和Ad-ΔN90都能增加β-catenin的蛋白表达量,TOPFlash检测到Ad-β-catenin和Ad-ΔN90都能增加β-catenin的转录活性。说明成功构建Ad-β-catenin和Ad-ΔN90腺病毒载体,并获得高滴度病毒子。To construct the recombinant adenovirus vector containing β-catenin or AN90 gene for later research of the mechanism of β-catenin in MSCs migration,β-catenin or AN90 cDNA was inserted into a pAdTrack-CMV transfer vector, linearized with Pme I digestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination adenoviruse Ad- β-catenin or Ad-AN90. Then they were linearized and transfected into QBI-293A cells by LipofectamineTU2000. Through PCR, endonuclease culling and gene sequencing, the target gene was verifed to be correctly cloned in adenovirus vector. The high expression of green fluorescence protein in QBI-293A cell line was found under fluorescent microscope. Compared with control group, the β-catenin expression was obviously increased 48 h after infection with Ad-β-catenin or Ad-AN90. TOPFlash assay confirmed that Ad-β--catenin and Ad-AN90 results in a dramatic increase in signaling activity. The recombinant adenovirus vectors ofβ-catenin and AN90 were successfully constructed and the adenovirus was packaged in QBI-293A cell line.

关 键 词：腺病毒载体 Β-CATENIN ΔN90 骨髓间充质干细胞 构建 

分 类 号：R346[医药卫生—基础医学]