pBiG-CAR真核表达载体的构建及鉴定  

Construction and identification of pBiG-CAR eukaryotic expression vector

作　　者：姜苗苗[1] 申景岭[2] 孙唐娜 王凤梅[1] 王帆[1] 白景玉 卢少玲[1] 张烁[1] 

机构地区：[1]哈尔滨医科大学附属第二医院心血管病科 [2]哈尔滨医科大学组织学与胚胎学教研室,黑龙江哈尔滨150081

出　　处：《哈尔滨医科大学学报》2013年第3期201-204,共4页Journal of Harbin Medical University

基　　金：国家自然科学基金资助项目(81170200)

摘　　要：目的构建及鉴定pBiG-CAR真核表达载体。方法提取C57BL/6胎鼠总RNA,逆转录合成cDNA,经PCR扩增获得CAR基因,与双酶切的pBlue质粒连接,经转化、蓝白筛选及测序鉴定pBlue-CAR载体构建成功。扩增pBlue-CAR的CAR基因,经双酶切后与pBiG质粒连接,构建pBiG-CAR真核表达载体。结果经琼脂糖凝胶电泳,获得目的基因CAR条带,pBlue-CAR目标条带及pBiG-CAR目标条带。基因测序证实所检测重组质粒序列与pBiG-CAR序列完全一致。结论克隆C57BL/6鼠CAR基因、pBlue-CAR重组质粒的构建获得成功,并初步证实pBiG-CAR真核表达载体的构建获得成功,为进一步构建可控心肌特异性CAR过表达转基因鼠提供基础。Objective To construct pBiG-CAR eukaryotic expression vector and identify it.Methods Total RNA was prepared from hearts of fetal C57BL/6 mice to obtain the cDNA by reverse transcription polymerase chain reaction （PCR）.The DNA of coxsckievirus-adenovirus receptor（CAR） was amplified using PCR,and was double digested by enzymes to be ligated with pBlue.The recombinant vector pBlue-CAR was identified by blue white screening with transfromation and DNA sequencing.To ligate DNA with pBiG,CAR was amplified from pBlue-CAR using PCR and double digested by enzymes.Results The obtaining of target gene CAR was confirmed through electrophoresis analysis.The gene band of recombinant vector pBlue-CAR and pBiG-CAR was confirmed by restriction enzyme digestion and electrophoresis analysis.The sequence of recombinant vector showed on difference,as compared with pBiG-CAR by DNA sequescing.Conclusion The DNA of CAR is successfully cloned.The pBlue-CAR and pBiG-CAR are both successfully constructed.It can provide the basis for construction of conditional cardiomyocyte-targeted CAR overexpressing mice.

关 键 词：柯萨奇病毒-腺病毒受体 C57BL 6鼠 pBiG质粒 载体构建 

分 类 号：R394[医药卫生—医学遗传学]