上海交通大学学报（医学版）

›› 2010, Vol. 30 ›› Issue (12): 1501-.doi: 10.3969/j.issn.1674-8115.2010.12.013

• 论著（基础研究） • 上一篇    下一篇

小鼠骨生成诱导因子基因逆转录病毒载体的构建及其在293T细胞中的表达

郑翠侠1, 张晓娜2, 韩 兵1, 宋怀东2, 马勤耘2   

  1. 上海交通大学 1.医学院附属第九人民医院呼吸病科, 上海 200011, 2.医学院附属瑞金医院分子医学中心 上海市内分泌代谢病研究所, 上海 200025
  • 出版日期:2010-12-25 发布日期:2010-12-31
  • 通讯作者: 马勤耘, 电子信箱: qinyunma@hotmail.com。
  • 作者简介:郑翠侠（1966—）, 主任医师, 博士生；电子信箱: zcx9566@163.com。
  • 基金资助:

    上海市自然科学基金（10ZR1418300）

Construction of mouse osteoinductive factor gene retroviral vector and its expression in 293T cells

ZHENG Cui-xia1, ZHANG Xiao-na2, HAN Bing1, SONG Huai-dong2, MA Qin-yun2   

  1. 1.Department of Respiratory Medicine, The Ninth People's Hospital,Shanghai Jiaotong University School of Medicine, Shanghai 200011, China；2.Molecular Medicine Center, Shanghai Institute of Endocrinology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2010-12-25 Published:2010-12-31
  • Supported by:

    Shanghai Natural Science Foundation, 10ZR1418300

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摘要：

目的 构建小鼠骨生成诱导因子(OIF)基因重组逆转录病毒载体，鉴定其在293T细胞中的表达。方法 PCR法扩增OIF-3FLAG基因，测序后克隆入逆转录病毒载体pMSCV PIG（Puro-IRES-GFP）的相应位点，构建重组逆转录病毒表达载体pMSCV PIG-OIF-3FLAG，并对重组体进行测序鉴定。应用脂质体分别将PMSCV PIG-OIF-3FLAG和VSVG、GAG-POL辅助病毒包装载体共转染至293T包装细胞，48 h后荧光显微镜下观察绿色荧光蛋白（GFP）表达；收集包装细胞上清液，进一步用含有包装病毒的上清液再感染293T细胞，48 h后加入3 μg/mL嘌呤霉素（puromycin），连续7 d，筛选病毒感染的稳定表达pMSCV PIG-OIF-3FLAG 的293T细胞株，分别采用Real-time PCR技术和Western blotting方法检测293T细胞OIF mRNA及其蛋白表达。结果 构建OIF基因逆转录病毒表达载体PMSCV PIG-OIF-3FLAG，经酶切及测序鉴定证实目的基因插入位点和读码框架正确；病毒感染293T细胞，经嘌呤霉素筛选稳定表达小鼠OIF的293T细胞株；Real-time PCR和Western blotting鉴定证实OIF mRNA和蛋白在该细胞株中呈高表达。结论 成功构建小鼠OIF基因的逆转录病毒载体，并获得稳定高表达OIF基因的293T细胞株。

关键词: 逆转录病毒载体, 小鼠骨生成诱导因子, 293T细胞

Abstract:

Objective To construct mouse osteoinductive factor (OIF) gene recombinant retroviral vector, and investigate its expression in 293T cells. Methods OIF-3FLAG gene was amplified with PCR and cloned into retroviral vector (Puro-IRES-GFP) after sequencing, and recombinant retroviral vector pMSCV PIG-OIF-3FLAG was constructed and identified by sequencing. PMSCV PIG-OIF-3FLAG retroviral vector with helper vectors of VSAG and GAG-POL were co-transfected into 293T cells by lipofectamine2000. Forty-eight hours after transfection, the expression of green fluorescent protein (GFP) was examined by fluorescence microscopy. The supernatant of 293T cells, which were tansfected with pMSCV PIG-OIF-3FLAG retroviral vector and helper vectors of VSAG and GAG-POL, was used to reinfect 293T cells. After 48 h, 293T cells stably expressing pMSCV PIG-OIF-3FLAG retroviral vector were screened by 3 μg/mL puromycin for 7 d, and the expression of OIF mRNA and protein of 293T cells was detected by Real-Time PCR and Western blotting, respectively. Results OIF gene retroviral vector pMSCV PIG-OIF-3FLAG was successfully constructed, and the cloning site and reading frame of objective gene were confirmed by enzyme digestion and sequencing. Then, 293T cells were infected by retrovirus supernatant, and 293T cells with stable expression of mouse OIF were obtained by puromycin screening. Real-Time PCR and Western blotting revealed high expression of OIF mRNA and protein in 293T cells. Conclusion Mouse OIF gene retroviral vector has been successfully constructed, and 293T cells with stable expression of OIF gene are obtained.

Key words: retroviral vector, mouse osteoinductive factor, 293T cell