β-catenin和突变体ΔN90腺病毒载体的构建及其在MSCs中的表达

生物技术通报 ›› 2013, Vol. 0 ›› Issue (11): 153-158.

β-catenin和突变体ΔN90腺病毒载体的构建及其在MSCs中的表达

崔恒进, 王春燕, 胡君, 谢桂琴, 徐晓静, 张焕相

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

收稿日期:2013-04-22 出版日期:2013-11-14 发布日期:2013-11-14
作者简介:崔恒进，男，硕士，研究方向：细胞生物学；E-mail：cuihengjin2008@163.com
基金资助:
国家自然科学基金资助项目(30870642，31071220)

Construction of Adenovirus Vector Containing Rat β-catenin or ΔN90 Gene and Its Expression in MSCs

Cui Hengjin, Wang Chunyan, Hu Jun, Xie Guiqin, Xu Xiaojing, Zhang Huanxiang

(Jiangsu Province Key Laboratory of Stem Cell Research，School of Biology and Basic Medical Sciences，Soochow University, Suzhou 215123)

Received:2013-04-22 Published:2013-11-14 Online:2013-11-14

摘要/Abstract

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。

关键词: 腺病毒载体, β-catenin, ΔN90, 骨髓间充质干细胞, 构建

Abstract: To construct the recombinant adenovirus vector containing β-catenin or ΔN90 gene for later research of the mechanism of β-catenin in MSCs migration. β-catenin or ΔN90 cDNA was inserted into a pAdTrack-CMV transfer vector, linearized with PmeⅠdigestion and pAdEasy-1 backbone vector was further cotransformed into the bacteria BJ5183 competent cells for homologous recombination adenoviruse Ad-β-catenin or Ad-ΔN90. Then they were linearized and transfected into QBI-293A cells by Lipofectamine^TM2000. Through PCR, endonuclease cutting 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-ΔN90. TOPFlash assay confirmed that Ad-β-catenin and Ad-ΔN90 results in a dramatic increase in signaling activity. The recombinant adenovirus vectors of β-catenin and ΔN90 were successfully constructed and the adenovirus was packaged in QBI-293A cell line.

Key words: Adenovirus vector, β-catenin, ΔN90, MSCs, Construction

崔恒进, 王春燕, 胡君, 谢桂琴, 徐晓静, 张焕相. β-catenin和突变体ΔN90腺病毒载体的构建及其在MSCs中的表达[J]. 生物技术通报, 2013, 0(11): 153-158.

Cui Hengjin, Wang Chunyan, Hu Jun, Xie Guiqin, Xu Xiaojing, Zhang Huanxiang. Construction of Adenovirus Vector Containing Rat β-catenin or ΔN90 Gene and Its Expression in MSCs[J]. Biotechnology Bulletin, 2013, 0(11): 153-158.

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