大鼠胱抑素C腺病毒高效表达载体的构建在大鼠心肌成纤维细胞中的表达  

Construction of recombinant adenovirus vector carrying CysC gene and its expression in rat cardiac fibroblast

作　　者：张书敏[1] 王永梅[1] 杜贻萌[1] 魏峰涛[1] 蒋卫东[1] 王欣[1] 董兆强[1] 姜红[2] 鹿庆华[1] 

机构地区：[1]山东大学第二医院心血管内科,济南250033 [2]教育部和卫生部心血管重构和功能研究重点实验室

出　　处：《中华老年心脑血管病杂志》2011年第3期252-255,共4页Chinese Journal of Geriatric Heart,Brain and Vessel Diseases

基　　金：山东省科学技术发展计划(2007GG20002028)

摘　　要：目的采用Gateway^(TM)技术构建携带胱抑素C(CysC)基因的重组腺病毒载体,并观察在大鼠心肌成纤维细胞中的表达。方法采用RT-PCR方法从大鼠心肌组织中扩增出CysC基因,将CysC基因经BP重组反应定向克隆至pDONR221载体,获得重组质粒pDONR221-CysC,经PCR及测序验证正确的pDONR221-CysC与腺病毒载体pAd/CMV/V_5-DEST在体外进行LR重组反应,CysC取代pAd/CMV/V_5-DEST中的ccdB-Cm^R基因,获得重组腺病毒载体pAd/CMV/V_5-DEST-CysC。采用Western blot技术检测CysC蛋白在293A细胞及大鼠心肌成纤维细胞中的表达。将培养的心肌成纤维细胞随机分为实验组、空载体组和对照组。结果 CysC腺病毒高效表达载体构建成功,测得病毒滴度为5.36×10^(10)ifu/ml,用重组CysC腺病毒转染心肌成纤维细胞24 h后,实验组的转染效率最高(≥90%);与对照组和空载体组比较,实验组CysC蛋白表达明显上调(P<0.05)。结论成功构建了大鼠CysC腺病毒高效表达载体,并成功包装了含有该基因的重组腺病毒,可有效转染心肌成纤维细胞。Objective To establish a recombinant adenovirus vector carrying CysC gene using Gateway TM technique and to observe its expression in rat cardiac fibroblast. Methods CysC gene of rat myocardium was amplified by RT-PCR and directly cloned into entry vector pDONR221 by BP reaction to generate the entry clone pDONR221 CysC. The recombinant plasmid pDONR221-CysC was identified by PCR and sequencing. Along with the LR recombination reactions, the pDONR221-CysC and the target vector pAd/CMV/V5-DEST was recombined together in vitro to create the expression clone（pAd/CMV/Vs-DEST-CysC）, The ccdB-CmR gene in pAd/CMV/V5- DEST was replaced by CysC gene. Rat cardiac fibroblasts were randomly divided into normal control group,Ad-GFP group and Ad-CysC group. Results The recombinant Ad-CysC was successfully constructed. The expression clone Ad CysC was packaged into maturated adenovirus successfully. The efficiency of Ad-CysC for infecting rat cardiac fibroblast was equal to 90% at 24 h after transfection. Compared with control group, the protein expression of CysC was increased significantly in experiment group （P 〈 0.05）. Conclusion The recombinant Ad-CysC was successfully constructed and it could effectively transfect the rat cardiac fibroblast.

关 键 词：腺病毒科 基因表达 转染 肌细胞 心脏 成纤维细胞 半胱氨酸蛋白酶抑制剂 逆转录聚合酶链反应 

分 类 号：R541[医药卫生—心血管疾病]