小鼠cyclin A2正义和反义真核表达载体的构建及鉴定  

Construction and identification of sense and antisense eukaryotic expression vector of mouse cyclin A2

作　　者：秦文华 付春景[2] 

机构地区：[1]中国平煤神马医疗集团总医院病理科,河南平顶山467000 [2]郑州大学基础医学院病理生理学教研室,河南郑州450001

出　　处：《中国现代医生》2012年第31期1-3,7,共4页China Modern Doctor

摘　　要：目的构建小鼠cyclin A2的正义和反义真核表达载体。方法采用小鼠胚胎组织取材,提取总RNA。设计引物,采用RT-PCR方法扩增小鼠cyclin A2 mRNA得到cyclin A2的DNA,将此DNA插入pGEM-T载体,转化入JM109感受态细菌。将鉴定得到的重组质粒进行扩增,EcoRⅠ酶切后,回收cyclin A2目的片段亚克隆入pcDNA3.1真核表达载体。酶切筛选鉴定重组的真核表达载体pcDNA3.1-cyclin A2。结果将小鼠基因的开放阅读框(ORF)重组到真核表达载体pcDNA3.1内,用EcoRⅠ酶切重组质粒后可得到一接近1660 bp的DNA条带,核酸测序证实和GenBank所公布序列相符。HindⅢ酶切鉴定重组质粒后,证明分别为正向和反向插入载体的质粒。DNA测序证明构建的真核表达载体pcDNA3.1-cyclin A2的序列是正确的。结论成功构建了小鼠cyclin A2的正义和反义真核表达载体。Objective To construct a mouse cyclin A2 eukaryotic expression vector. Methods Cyclin A2 mRNA was ex- tracted from embryo tissue, and primer was designed according to cyclin A2 DNA exon. The cyclin A2 mRNA was ampli- fied by RT-PCR. Inserted the fragment cyclin A2 into the pGEM-T vector. The plasmid was transformed into E.coli JM109. The mouse cyclin A2 gene was recombinanted with eukaryotic expression vector pcDNA3.1 through subcloning. Results The open reading frame of mouse cyclin A2 expression vector pcDNA3.1-cyclin A2 was constructed. Single re- striction endonuclease EcoR I enzyme digestion recombinant eukaryotic expression plasmid pcDNA3.1-cyclin A2. The 1660 bp fragment of DNA was successfully cloned. The sequence was demonstrated to be the same as in GenBank. Single restriction endonuclease Hind m enzyme digestion identification of recombinant eukaryotic expression plasmid pcDNA3.1- cyclin A2 and DNA sequencing analysis demonstrated total agreement of the sequence with that in GenBank. Conclusion We have successfully constructed sense and antisense eukaryotic expression vector pcDNA3.1-cyclin A2 of the mouse.

关 键 词：细胞周期蛋白A2 正义 反义 真核表达载体 

分 类 号：R346[医药卫生—基础医学]