诱导型一氧化氮合酶基因真核表达载体的构建与表达

CONSTRUCTION AND EXPRESSION OF EUKARYOTIC EXPRESSION VECTOR OF INDUCED NITRIC OXIDE SYNTHASE

目的 构建诱导型一氧化氮合酶基因真核表达载体 pc DNA3/ i NOS,并以脂质体为介导观察其在肝窦内皮细胞中的转染效率及表达情况。方法 先用限制性核酸内切酶 Hinc +Cla 酶切质粒 PSP/ i NOS和 PU C6 s,构建 PU C6 s/ i NOS中间质粒 ,再用 Hind +Xhol酶切质粒 PUC6 s/ i NOS和 pc DNA3,构建 pc DNA3/ i NOS重组质粒 ,鉴定正确后以 L ipofec-tam ine为介导将其与 p EGFP共转染分离培养的大鼠肝窦内皮细胞 ,在荧光显微镜下计算转染效率并用 RT- PCR和 West-ern blot检测 i NOS基因的表达。结果 成功构建了诱导型一氧化氮合酶基因真核表达载体 pc DNA3/ i NOS,以脂质体为介导转染肝窦内皮细胞后其转染效率为 35 .6 %± 3.4 % ,用 RT- PCR和 Western blot检测到相应的 i NOSm RNA和蛋白。结论 以脂质体介导的 i

Objective To construct the eukaryotic expression vector pcDNA 3/iNOS for detecting the expression of iNOS in isolated sinusoidal endothelial cells(SEC).Methods Plasmid of PSP/iNOS and PUC6s were digested with HincⅡ and ClaⅠto construct the intermedia plasmid PUC6s/iNOS for obtaining suitable multiple cloning site.PUC6s/iNOS and pcDNA 3 were further digested with HindⅢ and Xhol to construct recombinant pcDNA 3/iNOS.After co-transduction of pcDNA 3 /iNOS with pEGFP mediated by Lipofectamine into SEC,transfection rate was calculated and RT-PCR as well as Western blot were used to detect the expression of iNOS.Results pcDNA 3/iNOS was successfully constructed and can express corresponding mRNA and protein in SEC mediated by Lipofectamine.The transfection rate was about 35.6%±3.4%.Conclusion Eukaryotic expression vector pcDNA 3/iNOS can effectively transduct into SEC and express corresponding mRNA and protein with a relatively high transfection rate.