大鼠Ngb基因慢病毒表达载体构建及鉴定

摘要

目的：克隆大鼠脑红蛋白(Ngb)基因，构建其慢病毒表达载体Lentivirus-Ngb，并转染原代培养的大鼠骨髓间充质干细胞(BMSCs)。方法：人工合成大鼠Ngb cDNA，连接至pLenti6.3-IRES-EGFP慢病毒载体，选择阳性克隆进行酶切鉴定和测序，并将其包装至慢病毒，共转染293T细胞，收获病毒颗粒，检测病毒滴度，PCR检测Ngb的表达。用包装成功的病毒液感染BMSCs，显微镜下观察BMSCs细胞，Western-blot检测Ngb蛋白在BMSCs中的表达。结果：PCR产物双酶切和基因测序确定Ngb连接正确；感染72 h后，荧光显微镜下可以看到GFP阳性的BMSCs细胞，Westen-blot检测表明Ngb在感染慢病毒的BMSCs中表达。结论：成功构建大鼠Ngb基因慢病毒表达载体pLen-ti-Ngb-IRES-EGFP，并成功转染BMSCs。%Objective:To clone neuroglobin (Ngb) gene of rat and construct the lenti-viral expression vec-tors of Ngb, and then transfect the vectors into bone marrow mesenchymal stem cells (BMSCs). Methods:The Ngb gene was connected to the pLenti6.3-IRES-EGFP and the positive clones were selected for re-striction enzyme digestion and sequencing. The plasmids were inserted into lenti-virusus to construct the lenti-viral expression vectors, and then the vectors were identified by enzyme digestion and sequencing analysis. The lentivirus expression in transfected 293T cells was identified by PCR, and the Ngb protein in transfected BMSCs was detected by western-blot. Results:The products were identified by enzyme diges-tion and sequencing analysis. GFP positive BMSCs can be seen under fluorescence microscope 72 hours after transfection. Westen blot analysis showed the expression of Ngb protein in BMSCs. Conclusion:The lentiviral expression vectors of Ngb were constructed and transfected into BMSCs successfully.