大鼠HO-1基因慢病毒载体的构建与鉴定

Construct and Identification of Lentiviral Expression Vector Encoding Rat Heme Oxygenase-1(HO-1) Genes

目的构建携带大鼠血红素加氧酶-1(HO-1)的慢病毒表达载体。方法构建HO-1基因表达质粒GV208-EGFP-HO-1,并进行测序鉴定。重组质粒GV208-EGFP-HO-1与两种辅助包装原件载体质粒通过Lipofectamine 2000共转染293T细胞,培养48 h后,收集细胞培养上清液,将病毒浓缩后测定病毒滴度。结果成功构建HO-1基因表达质粒GV208-EGFP-HO-1,转化感受态DH5ɑ大肠杆菌。挑阳性单克隆PCR得到1074 bp的特异性条带,测序结果与目标序列完全一致。将HO-1基因过表达载体质粒与另外两种辅助包装原件载体质粒共转染293T细胞后在荧光显微镜下发出明显绿色荧光。Western Blot检测观察到58 kD附近处有特征条带,其大小和目的基因融合蛋白相吻合。病毒原液滴度为2×1012TU/L。结论成功构建HO-1的慢病毒表达载体,为研究HO-1基因对缺血性心脏病的影响打下基础。

Objective To construct a lentiviral expression vector encoding rat heme oxygenase-1（HO-1） genes.Methods Over-expression vector for GV208-EGFP-HO-1 was constructed and confirmed by sequencing.GV208-EGFP-HO-1 and a lentivirus packaging mix were co-transfected into 293T cells to obtain packaged lentivirus particles by Lipofectamine 2000.After 48 hours of culture,the supernatant was obtained to condense for viral titer determination.Results Over-expression plasmid of GV208-EGFP-HO-1 was constructed successfully.Expression plasmid transformed competent DH5ɑ Escherichia coli.Picking positive monoclonal for the PCR obtained specificity 1074bp stripes,sequencing results were exactly the same to the target packaging mix issued green fluorescence under fluorescence microscope.Western Blot detected a characteristic stripe near 58 kD,its size corresponds to the gene fusion protein.The concentrated titer of virus supernatant is 2×1012 TU/L.Conclusion The lentiviral expression vector GV208-EGFP-HO-1 was successfully constructed and provide basis for the study of the effect of the HO-1 gene on ischemic heart disease.