鼠NF～IL～6反义真核表达载体的构建和鉴定  

Construction and Identification of Recombinant Eukaryotic Expression Vector of Mouse Antisense NF-IL6 Gene

作　　者：郑小莉[1] 谢晓东[2] 罗波[1] 梅志强[3] 刘佳佳[4] 

机构地区：[1]泸州医学院基础医学院生物化学教研室四川,646000 [2]泸州医学院教务处 [3]泸州医学院基础医学研究中心 [4]泸州医学院基础医学院免疫学教研室

出　　处：《西南军医》2011年第5期810-813,共4页Journal of Military Surgeon in Southwest China

基　　金：四川省卫生厅项目（100208）

摘　　要：目的构建包含鼠细胞转录因子白介素6（NF～IL6）反义基因的部分功能区的重组反义真核表达载体pcDNA3.0/NF～IL6,为进一步研究鼠巨噬细胞中NF～IL6的功能奠定基础。方法 Trizol试剂法提取体外分离培养的鼠巨噬细胞总RNA,经逆转录聚合酶链式反应（RT～PCR）扩增NF～IL6基因目的片段,PCR产物及真核表达载体pcD-NA3.0分别双酶切并进行连接,转化入大肠杆菌Ecoli Dh5α扩增后获得重组载体。结果 RT～PCR获得预期大小的特异性NF～IL6 DNA片段,经PCR、双酶切及测序分析证实已将鼠NF～IL6 cDNA片段正确的反向插入真核表达载体中。结论成功获得反义pcDNA3.0/NF～IL6重组质粒。Objective To construct a recombinant eukaryotic expression vector pcDNA3.0/NF-IL6containing functional region of mouse antisense NF-IL6 gene so as to provide a tool for further study on the impact of NF-IL6 on mouse macrophage.Methods Total RNA was extracted by Trizol onestep method from mouse macrophages;Reverse transcriptasepolymerase chain reaction（RT-PCR） was applied in amplifying cDNA;the product of PCR,amplified by being transformed into E.coli Dh5α,was inserted reversely into the eukaryotic expression vector after digestion and ligation using restriction endonucleases and ligase.Results The correct mouse macrophage NF-IL6 cDNA clone was gained and PCR,digestion identification and sequencing confirmed that NF-IL6had been inserted reversely into the eukaryotic expression vector correctly.Conclusions The recombinant eukaryotic expression vector pcDNA3.0/NF-IL6 is successfully constructed.

关 键 词：NF^IL6 反义核酸 巨噬细胞 

分 类 号：R392.11[医药卫生—免疫学]