摘要
目的构建含有人髓细胞白血病基因-1(myeloid cell leukemia-1,MCL1)和绿色荧光蛋白基因(pEGFP)的真核共表达载体,为聋病的基因治疗奠定实验基础。方法应用基因重组技术和限制性内切酶酶切,及基因测序方法,构建并鉴定pEGFP-MCL1真核表达质粒。经脂质体介导转染293细胞系,荧光显微镜下观察pEGFP-MCL1真核表达质粒在293细胞系中的表达,利用逆转录-聚合酶链反应(Reverse transcription polymerase chain reaction,RT-PCR)检测MCL1mRNA的表达,Western Blot(蛋白质印迹)方法检测MCL1蛋白的表达。结果阳性重组子经酶切鉴定含有MCL1基因片段,基因测序结果与GenBank中MCL1序列相同。重组pEGFP-MCL1真核表达质粒转入293细胞系后24小时,荧光显微镜下可见绿色荧光表达,RT-PCR能够扩增出MCL1的条带,Western Blot检测出40kDa大小的蛋白。结论成功地构建了含有人全长MCL1基因和pEGFP基因的真核共表达载体,且能在哺乳动物293细胞系中表达。
Objective To construct an eukaryotic expression vector containing human MCL1 gene and EGFP gene for gene therapy for senile deafness.Methods The eukaryotic pEGFP-MCL1 expression plasmid was constructed and identified using Gene recombination and restriction enzyme digestion and gene sequencing.Fluorescence microscopy was used to examination expression in 293 cells transfected by liposome.mRNA was tested using reverse transcription-polymerase chain reaction（RT-PCR）,and Western Blot was used to detect MCL1 protein expression.Results The positive recombinant gene contained the MCL1 fragment with the same sequence as in the gene bank.Transfection into 293 cell was verified by green fluorescent staining and success in amplification of the MCL1 bands.The 40 kDa-sized protein was detected by Western Blot.Conclusions We successfully constructed the eukaryotic expression vector containing human full MCL1 and EGFP gene which expressed in mammalian 293 cell lines。
出处
《中华耳科学杂志》
CSCD
2010年第4期466-469,共4页
Chinese Journal of Otology
基金
国家自然基金资助项目(No:30973304)
十一五国家科技支撑计划项目(No:2006BAI02B06)
解放军总医院苗圃基金(No.06MP29)
