SOX9慢病毒表达载体的构建及其在LNcap细胞中的稳定表达

王江; 段君君; 张小玉; 贾霄; 汪小波; 何颖红

SOX9慢病毒表达载体的构建及其在LNcap细胞中的稳定表达

王江^1,2,
段君君^1,2,
张小玉^1,2,
贾霄^1,2,
汪小波^1,2,
何颖红^1,2

1. 大理大学基础医学院
2. 云南省高校细胞生物学重点实验室

基金项目:

基金：国家自然科学基金资助项目 (31360288);

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出版历程
- 收稿日期: 2017-10-23

Construction of Lentiviral Expression Vector Carrying SOX9 Gene and Its Stable Expression in LNcap Cell

Wang Jiang ^1,2,
Duan Jun Jun ^1,2,
Zhang Xiao Yu ^1,2,
Jia Xiao ^1,2,
Wang Xiao Bo ^1,2,
He Ying Hong ^1,2

Funds:

基金：国家自然科学基金资助项目 (31360288);

摘要

摘要: 目的构建SOX9基因慢病毒表达载体, 建立稳定表达SOX9的LNCa P细胞株.方法 PCR扩增SOX9基因, 将其克隆到慢病毒表达载体p LVX-IRES-Puro中, 双酶切和测序鉴定重组质粒.用HEK293细胞包装重组病毒颗粒并感染LNcap细胞, 经puromycin筛选获得稳定转染细胞株.q RT-PCR和Western Blot分别检测SOX9 m RNA水平及蛋白表达.结果经双酶切和测序鉴定证实目的片段已插入到慢病毒表达载体上.重组质粒转入LNCa P细胞后经puromycin抗性筛获得稳定表达的细胞株, q RT-PCR检测到SOX9基因在转录水平的表达, Western blot分析显示SOX9蛋白高表达.结论慢病毒表达载体p LVX-IRES-FLAG-SOX9构建成功, 实现了SOX9在LNcap细胞中的外源性表达.
- SOX9 /
- 慢病毒表达载体 /
- LNcap细胞 /
- 脂质体转染
Abstract: Objective To construct the lentiviral expression vector of SOX9 gene and establish a LNcap cell strain with stable expression of SOX9. Me thods SOX9 gene was amplified by PCR and cloned into lentiviral expression vector p LVX-IRES-Puro. p LVX-IRES-FLAG-SOX9 recombinant plasmid was verified by restriction enzyme digestion and DNA sequencing. Then we gained recombinant virus particles in packaging cell HEK 293 and infected LNcap cell. The monoclonal LNcap cell strain stably expressed were obtained through puromycin screening.The m RNA level and protein level in the infected LNcap cell were detected by q RT-PCR and Western blot respectively. Re s ults Restriction enzyme digestion and sequencing demonstrated that SOX9 c DNA was successfully cloned into p LVX-IRES-FLAG lentiviral vector. After the transfection to LNCa P cells, the monoclonal cell strain of stably expressed SOX9 were obtained by puromycin screening, which showed expression of SOX9 m RNA detected by q RT-PCR. Western blot analysis revealed that SOX9 protein expressed markedly. Conclus ion The recombinant lentiviral vector bearing human SOX9 c DNA has been successfully constructed, and the exogenous expression of SOX9 in LNcap cells was achieved.
- SOX9 /
- Lentiviral vector /
- LNcap cell /
- Liposome tansfection

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参考文献(15)

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