大鼠CD40基因RNA干扰真核表达载体的构建及鉴定  

Construction and identification of the RNA interference eukaryotic expression vector targeting the CD40 gene in rats

作　　者：王楠[1] 马庆久[1] 鲁建国[1] 何显力[1] 邹爱民[2] 李坤[2] 

机构地区：[1]第四军医大学唐都医院普通外科,陕西西安710038 [2]第四军医大学唐都医院中心实验室,陕西西安710038

出　　处：《医学研究生学报》2008年第3期259-262,I0003,共5页Journal of Medical Postgraduates

基　　金：陕西省科技攻关项目资助[批准号:2005K09-G12(4)]

摘　　要：目的:构建靶向大鼠CD40基因小分子干扰RNA(siRNA)真核细胞表达载体。方法:利用Ambion公司的网上设计工具,设计2个靶向CD40基因的发夹状siRNA,通过化学合成的方法合成2对分别互补的寡核苷酸链,退火后将双链DNA片段连接至pSilencer4.1-CMV neo vector的多克隆位点,转化扩增提取质粒,对重组质粒进行BamHⅠ和HindⅢ双酶切电泳鉴定和DNA序列分析鉴定。结果:经酶切电泳鉴定和DNA序列分析鉴定,目的片段与pSilencer4.1-CMV neo vector连接正确,靶向大鼠CD40基因的siRNA重组表达质粒构建成功。结论:成功构建了大鼠CD40基因的RNA干扰真核表达载体pSilencer4.1-CMV-CD40.1和pSilencer4.1-CMV-CD40.2,为进一步研究CD40基因在同种器官移植排斥反应中的作用奠定基础。Objective： To construct and identify the eukaryotic expression vector targeting the CD40 gene in rats. Methods ： Two sequences corresponding to the rat CD40 gene were designed on Ambion＇ s Web. The two complementary oligonucleotide strands of DNA fragments were synthesized by chemosynthesis. After annealing of the complementary strands, the DNA fragments were connected to the polyclone sites of the pSilencer 4.1-CMV neo vector, followed by transformation, amplification, plasmid extraction and identification of the recombinant plasmids by BamH Ⅰ and Hind Ⅲ digestion and DNA sequence analysis. Results： The connections between the DNA fragments encoding CD40-targeted siRNA and the pSilencer 4.1-CMV neo vector were correct, as confirmed by agarose gel electrophoresis and DNA sequence analysis. Conclusion： The two recombinant plasmids expressing siRNA of the rat CD40 gene were successfully constructed, which prepared the groundwork for future research on the role of the CD40 gene in homograft rejection.

关 键 词：RNA干扰 CD40 重组载体 

分 类 号：Q782[生物学—分子生物学]