KLF9基因腺病毒载体的构建及其抗ROS功能

基础医学与临床 ›› 2014, Vol. 34 ›› Issue (6): 762-766.

KLF9基因腺病毒载体的构建及其抗ROS功能

焦涛¹,崔安芳¹,常永生²

1. 中国医学科学院基础医学研究所
2. 中国医学科学院基础医学研究所北京协和医学院基础学院

收稿日期:2014-04-08 修回日期:2014-04-14 出版日期:2014-06-05 发布日期:2014-05-26
通讯作者: 常永生 E-mail:changy@ibms.pumc.edu.cn
基金资助:
国家自然科学基金

KLF9 adenoviral vector construction and it’s function in anti-ROS

Received:2014-04-08 Revised:2014-04-14 Online:2014-06-05 Published:2014-05-26
Contact: Yong-sheng CHANG E-mail:changy@ibms.pumc.edu.cn
Supported by:
the Chinese National Natural Science Foundation

摘要/Abstract

摘要： 目的构建及鉴定KLF9基因过表达的腺病毒载体，初步研究KLF9在对抗反应活性氧方面的功能。方法克隆KLF9过表达质粒，定向克隆入穿梭载体pAdTrack-CMV，Pme I 酶切线性化，转化E.coli. BJ5183（含腺病毒载体pAdEasy-1）感受态细菌，产生重组腺病毒载体。重组载体经过卡那霉素抗性筛选和限制性内切酶分析确认重组后，再用Pac I 线性化重组质粒，回收，转染293A细胞，7-12 d包装产生病毒颗粒。利用H2O2和过表达KLF9的腺病毒处理体外培养的C57BL/6J小鼠原代肝脏细胞，实时定量PCR检测KLF9和抗ROS基因（CAT、SOD2和GPx1）的表达。结果 H2O2可以刺激C57BL/6J小鼠原代肝脏细胞KLF9和抗ROS基因的表达上调（P<0.05）。定量PCR以及Western blot结果显示成功包装过表达KLF9腺病毒，感染效率达90％以上。过表达KLF9可以上调抗ROS基因的表达水平（P<0.05）。结论初步认定KLF9基因可参与C57BL/6J小鼠原代肝脏细胞中抗ROS基因的调节。

关键词: 关键词：KLF9, 过表达, 腺病毒, 反应活性氧

Abstract: Objective To construct the adenoviral overexpressed vector of Krüppel-like factor 9(KLF9) and to explore the role of KLF9 in the regulation of anti-ROS genes expression. Methods The KLF9 gene is first cloned into the shuttle vector pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease Pme I, and subsequently cotransformed into E. coli. BJ5183 competent cells with pAdEasy-1.Recombinants are selected for kanamycin resistance and recombination confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into 293A cells. Recombinant adenoviruses are typically generated within 7 to 12 days. The mouse primary hepatocytes are isolated from the C57BL/6J mouse and subsequently H2O2 treatment is conducted. We aslo performed KLF9 overexpression by using Ad-virus system (Ad-KLF9) in mouse primary hepatocytes isolated from.Quantitative real-time PCR (qRT-PCR) analysis was further performed to determine the expression of anti-ROS genes including catalase(CAT), manganese superoxide dismutase(SOD2) and genes were upregualted in C57BL/6J mouse primary hepatocytes following H2O2 treatment (P<0.05).The results of qRT-PCR and western blot replied that adenoviral vector of KLF9 was successfully produced with 90% infection efficiency. The expression of anti-ROS genes were also upregualted in C57BL/6J mouse primary hepatocytes after infecting with Ad-KLF9(P<0.05).Conclusion KLF9 represents its impact on the expression of anti-ROS genes.

Key words: Key words: KLF9, Overexpression, Adenovirus, ROS

中图分类号:

R73-3

焦涛崔安芳常永生. KLF9基因腺病毒载体的构建及其抗ROS功能[J]. 基础医学与临床, 2014, 34(6): 762-766.

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