摘要
目的构建含有突变型SLC26A4基因和绿色荧光蛋白基因(PEGFP)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。方法通过快速定点诱变试剂盒定点突变SLC26A4基因,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(1517T>G)真核表达质粒。经脂质体介导转染HELA细胞系,共聚焦显微镜下观察突变型pEGFP-SLC26A4(1517T>G)真核表达质粒在HELA细胞系中的表达。结果阳性重组子经酶切鉴定含有突变型SLC26A4基因片段,基因测序结果正确。重组突变型PEGFP-SLC26A4(1517T>G)真核表达质粒转入HELA细胞系后24小时,荧光显微镜下可见绿色荧光表达,并且在细胞膜和胞浆内都有表达。结论本文成功地构建了突变型SLC26A4基因(1517T>G)和PEGFP基因的真核共表达载体,且能在HELA细胞系中表达。荧光显微镜下可见细胞膜和胞浆内都有表达,提示该突变型表达蛋白的转运功能可能发生了改变。
Objective cTo construct a eukaryotic expression vector containing the mutation-type SLC26A4 gene and green fluorescent protein gene for potential utility in genetic and pre-natal diagnosis of SLC26A 4 gene-related deafness. Methods A fast site-directed mutagenesis test kit was used to locate fixed-point SLC26A4 gene mutations. pEGFP-SLC26A4 eukaryotic expression plasmid was constructed and verified via gene recombination, restriction enzyme digestion and gene sequencing. This was used to transleectffELA cells via liposomes and SLC26A4 gene expression was examined using a confocal microscope. Results The recombinant gene was confirmed to contain the SLC26A4 gene with correct sequence. Following transduction to the HELA cells, green fluorescent signals were visible in cell membrane and cytoplasm. Conclusion We have successfully constructed the eukaryotic expression vector containing mutation-type SLC26A4 and EGFP genes which can be expressed in HELA cells. The presence of fluorescent signals in both the cell membrane and cytoplasm indicates abnormal transportation of expression proteins.
出处
《中华耳科学杂志》
CSCD
2011年第3期264-268,共5页
Chinese Journal of Otology
基金
国家自然科学基金青年项目(30801285)
北京市科技新星计划(2009B34)资助
