中文摘要：

目的构建含有小鼠Smad4基因和增强型绿色荧光蛋白基因（EGFP）的慢病毒病毒表达载体,为今后应用该重组慢病毒介导的内耳基因导入和相关的聋病基因治疗奠定实验基础.方法利用基因重组、限制性内切酶酶切及基因测序的方法,构建并鉴定pLenti6.3-Smad4-IRES2-EGFP真核表达质粒;利用脂质体介导转染的方法,将pLenti6.3-Smad4-IRES2-EGFP转染导入293T细胞,荧光显微镜下观察EGFP基因表达情况,利用实时荧光PCR的方法检测小鼠Smad4基因mRNA水平的表达情况.结果经PCR鉴定和基因测序证实了小鼠Smad4基因序列与基因bank中的序列相一致.pLenti6.3-Smad4-IRES2-EGFP质粒转入293T细胞后,荧光显微镜下可见有绿色荧光蛋白表达.293T细胞经慢病毒感染后,其小鼠Smad4基因mRNA表达量增加了66427倍.病毒滴度经测定为2.5×108TU/ml.结论成功地构建了含有小鼠Smad4基因的慢病毒表达载体,并能在293T细胞中表达.

英文摘要：

Objective To construct a lentivirus expression vector containing the mouse smad4 gene and EGFP gene for possible application in recombinant lentivirus-mediated inner ear gene transfer and gene therapy for deafness. Methods The eukaryotic expression plasmid pLenti6.3-Smad4-IRES2-EGFP was constructed and identified by gene recombination, restriction enzyme digestion and gene sequencing methods. The 293T cells were transfected with the pLenti6.3-Smad4-IRES2-EGFP plasmid by the lipofectamine-mediated transfection method EGFP gene expression was examined under a fluorescence microscope. Mouse Smad4 mRNA expression was detected by real-time PCR. Results The PCR and sequencing analysis confirmed that the mouse smad4 gene sequence was consistent with the reference in the gene bank. After 293T cells were transfected with the pLenti6.3-Smad4-IRES2-EGFP plasmid, the green fluorescent protein was visible. After 293T cells were infected with lentivirus, Smad4 gene mRNA expression increased by 66,427 times, reaching to a titer of 2.5 ×108TU/ml. Conclusions The recombinant lentivirus expression vector for mouse smad4 gene can be constructed with successful vector expression in 293T cells.