结缔组织生长因子全长及其缺失突变体重组腺病毒的构建和鉴定  

Establishment of six adenoviruses that express the full length of connective tissue growth factor and its deletion mutants

作　　者：孙双双[1] 陈红[1] 唐敏[1] 施琼[1] 黎玉叶[1] 王建伟[2] 周兰[1] 何通川[1] 左国伟[1] 

机构地区：[1]重庆医科大学临床检验诊断学教育部重点实验室 [2]重庆医科大学基础医学院组织学与胚胎学教研室,重庆400016

出　　处：《重庆医科大学学报》2011年第8期949-953,共5页Journal of Chongqing Medical University

基　　金：国家自然科学基金资助项目(编号:30970872)

摘　　要：目的:结缔组织生长因子(Connective tissue growth factor,CTGF)含有4个不同功能结构域,构建表达带有3×Flag标签的全长基因及5个缺失突变体的重组腺病毒,以研究CTGF各结构域的功能和作用机制。方法:通过聚合酶链反应(Polymerasechain reaction,PCR)等克隆方法制备目标质粒DNA,然后通过腺病毒穿梭载体与骨架载体的重组、包装构建全长CTGF和缺失突变体重组腺病毒。用anti-Flag抗体作为一抗做Western blot检测目的基因表达情况。结果:通过腺病毒基因组DNA PCR确认有CTGF全长和缺失突变体的存在;Western blot法验证了CTGF全长和缺失突变体的表达。结论:成功构建了6个表达CTGF全长和缺失突变体的重组腺病毒,为进一步研究CTGF的结构域功能奠定了基础。Objective：Connective tissue growth factor（CTGF）,which contains four modules,plays an important role in both cell proliferation and differentiation.This study is to elucidate the distinctive function of each module,and to establish recombinant adenoviruses that express full length and deletion mutants of CTGF gene.Methods：Six plasmids containing full-length CTGF,CTGFCD1（no module Ⅳ）,CTGFCD2（no module Ⅲand Ⅳ）,CTGFID1（no module Ⅱ）,CTGFID2（no module Ⅲ）,CTGFID3（no module Ⅱ or Ⅲ） were constructed with Polymerase chain reaction（PCR） and gene cloning techniques.The corresponding adenoviruses were generated by recombination and packaging,and evaluated by PCR and Western blot.Results：The presence of full-length CTGF or CTGF deletion mutants in these recombinant adenoviruses was confirmed by DNA sequencing and PCR,and the expression of these CTGF constructs was confirmed by Western blot with anti-Flag antibody to the fusion 3×Flag tag at the C-terminus of these constructs.Conclusion：Six recombinant adenoviruses that express full-length CTGF and deletion mutants have been successfully established,which could facilitate the dissection of CTGF protein function.

关 键 词：结缔组织生长因子 缺失突变体 重组腺病毒 

分 类 号：Q782[生物学—分子生物学]