中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (41): 7701-7705.doi: 10.3969/j.issn.1673-8225.2011.41.025

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

单纯疱疹病毒Ⅰ型基因治疗载体的快速构建

蔡晓辉，张钦宪   

  1. 郑州大学基础医学院，河南省郑州市  450052
  • 收稿日期:2011-07-14 修回日期:2011-08-29 出版日期:2011-10-08 发布日期:2011-10-08
  • 通讯作者: 张钦宪，教授，博士生导师，郑州大学基础医学院，河南省郑州市 450052
  • 作者简介:蔡晓辉★，女，1977年生，河南省洛阳市人，汉族，郑州大学在读硕士，主要从事肿瘤分子生物学研究。 huixiaocai@126.com

Rapid construction of herpes simplex virus type I vector for gene therapy 

Cai Xiao-hui, Zhang Qin-xian   

  1. Basic Medical College of Zhengzhou University, Zhengzhou   450052, Henan Province, China
  • Received:2011-07-14 Revised:2011-08-29 Online:2011-10-08 Published:2011-10-08
  • Contact: Zhang Qin-xian, Professor, Doctoral supervisor, Basic Medical College of Zhengzhou University, Zhengzhou 450052, Henan Province, China
  • About author:Cai Xiao-hui★, Studying for master’s degree, Basic Medical College of Zhengzhou University, Zhengzhou 450052, Henan Province, China huixiaocai@126.com

PDF

465

被引次数

9

摘要：

背景：单纯疱疹病毒Ⅰ型载体因具有独特的优点目前被广泛应用，但其构建尚缺乏一种快速有效的方法。
目的：利用Cre/Loxp高效重组系统构建单纯疱疹病毒Ⅰ型载体。
方法：分离单纯疱疹病毒HSV-1，将含Cre重组酶的c66-SV40-cre质粒转染Vero细胞，构建一株带有Loxp位点的重组HSV-1框架载体HSVLoxp。构建穿梭载体pShuttle- SV40-Cre-Loxp-IRES及重组单纯疱疹病毒Ⅰ型载体HSV-GDNF，用HAT培养基筛选出阳性毒株后用GDNF引物做PCR鉴定，扩增培养后测定滴度。
结果与结论：成功构建pHV-TK-GFP质粒，并在Vero细胞内发生重组，分离出缺失了Us3基因的重组病毒HSVtk-Loxp-GFP01。成功构建HSV-1框架载体HSVLoxp及穿梭载体pShuttle- SV40-Cre-Loxp-IRES，成功获得GDNF基因，并将其转移到了HSV-1基因组上，成功构建了表达GDNF的单纯疱疹病毒HSV-1载体，测定其滴度约为2.25×106 IU/mL。

关键词: 单纯疱疹病毒Ⅰ型(HSV-1)载体, 基因治疗, GDNF, Cre重组酶, 构建

Abstract:

BACKGROUND: Herpes simplex virus typeⅠ(HSV-1) vector has been currently widely used due to its unique advantages, but a rapid and effect method is needed to construct this vector.
OBJECTIVE: To develop a rapid method using Cre/loxp site specific recombination system to construct HSV-1 vector.
METHODS: HSV-1 was isolated and c66-SV40-cre plasmid containing Cre recombinase was used to transfect Vero cells. Then HSV-1 HSVLoxp was constructed. Shuttle vector pShuttle-SV40-Cre-Loxp-IRES and HSV-I vector HSV-GDNF were constructed. The positive strains screened by HAT culture medium were identified by PCR taking GDNF as primer. After amplification, titer was determined.
RESULTS AND CONCLUSION: A strain of HSV-1 was isolated and pHV-TK-GFP was constructed successfully. Recombinant virus HSVtk-Loxp-GFP01 without Us3 gene was isolated. HSV-1 HSVLoxp and shuttle vector pShuttle- SV40-Cre-Loxp-IRES were successfully constructed. GDNF gene was successfully obtained and transferred into HSV-1 vector. Thus, HSV-1 vector expressing GDNF was successfully constructed and the titer was 2.25×106 IU/mL.

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