激光生物学报Issue(3)：246-250,5.DOI:10.3969/j.issn.1007-7146.2014.03.010
小鼠 LRRC 10腺病毒载体构建及其在心肌细胞中的表达
Construction of Mouse Lrrc 1 0 Adenovirus Vector and Analysis of Its Expression in Cadiomyocytes
摘要
Abstract
Objective:To construct the recombinant adenovirus expression vector of mouse Leucine-rich Repeat Contai-ning protein 10 (Lrrc10).Methods:Specific primers for the mouse Lrrc10 gene were designed and were employed to amplify the coding sequence using mouse cDNA as template.HA-tagged protein and a SalΙrestriction enzyme site were induced into the PCR primers.The PCR product was purified after gel electrophoresis and inserted into the pMD-18T vector.After sequencing,the positive plasmid was digested with Sal Ιand Hind III and the target fragment was sub-cloned into pAd-track-cmv shuttle vector.The recombinant plasmid was linearized with restriction enzyme PmeΙ.About 100 ng of the purified linear fragment was transformed into bacteria BJ5183.After homologous recombination in the bac-teria,plasmid pAd-Lrrc10 was obtained.pAd-Lrrc10 was linearized with restriction enzyme PacⅠ and transfected into 293A cells with LipofectamineTM2000.Then,the recombinant virus containing Lrrc10 was obtained after packaging. The virus was amplified in 293A cells and then the cells were repeatedly freezed and thawed to get higher titers virus flu-id containing Lrrc10.Cardiomyocytes were infected with the collected virus fluid.To detect the expression of Lrrc10-HA,green fluorescence protein (GFP)was observed by green fluorescence and immunoblotting was performed.Re-sults:Green fluorescent could be detected by fluorescence microscope in the primary cardiomyocytes infected with the vi-rus fluid before 24 h.The total protein of myocardial cells was extracted and the expression of Lrrc10-HA fusion protein could be detected by Western blot..Conclusion:The mouse Lrrc10 adenovirus vector was constructed successfully,and it could induce the sequence coding Lrrc10-HA into cardiomyocytes.关键词
腺病毒载体/Lrrc 1 0/心肌细胞Key words
adenovirus vector/Lrrc10/cardiomyocyte分类
生物科学引用本文复制引用
周作琼,彭夕洋,肖丽娜,李佑锋,彭浩,吴秀山,范雄伟,李永青..小鼠 LRRC 10腺病毒载体构建及其在心肌细胞中的表达[J].激光生物学报,2014,(3):246-250,5.基金项目
国家自然科学基金资助项目（81270156）；湖南省社会发展支撑计划项目（2014SK3099）；湖南省生物发育工程及新产品研发协同创新中心 （）
