PDF《的表达》

%% 收稿日期:2009-10-24 基金项目:国家自然科学基金(30772232) 作者简介:李明(1983-),男,博士,E-mail:%liming1142@yahoo.

com.cn 通讯作者:张世忠,教授,主任医师,博士生导师,

电话:020-61643267, E-mail:%shizhongzh@163.com pDsRed2-N1-SDF-1α 真核表达载体的构建及在小鼠骨髓间充质干细胞中 的表达 李明,张世忠,邹志浩,郭燕舞,卢凤飞,姜晓丹,寇盛斌,卢国辉,李振勇(南方医科大学珠江医院神经外科 / 广 东神经外科研究所 / 广东省脑功能修复与再生重点实验室,广东 广州 510282) 摘要:目的 构建 pDsRed2-N1-SDF-1α 真核表达载体并转染骨髓间充质干细胞(MSCs),观察其在 MSCs 内的表达. 方法设计并合成 SDF-1α 基因引物,用RT-PCR 法从小鼠平滑肌细胞中扩增出带有 XhoI 与EcoRI 酶切位点的 SDF-1α 基因片段,将SDF-1α 基因片段克隆到真核表达载体 pDsRed2-N1 上,酶切和测序鉴定. 培养小鼠 MSCs 并Vimentin 蛋 白免疫荧光鉴定. 通过脂质体将 pDsRed2-N1-SDF-1α 转染入小鼠 MSCs. 免疫荧光检测转染后的 MSCs 表达 SDF-1α 蛋白的情况. 结果 扩增出的基因片断大小与基因文库中已知的 SDF-1α 序列大小完全相符,酶切也得到目的基因片 断,测序结果显示与已知的 SDF-1α 序列相同,成功构建 pDsRed2-N1-SDF-1α 真核表达载体. 培养的细胞经 Vimentin 蛋白免疫荧光检测为阳性,pDsRed2-N1-SDF-1α 表达载体转染到 MSCs,24%h 后细胞免疫荧光检测可见 SDF-1α 融合蛋 白表达. 结论 pDsRed2-N1-SDF-1α 真核表达载体能够在小鼠 MSCs 中表达 SDF-1α 蛋白. 关键词:SDF-1α 蛋白;

骨髓间充质干细胞;

真核表达载体 中图分类号:R331.22;

R34 文献标识码:A 文章编号:1673-4254(2010)03-0439-04 Construction of a mouse pDsRed2-N1-SDF-1α eukaryotic expression vector and its expression in mouse bone marrow mesenchymal stem cells LI%Ming,%ZHANG%Shi-zhong,%ZOU%Zhi-hao,%GUO%Yan-wu,%LU%Feng-fei,%JIANG%Xiao-dan,%KOU%Sheng-bin,%LU%Guo-hui,%LI%Zhen-yong Department%of%Neurosurgery/Neurosurgery%Institute%of%Guangdong%Province,%Zhujiang%Hospital,%Southern%Medical%University,%Guangzhou% 510282,%China Abstract: Objective To%construct%the%eukaryotic%expression%vector%pDsRed2-N1-SDF-1α and%observe%its%expression%in%the% mouse%bone%marrow%mesenchymal%stem%cells. %Method SDF-1α gene%sequence%with%XhoI, %EcoRI%restriction%enzyme%cutting% site% was% amplified% from% the% total% RNA% of% mouse% smooth% muscle% cells% by% reverse% transcription-polymerase% chain% reaction% (RT-PCR) %and%inserted%into%the%eukaryotic%expression%vector%pDsRed2-N1%encoding%red%fluorescent%protein%gene, %and%the% insertion% was% verified% by% endonuclease% digestion% and% DNA% sequencing. % Mouse% bone% marrow% mesenchymal% stem% cells% identified% with% immunofluorescence% assay% for% vimentin% expression% were% transfected% with% the% constructed% plasmid% pDsRed2-N1-SDF-1α, % and% the% expression% of% sdf-1α was% detected% using% immunofluorescence% assay. % Results The% DNA% fragment% amplified% by% PCR% from% the% total% RNA% was% identical% to% SDF-1α from% the% gene% library, % and% an% identical% DNA% fragment%was%also%amplified%from%the%recombinants. %Sequence%analysis%confirmed%the%successful%insertion%of%SDF-1α into%the% pDsRed2-N1%vector%and%the%eukaryotic%expression%vector%pDsRed2-N1-SDF-1α was%successfully%constructed. %The%cultured% mouse%bone%marrow%mesenchymal%stem%cells%positive%for%vimentin%protein%showed%SDF-1α expression%24%h%after%transfection% with%the%recombinant%vector. %Conclusion The%pDsRed2-N1-SDF-1α eukaryotic%expression%vector%constructed%is%capable%of% expression%of%SDF-1α fusion%protein%in%the%mouse%bone%marrow%mesenchymal%stem%cells. Key words: SDF-1α protein;