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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 construct a recombinant adenovirus carrying Cystatin C (CysC) gene by Gateway ~ (TM) technique and observe its expression in rat cardiac fibroblasts. Methods The CysC gene was amplified by RT-PCR from rat myocardial tissue. The CysC gene was cloned into pDONR221 vector by BP recombination, and the recombinant plasmid pDONR221-CysC was obtained. The correct pDONR221-CysC and adenosine Recombinant adenoviral vector pAd / CMV / V_5-DEST-CysC was obtained by carrying out LR recombination reaction in vitro and CysC replacing ccdB-Cm ~ R gene in pAd / CMV / V_5-DEST. Western blot was used to detect the expression of CysC in 293A cells and rat cardiac fibroblasts. The cultured cardiac fibroblasts were randomly divided into experimental group, empty vector group and control group. Results The CysC adenovirus expression vector was constructed successfully. The titer of the virus was 5.36 × 10 ~ (10) ifu / ml. After transfection of cardiac fibroblasts with recombinant CysC for 24 hours, the transfection efficiency of the experimental group was the highest ≥90%). Compared with control group and empty vector group, the expression of CysC in experimental group was significantly increased (P <0.05). Conclusion The rat CysC adenovirus expression vector was successfully constructed and the recombinant adenovirus containing this gene was successfully packaged for efficient transfection of cardiac fibroblasts.