上海交通大学学报（医学版）

›› 2009, Vol. 29 ›› Issue (7): 821-.

• 论著（基础研究） • 上一篇    下一篇

GDNF和EGFP双基因共表达重组杆状病毒载体的构建及鉴定

陈艳春1, 王 俊1, 王士礼1, 蔡昌枰1, 李 彪2, 张一帆2, 郭 睿2   

  1. 上海交通大学 医学院瑞金医院 1. 耳鼻咽喉科,  2. 核医学科, 上海 200025
  • 出版日期:2009-07-25 发布日期:2009-09-16
  • 通讯作者: 王士礼, 电子信箱: shiliwang@online.sh.cn。
  • 作者简介:陈艳春（1980—）, 女, 硕士生；电子信箱: md.chen@163.com
  • 基金资助:

    上海市教委基金 (06BZ036)

Construction and identification of recombinant baculovirus vector to coexpress GDNF and EGFP gene

CHEN Yan-chun1, WANG Jun1, WANG Shi-li1, CAI Chang-ping1, LI Biao2, ZHANG Yi-fan2, GUO Rui2   

  1. 1. Department of Otolaryngology, 2. Department of Nuclear Medicine, Ruijin Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200025, China
  • Online:2009-07-25 Published:2009-09-16
  • Supported by:

    Shanghai Education Committee Foundation (06BZ036)

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摘要：

目的 构建携带增强型绿色荧光蛋白（EGFP）和胶质细胞源性神经营养因子（GDNF）的重组杆状病毒载体。方法 将目的基因（EGFP和GDNF）克隆入杆状病毒表达载体pFastBacDual中，构建重组质粒pFB-EGFP-GDNF并予酶切鉴定；将pFB-EGFP-GDNF转化到含杆状病毒穿梭载体Bacmid的DH10Bac感受态菌中，获得重组杆状病毒载体Bacmid-EGFP-GDNF，抽提质粒并行PCR鉴定；脂质体转染法将Bacmid-EGFP-GDNF转染Sf9细胞包装病毒；免疫荧光法检测Sf9细胞EGFP和GDNF蛋白表达。结果 目的基因片段正确插入pFastBacDual载体中；重组Bacmid正确；Bacmid-EGFP-GDNF包装转染成功，获得较高病毒滴度；免疫荧光检测表明，Sf9细胞中GDNF和EGFP蛋白共表达。结论 成功构建重组杆状病毒Bacmid-EGFP-GDNF，转染Sf9细胞共表达GDNF和EGFP蛋白，为进一步研究GDNF蛋白对内耳的保护作用奠定了实验基础。

关键词: 胶质细胞源性神经营养因子, 增强型绿色荧光蛋白, 杆状病毒, 蛋白表达

Abstract:

Objective To construct a novel enhanced green fluorescent protein (EGFP) and glial cell line-derived neurotrophic factor (GDNF) recombinant baculovirus. Methods The target gene（EGFP and GDNF）was cloned into baculovirus transfer vector pFastBacDual, pFB-EGFP-GDNF was constructed and restriction enzyme analysis was conducted. pFB-EGFP-GDNF was transposited with baculovirus shuttle vector (Bacmid) into DH10Bac competent cells, and recombination baculovirus vector Bacmid-EGFP-GDNF was constructed. The plasmid was extracted and PCR was performed for identification. Bacmid-EGFP-GDNF was transfected with Sf9 insect cell package virus by liposomal transfection method. Immunofluorescent staining was employed to detect the expression of EGFP and GDNF protein in Sf9 cells. Results The target gene fragment was correctly cloned into pFastBacDual vector, and recombinant Bacmid was constructed. Bacmid-EGFP-GDNF was successfully transfected, and higher virus titer was obtained. The coexpression of GDNF and EGFP protein in Sf9 cells was identified by immunofluorescent staining. Conclusion The recombinant baculovirus Bacmid-EGFP-GDNF can be successfully constructed, and the protein of EGFP and GDNF is coexpressed in Sf9 cells, which paves a way for the research of GDNF gene therapy.

Key words: glial cell line derived neurotrophic factor, enhanced green fluorescent protein, baculovirus, protein expression