新基因PRR11的克隆、真核表达载体的构建及鉴定  

CLONING,EUKARYOTIC EXPRESSION AND IDENTIFICATION OF A NOVEL GENE PRR11

作　　者：徐瑲[1] 卜友泉[2] 郭勇[1] 崔涛[3] 兰欢[4,5] 

机构地区：[1]泸州医学院组织胚胎学教研室,四川泸州646000 [2]重庆医科大学生物化学与分子生物学教研室 [3]重庆医科大学分子医学与肿瘤研究中心 [4]重庆医科大学 [5]泸州医学院心肌电生理学研究室

出　　处：《泸州医学院学报》2011年第5期475-479,共5页Journal of Luzhou Medical College

基　　金：国家自然科学基金(30801356;30871237;30872758);重庆市科委自然科学基金(2007BB5302);重庆医科大学重点课题资助项目(XBZD200707)

摘　　要：目的:克隆新基因PRR11的开放阅读框区,构建其真核表达载体,并进行表达检测及鉴定。方法:以HeLa细胞cDNA为模板,半定量RT-PCR扩增PRR11基因,克隆入真核表达载体pcDNA3.1中,酶切、测序鉴定确认获得PRR11基因的重组真核表达载体pcDNA3.1-PRR11。然后将pcDNA3.1-PRR11重组载体转染入HeLa细胞中,收集细胞,制备总RNA和细胞裂解液,采用半定量RT-PCR和蛋白质免疫印迹法检测目的基因表达情况。结果:成功扩增了PRR11基因,双酶切、测序鉴定证实目的基因成功克隆到真核表达载体pcDNA3.1中,测序结果表明序列完全正确,pcDNA3.1-PRR11真核重组表达载体构建成功。半定量RT-PCR和蛋白质免疫印迹法证实了转染入HeLa细胞中的目的基因表达成功。结论:成功构建了PRR11基因的真核表达载体,并可在HeLa细胞中成功表达,为进一步研究PRR11基因的功能奠定了基础。Objective： To clone PRR11 gene,construct the recombinant eukaryotic expression vector of human PRR11,and identify its exogenous expression in HeLa cell.Methods： PRR11 gene was amplified by RT-PCR from HeLa cell cDNA,and cloned into eukaryotic expression vector pcDNA3.1 to construct pcDNA3.1-PRR11.The recombinant plasmid was subsequently transfected into HeLa cell.Forty-eight hours after transfection,total RNA and whole cell lysates were prepared and subjected to RT-PCR and Western blot.Results： PRR11 gene was successfully amplified by PCR and cloned into pcDNA3.1 vector by restriction endonuclease and sequencing analysis.RT-PCR and Western blotting demonstrated that the PRR11 was exogenously expressed in HeLa cell with a relatively high level.Conclusion： The PRR11 was successfully expressed in HeLa cell,which could provide the basis for further research of PRR11 function.

关 键 词：PRR11 PCDNA3.1 真核表达 

分 类 号：R512.6[医药卫生—内科学]