小鼠NF-κBp65亚基真核表达质粒的构建  

Construction of expression system of NF-κBp65 subunit of mouse

作　　者：李雄[1] 罗成群[1] 周建大[1] 贺全勇[1] 徐阳成[1] 彭浩[1] 陈铁夫[1] 欧阳华伟[1] 

机构地区：[1]中南大学湘雅三医院烧伤整形外科,湖南长沙410013

出　　处：《中国现代医学杂志》2010年第9期1281-1283,1288,共4页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:30672035)

摘　　要：目的构建小鼠NF-κBp65亚基真核表达质粒。方法从培养的小鼠腹腔巨噬细胞RAW264.7中提取总RNA,逆转录得到DNA,利用PCR方法钓取目的基因,将目的基因与目的载体pcDNA3.1(-)分别进行酶切。酶切产物电泳回收后进行定向连接,其产物转化细菌感受态。对长出的克隆先进行菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析,比对正确的即为构建成功的目的质粒。结果经过PCR鉴定和酶切鉴定,证实DNA片段长度和序列正确。结论通过RT-PCR克隆出小鼠NF-κBp65亚基基因,并成功连接于目的载体,为进一步研究NF-κB在烧伤后免疫反应中的作用机制打下实验基础。【Objective 】To construct the mouse NF-κ B p65 subunit expression plasmids.【Methods】The cDNA encoding NF-κ B p65 was isolated by using RT-PCR method from the mouse macrophage cells RAW264.7.P65 subunit was then cloned into vector pcDNA3.1（-）,and identified by restriction enzyme analysis and sequencing.【Results】The NF-κ B p65 gene were successfully cloned into pcDNA3.1（-）.The sequence showed the same to that in the GenBank.【Conclusion】The construction of an expression system of NF-κ B p65 subunit was constructed,which established the foundation for the research on the role of NF-κ B post-burned.

关 键 词：核因子ΚB pcDNA3.1(-) 表达质粒 

分 类 号：R-332[医药卫生]