目的 采用GatewayTM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/V5-DEST在体外进行LR重组反应,CysC取代pAd/CMV/V5-DEST中的ccdB-CmR基因,获得重组腺病毒载体pAd/CMV/V5-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5.36×1010ifu/ml.用重组CysC腺病毒转染心肌成纤维细胞24h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 GatewayTM 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/V5-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 transfeet the rat cardiac fibroblast.
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