RMND5B基因的克隆及其腺病毒载体的构建  

Cloning of RMND5B Gene and Construction of RMND5B Adenovirus Vector

作　　者：张仕超[1] 陈宇[1] 尹丽阳 刘文邦[1] 崔俊毅 蔡英桂 朱旋[1] 陈辉[1] 吴秀山[1] 范雄伟[1] 王跃群[1] ZHANG Shichao CHEN Yu YIN Liyang LIU Wenbang CUI Junyi CAI Yinggui ZHU Xuan CHEN Hui WU Xiushan FAN Xiongwei WANG Yuequn＂(The Center for Heart Development, Key Lab of MOE for Department Biology and Protein Chemistry, I-Iunan Normal University, Changsha 410081, Hunan, China)

机构地区：[1]湖南师范大学蛋白质化学及鱼类发育生物学教育部重点实验室,心脏发育研究中心,湖南长沙410081

出　　处：《激光生物学报》2016年第4期350-355,共6页Acta Laser Biology Sinica

基　　金：国家自然科学基金面上项目(31272396;31572349;81400304;2015JJ3087);湖南省生物发育工程及新产品研发协同创新中心(2013-448-6)

摘　　要：已有研究表明RMND5B可能与心肌肥大相关,但具体机制不明,RMND5B的重组腺病毒载体的构建和鉴定为研究RMND5B功能提供了基础工具。首先,设计小鼠RMND5B基因的特异性引物,以c DNA为模板,PCR扩增RMND5B ORF区,并在两端各加入HindⅢ及SalⅠ的酶切位点。将此片段插入到p MD18-T载体,再亚克隆至线性化的穿梭质粒p Ad Track-CMV中。PmeⅠ酶切线性化之后,电转化到含p Ad Easy-1的BJ5183感受态细菌中。在BJ5183细菌中发生同源重组获得了重组质粒p Ad-RMND5B质粒。PacⅠ酶切线性化之后转染293A细胞,经过包装获得腺病毒AdRMND5B。将此腺病毒感染新生大鼠原代心肌细胞,并在一段时间后观察绿色荧光并通过RT-PCR检测RMND5B的表达情况。结果表明,成功构建了RMND5B的重组腺病毒载体并实现了AdRMND5B在新生大鼠原代心肌细胞中表达,为进一步研究RMND5B基因在心肌肥大中的作用奠定了良好的实验基础。Studies have proved that RMND5B might be associated with myocardial hypertrophy but specific mechanism was not clear yet.The construction of RMND5B recombinant adenovirus vector provided the basis tools for RMND5B function research.First of all,specific primers for the mouse RMND5B gene were designed and employed to amplify RMND5B ORF area using cDNAs as the template.At the same time joined Hind Ⅲ and SalⅠenzyme loci at each end.This fragment was inserted into the pMD18-T carrier and subcloned to linearized pAdTrack-CMV shuttle plasmid.It was electroporated into the competence of BJ5183 bacteria with pAdEasy-1 after linearized by PmeⅠenzyme.Homologous recombination occurred during BJ5183 bacteria and finally obtained the recombinant pAd-RMND5B plasmid.It was used to infect 293A cells after linearized by PacⅠenzyme and adenovirus AdRMND5B was obtained after packaging.The ad-enovirus infected primary myocardial cells of neonatal rat,and after a period of time the green fluorescence was ob-served.Then the expression of RMND5B was detected by RT-PCR and agarose gel electrophoresis.Above results proved that we successfully built the mice RMND5B recombinant adenovirus vector and implemented the AdRMND5B expressed in primary myocardial cells of neonatal rat laid a solid experimental foundation for the further studies on the function of RMND5B gene of myocardial hypertrophy.

关 键 词：腺病毒 RMND5B 心肌细胞 

分 类 号：Q782[生物学—分子生物学]