目的:构建并鉴定含14-3-3蛋白抑制肽R18的重组腺病毒,为研究14-3-3蛋白的功能提供基础工具。方法:用同源重组方法构建含14-3-3蛋白抑制肽R18的复制缺陷型腺病毒载体(AdR18),并加以鉴定、扩增,以获得高滴度AdR18病毒液,体外感染乳大鼠心肌细胞,检测目的基因表达。结果:将构建的重组腺病毒载体AdR18感染乳大鼠心肌细胞并表达48h后,蛋白印迹结果显示AdR18感染组有明显R18的表达,对照组无表达。结论:腺病毒载体可高效率导入外源基因在心肌细胞中高表达。
Objective:To construct a recombinant adenovirus expressing R18,an inhibitory peptide of 14-3-3 protein.Methods: The adenovirus expressing R18 (AdR18) was prepared by the homorecomnination method,then identified and amplified.The high titer adnovirus product was used to infect neonatal cardiac myocyte,and the expression of R18 in cardiac myocyte was detected by PCR and Western blot.Results:48 hours after infected,the expression of R18 was detected in the AdR18 transfected group by western blot assay, but it was not found in the control Ad group.Conclusion:Adenovirus can high efficiently induce the expression of exogene in cardiac myocyte.