中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (33): 6177-6181.doi: 10.3969/j.issn.1673-8225.2011.33.024

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

Beclin1 基因慢病毒表达载体的构建和鉴定

王文玉1, 2，樊红琨3，赵国强4，乔  鹏3，王书春3，伍  钢1   

  1. 1华中科技大学同济医学院附属协和医院肿瘤中心，湖北省武汉市 430023
    2河南省人民医院肿瘤科，河南省郑州市 450003
    3郑州大学基础医学院生理教研室，河南省郑州市 450052
    4郑州大学基础医学院微生物学与免疫学教研室，河南省郑州市450052
  • 收稿日期:2011-03-08 修回日期:2011-06-12 出版日期:2011-08-13 发布日期:2011-08-13
  • 通讯作者: 伍钢，教授，主任医师，华中科技大学同济医学院附属协和医院肿瘤中心，湖北省武汉市 430023
  • 作者简介:王文玉☆，1972年生，男，山西省大同市人，汉族，华中科技大学同济医学院在读博士，河南省人民医院肿瘤科，副主任医师，主要从事非小细胞肺癌的耐药及化疗增敏研究。 wangwy0712@163.com
  • 基金资助:

    课题受河南省科技创新人才工程项目(0623060900) 的支持，课题名称：非小细胞肺癌耐药机理及临床药敏试验和化疗增敏研究。

Construction and identification of lentiviral vector for Beclin1 gene

Wang Wen-yu1, 2, Fan Hong-kun3, Zhao Guo-qiang4, Qiao Peng3, Wang Shu-chun3, Wu Gang1   

  1. 1Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan  430023, Hubei Province, China；2Department of Medical Oncology, Henan Province People’s Hospital, Zhengzhou  450003, Henan Province, China; 3Department of Physiology, 4Department of Microbiology and Immunology, School of Medicine, Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • Received:2011-03-08 Revised:2011-06-12 Online:2011-08-13 Published:2011-08-13
  • Contact: Wu Gang, Professor, Chief physician, Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430023, Hubei Province, China
  • About author:Wang Wen-yu☆, Studying for doctorate, Associate chief physician, Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430023, Hubei Province, China; Department of Medical Oncology, Henan Province People’s Hospital, Zhengzhou 450003, Henan Province, China wangwy0712@163.com
  • Supported by:

    the Science and Technology Innovation Talent Engineering Program of Henan Province, No.0623060900*

PDF

401

被引次数

10

摘要：

背景：Beclin1基因是哺乳动物的自噬调控基因。
目的：实验拟构建Beclin1 基因慢病毒过表达载体。
方法：聚合酶链反应扩增目的基因Beclin1 后插入慢病毒表达载体pLenex中，构建重组载体pLenex-Beclin1。使用聚合酶链反应、双酶切和DNA的测序方法对其进行鉴定，并与辅助包装质粒共感染293T细胞。慢病毒颗粒转染非小细胞肺癌A549细胞后，用蛋白质印迹法检测Beclin1 基因的过表达效率。
结果与结论：聚合酶链反应鉴定结果显示扩增的阳性片段已插入pLenex载体，聚合酶链反应、双酶切和DNA测序结果表明，重组慢病毒载体pLenex-Beclin1 的插入序列完全正确，重组慢病毒载体感染A549细胞后，细胞内Beclin1蛋白高效表达。结果证实，实验成功构建了Beclin1 基因慢病毒过表达载体。

关键词: 慢病毒载体, Beclin1 基因, A549细胞, 过表达, 组织构建

Abstract:

BACKGROUND: Beclinl is an essential autophagy gene.
OBJECTIVE: To construct lentiviral vector pLenex-Beclin1 gene.
METHODS: Beclin1 gene amplification was used by real-time polymerase chain reaction. Gene amplification products were inserted in the lentiviral vector pLenex, and constructed lentiviral vector pLenex-Beclin1. Polymerase chain reaction analysis, double digests and DNA sequencing were used to confirm the constructed vectors whether or not success recombinant vector plasmid and to coinfect 293T cells, and then was transfected into non small cell lung cancer A549 cells. Western blot was then used to investigate the interfering efficiency of Beclin1 gene. 
RESULTS AND CONCLUSION: Polymerase chain reaction tests showed amplified positive fragments were inserted in pLenex vectors. Results of polymerase chain reaction analysis, double digestion and DNA sequencing showed that recombinant lentivirus plasmids pLenex-Beclin1 were constructed successfully. In transfected A549 cells, Beclin1 protein was over-expressed. Results verified that lentiviral vectors of Beclin1 gene over-expression were successfully constructed.

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