腺病毒相关病毒增强型mIL-10质粒的构建及表达

Construction of the Adeno-Associated Virus Enhanced Plasmid of Murine IL-10 and Its Expression in Murine Cardiomycocytes

构建腺病毒相关病毒增强型mIL 10质粒 ,并在心肌细胞内进行表达。利用分子克隆技术 ,将腺病毒相关病毒 (AAV )中的末端重复序列 (ITR )插在pORF5 mIL 10质粒启动子的上游 ,构建pORF AAV mIL 10质粒。在阳离子脂质体Lipofectin介导下 ,pORF5 mIL 10和pORF AAV mIL 10分别转染次代培养心肌细胞 ,TRIzol一步法提取总RNA ,RT PCR检测mIL 10的mRNA ;ELISA检测培养细胞上清中mIL 10的表达。结果是用双酶切证实成功构建质粒 ,分别在转染 2 4h、 4 8h、 72h、 96h、 12 0h后 ,用RT PCR在心肌细胞中检测到相应的目的条带 ,ELISA检测各组mIL 10的表达量。证实在阳离子脂质体Lipo fectin介导下 ,pORF AAV mIL 10能在一定时间内稳定的增加mIL 10在心肌细胞内的表达。

To construct the adeno-associated virus(AAV) enhanced plasmid of murine IL-10 (mIL-10)and to study its expression in cardiomyocytes of mice ex vivo,the inverted terminal repeat (ITR) of AAV was inserted in the up-stream of the promoter in the plasmid pORF5-mIL-10 by using the molecular cloning technique to construct the plasmid pORF5-AAV-mIL-10,pORF5-mIL-10 and pORF5-AAV-mIL-10 were used to transfect the cardiomyocyte cell line under the mediation of lipofection. The mRNA of mIL-10 was extracted by one step extraction with TRIzol and was detected by RT-PCR. The expression of mIL-10 in the supernatants of cell cultures was determined by ELISA. It was found that the plasmid pORF5-AAV-mIL-10 was successfully constructed by double digestion procedure. The corresponding target bars could be observed in RT-PCR,24 h,48 h,72 h,96 h and 120 h after transfection and the higher expression quantity was found in case of the pORF5-AAV-mIL-10 as detected by means of ELISA. It concludes that under the mediation of lipofection,plasmid pORF5-AAV-mIL-10 can steady improve the expression of mIL-10 in cardiomyocytes within a certain time limit.