野生型SLC26A4基因表达载体的构建及在HEK293细胞中的表达

摘要

Objective To construct a eukaryotic expression vector containing the wild-type SLC26A4 gene and green fluorescent protein gene for clinical deafness diagnosis and prenatal diagnosis about SLC26A4-related deafness. Methods Using the Gene recombination and restriction enzyme digestion and gene sequencing, to construct and identify the eukaryotic expression plasmid of pECFP-SLC26A4. Using fluorescence microscope to observe the expression of trans-fected HEK293 cells by liposome, using reverse transcription-polymerase chain reaction (RT-PCR) to detect the mRNA, and using Western Blot to detect SLC26A4 protein expression. Results The positive recombinant gene contain the SLC26A4 fragment, and the sequence were the same sequence as on the GenBank. After being transfected Into HEK293 cells, The green fluorescent can be observed, and The SLC26A4 bands can be amplified. The 87kDa -sized protein was detected by Western Blot. Conclusion we successfully constructed the eukaryotic expression vector containing wild-type SLC26A4 and EGFP gene and the vector can express in mammalian HEK293 cell lines.%目的 构建含有野生型SLC26A4基因和绿色荧光蛋白基因(pEGFP,enhanced green fluorescent protein)的真核共表达载体,为指导临床SLC26A4相关耳聋的基因诊断及产前诊断奠定实验基础.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-SLC26A4真核表达质粒.经脂质体介导转染HEK293细胞系,荧光显微镜下观察pEGFP-SLC26A4真核表达质粒在HEK293细胞系中的表达,利用逆转录-聚合酶链反应(RT-PCR)检测SLC26A4 mRNA的表达,利用Western Blot(蛋白印迹)方法 检测Pendrin的表达.结果 阳性重组子经酶切鉴定含有SLC26A4基因片段,基因测序结果与GenBank中SLC26A4序列相同.重组pEGFP-SLC26A4真核表达质粒转入HEK293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SLC26A4的条带,Western Blot检测出87 kDa大小的蛋白.结论 本文成功地构建了含有人全长SLC26A4基因和pEGFP基因的真核共表达载体,且能在哺乳动物HEK293细胞系中表达.