中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (41): 7691-7695.doi: 10.3969/j.issn.1673-8225.2010.41.022

• 组织构建实验造模 experimental modeling in tissue construction • 上一篇    下一篇

人树突状细胞特异性细胞间黏附分子-3-结合非整合素基因RNAi慢病毒载体的构建及外源性筛选与鉴定

李  兰，郭述良，王建军，邬亭亭   

  1. 重庆医科大学附属第一医院呼吸内科，重庆市  400016
  • 出版日期:2010-10-08 发布日期:2010-10-08
  • 通讯作者: 郭述良，博士，教授，重庆医科大学附属第一医院呼吸内科，重庆市 400016 guosl999@sina.com
  • 作者简介:李兰★，女，生于1983年生，汉族，四川省西昌市人，硕士，主要从事呼吸系统感染性疾病研究。 lilan_203@163.com
  • 基金资助:

    国家自然科学基金资助项目(30571653)，重庆市卫生局科研项目(05-2-113)，课题名称：DC-SIGN分子删除对结核病发生与转归影响研究。

Construction, exogenous selection and identification of lentiviral vector targeting human dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintergrin gene

Li Lan, Guo Shu-liang, Wang Jian-jun, Wu Ting-ting   

  1. Department of Respiratory, First Affiliated Hospital of Chongqing Medical University, Chongqing  400016, China
  • Online:2010-10-08 Published:2010-10-08
  • Contact: Guo Shu-liang, Doctor, Professor, Department of Respiratory, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China guosl999@sina.com
  • About author:Li Lan★, Master, Department of Respiratory, First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China lilan_203@163.com
  • Supported by:

    the National Natural Science Foundation of China, No. 30571653*; the Science and Technology Foundation of Health Bureau of Chongqing City, No. 05-2-113*

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被引次数

1

摘要：

背景：慢病毒载体是一个能够在原代培养的人树突状细胞中进行RNA干扰的有效工具，但目前国内对RNA干扰技术应用于人树突状细胞特异性细胞间黏附分子-3-结合非整合素分子的研究却少有报道。
目的：拟构建人树突状细胞特异性细胞间黏附分子-3-结合非整合素基因RNA干扰慢病毒载体，为调控人树突状细胞特异性细胞间黏附分子-3-结合非整合素表达水平提供有利的工具。
方法：根据人树突状细胞特异性细胞间黏附分子-3-结合非整合素基因的mRNA序列选择4条靶序列，设计合成靶序列的Oligo DNA，退火形成双链DNA，与经Age Ⅰ和EcoR Ⅰ双酶切后的pGCSIL-GFP载体连接产生DC-SIGN慢病毒载体，采用PCR及测序鉴定，应用Western blot技术外源性筛选干扰效率高的有效靶序列。用慢病毒包装系统pGCSIL-GFP、pHelper 1.0和pHelper 2.0共转染包装293T细胞，包装产生慢病毒，以系列稀释法测定病毒滴度。
结果与结论：PCR扩增和测序结果显示，人树突状细胞特异性细胞间黏附分子-3-结合非整合素核苷酸链序列插入正确，外源性筛选确定两条干扰效率高的有效靶序列。利用筛选确定的有效靶序列，包装产生慢病毒，其浓缩病毒悬液的滴度为1× 109 TU/mL。证实实验成功构建了DC-SIGN基因慢病毒载体。

关键词: 慢病毒载体, RNA干扰, DC-SIGN基因, 树突状细胞, 结核病

Abstract:

BACKGROUND: The lentiviral vector is an effective tool to execute RNAi in the human dendritic cells. However, there are few reports addressing applying RNAi technology to the human dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintergrin (DC-SIGN).
OBJECTIVE: To construct a lentiviral vector expressing small-hairpin RNA (shRNA) targeting human DC-SIGN gene, and to provide a useful tool to regulate the expression level of DC-SIGN.
METHODS: Four target sequences were selected according to human DC-SIGN mRNA sequence．The cDNA target sequence containing both sense and antisense Oligo DNA were designed. The obtained lentiviral vector containing DC-SIGN shRNA was confirmed by PCR, sequencing and exogenous selection. 293T cells were cotransfected with lentiviral vector pGCSIL-GFP, pHelper1.0 and pHelper 2.0. The titer of virus was tested by serial dilution．
RESULTS AND CONCLUSION: PCR and DNA sequencing demonstrated that the inserted sequences were correct. Two effective targeting sequences were determined by exogenous selection. The titer of concentrated virus was 1×109 T U/mL. The lentivirus vector targeting DC-SIGN has been successfully constructed.

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