RhoA突变体的构建及稳定表达  

Establishment of cell lines expressing mutant RhoA proteins

作　　者：宋富强[1] 陈炜[1] 王嘉丽[1] 张俊磊[1] 胡晓梅[1] 

机构地区：[1]第三军医大学基础医学部微生物学教研室重庆市微生物工程实验室,重庆400038

出　　处：《免疫学杂志》2010年第10期838-841,共4页Immunological Journal

基　　金：国家自然科学基金资助项目(30972596;30800983);第三军医大学青年创新人才基金(2009XQN10)

摘　　要：目的构建pCI-V14RhoA、pCI-N19RhoA、pCI-WtRhoA真核表达载体,筛选出稳定表达细胞克隆株,为进一步研究RhoA在登革病毒感染过程的作用奠定工作基础。方法构建活化突变型的pCI-V14RhoA、显性负性突变型pCI-N19RhoA及野生型pCI-WtRhoA真核表达载体,稳定转染至ECV304细胞,通过间接免疫荧光染色和免疫印迹实验进行鉴定。结果经PCR扩增、酶切和测序验证,重组质粒pCI-WtRhoA、pCI-V14RhoA、pCI-N19RhoA构建正确;经间接免疫荧光染色和免疫印迹实验验证,稳定表达细胞克隆株命名为ECVWtRhoA、ECVV14RhoA、ECVN19RhoA。结论成功建立了真核表达质粒pCI-WtRhoA、pCI-V14RhoA、pCI-N19RhoA;成功建立了稳定表达细胞克隆株ECVWtRhoA、ECVV14RhoA、ECVN19RhoA,为后续研究积累了有价值的实验资料。This study aimed to construct pCI-V14RhoA,pCI-N19RhoA and pCI-WtRhoA recombinant plasmids,and express them in ECV304 cells stablg.WtRhoA（wild-type）,V14RhoA（active） and N19RhoA（dominant-negative） sequences were cloned into pCI-neo vector and named pCI-WtRhoA,pCI-V14RhoA and pCI-N19RhoA respectively.ECV304 cells were transfected with above plasmids or pCI-neo vector.Following selection with G418 sulfate,cell colonies that had stably integrated the construct were picked and expanded into clonal cell lines,named ECVWtRhoA,ECVV14RhoA and ECVN19RhoA respectively.Then,these cell lines were examined for total RhoA expression by Western blotting and immunofluorescence staining.In this study,we successfully establish cell lines expressing mutant RhoA proteins,which lays a base for further study on role of RhoA in virus infection.

关 键 词：病毒 细胞骨架 RHOA 

分 类 号：R373.33[医药卫生—病原生物学]