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中国癌症防治杂志

中国癌症防治杂志 ›› 2016, Vol. 8 ›› Issue (4): 207-211.doi: 10.3969/j.issn.1674-5671.2016.04.02

• 基础研究 • 上一篇    下一篇

携带绿色荧光报告基因EBV-LMP2A真核表达载体构建及转染鼻咽癌细胞

  

  1. 广西医科大学生物化学与分子生物学教研室；广西医科大学医学科学实验中心 
  • 出版日期:2016-08-25 发布日期:2016-09-12
  • 通讯作者: 黄元姣 hyjgxmu@126.com
  • 基金资助:

    国家自然科学基金资助项目（81260405）；广西区域性高发肿瘤早期防治研究重点实验室基金项目（GK2013-A-02-02）

Construction of an EBV-LMP2A eukaryotic expression plasmid with a green fluorescent protein reporter and transfection into nasopharyngeal carcinoma cells

  • Online:2016-08-25 Published:2016-09-12

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摘要：

目的 构建携带绿色荧光基因的真核表达载体pIRES2-Zs-Green1-LMP2A，并转染至鼻咽癌CNE2细胞。方法 从EB病毒阳性的狨猴淋巴瘤细胞B95-8中克隆EB病毒编码的ＥＢＶ潜伏膜蛋白2A（latent membrane protein 2A，LMP2A）序列，并定向克隆入pIRES2-Zs-Green1载体，双酶切及测序鉴定重组的真核表达载体pIRES2-Zs-Green1-LMP2A；通过脂质体转染将重组的真核表达载体pIRES2-Zs-Green1-LMP2A转染至鼻咽癌CNE2细胞（实验组），同时另设转染pIRES2-Zs-Green1载体的阴性对照组及未转染的空白对照组。利用质粒所携带的绿色荧光蛋白表达计算细胞转染效率，RT-PCR检测目的基因LMP2A在鼻咽癌CNE2细胞中的表达。结果 双酶切及测序鉴定证实真核表达载体pIRES2-Zs-Green1-LMP2A构建成功，荧光显微镜下发现实验组和阴性对照组细胞均发出绿色荧光，实验组细胞转染率约为75%；RT-PCR检测发现实验组细胞中有目的基因LMP2A表达，但阴性对照组和空白对照组均未检测到目的基因表达。结论 成功构建了pIRES2-Zs-Green1- LMP2A真核表达载体并转染鼻咽癌CNE2细胞，目的基因LMP2A可在转染的鼻咽癌CNE2细胞中稳定表达。

关键词: 鼻咽肿瘤, EBV潜伏膜蛋白 2A, 序列克隆, pIRES2-Zs-Green1载体, pIRES2 -Zs-Green1- LMP2A真核表达载体, 基因转染, 基因表达

Abstract:

objective To construct the eukaryotic expression plasmid pIRES2-Zs-Green1-LMP2A encoding green fluorescent protein，and to transfect it into CNE2 nasopharyngeal carcinoma cells. Methods The LMP2A sequence from EBV-positive B95-8 cells was cloned into the vector pIRES2-Zs-Green1. The identity of the resulting pIRES2-Zs-Green1-LMP2A was confirmed using restriction enzymes and sequencing. The expression plasmid was transfected into CNE2 cells using a liposome-based method. The resulting transfectants were compared with negative controls transfected with pIRES2-Zs-Green1 and with untransfected cells in terms of expression of green fluorescent protein and LMP2A transcription. Results Restriction enzyme digestion and sequencing showed that pIRES2-Zs-Green1-LMP2A was constructed successfully. Transfection of pIRES2-Zs-Green1-LMP2A or empty vector pIRES2-Zs-Green1 led to expression of green fluorescent protein，and fluorescence microscopy indicated transfection efficiency of approximately 75%. Only cells transfected with the complete plasmid pIRES2-Zs-Green1-LMP2A produced LMP2A mRNA. Conclusion The eukaryotic expression plasmid pIRES2-Zs-Green1-LMP2A has been successfully constructed，and it can drive steady expression of LMP2A in CNE2 cells，making this system useful for studies of nasopharyngeal carcinoma.

Key words:  Nasopharyngeal neoplasm, EBV latent membrane protein 2A, Sequence clone, Vector pIRES2-Zs-Green1, Eukaryotic expression vector pIRES2-Zs-Green1-LMP2A, Gene transfection, Gene expression