摘要
目的构建含有全长WTX(Wilms tumor gene on the X chromosome)目的基因的表达载体,建立稳定表达WTX基因的胃癌BGC-823/WTX-EGFP细胞系,为WTX基因功能研究提供保障。方法以人胚肾293FT细胞为模板,经PCR扩增获得全长WTX cDNA序列,将全长WTX目的基因序列克隆入带有绿色荧光蛋白报告基因的pEGFP-N1表达载体,建立pEGFP-WTX表达载体。经限制性内切酶酶切鉴定、测序并转染293FT细胞观察报告基因表达情况,判断pEGFP-WTX表达载体构建是否成功。构建成功的pEGFP-WTX表达载体转染人胃癌BGC-823细胞,建立稳定表达WTX的BGC-823/WTX-EGFP细胞。qRT-PCR及免疫组化检测转染细胞WTX表达。结果 PCR方法从人胚肾293FT细胞扩增获得全长WTX cDNA,经测序鉴定序列完全正确;pEGFP-WTX质粒DNA经酶切鉴定片段大小正确、转染294FT细胞可见清晰的绿色荧光表达。BGC-823/WTX-EGFP细胞能够稳定表达WTX。结论本研究成功构建了含有全长WTX cDNA的表达载体系统,建立了稳定表达WTX的BGC-823/WTX-EGFP细胞,为WTX基因功能研究奠定了良好基础。
Objective To establish the BGC-823/WTX-EGFP gastric cancer cell line with stable expression of Wilms tumor gene on the X chromosome（MTX） for functional analysis of WTX gene.Methods The full-length WTX cDNA was amplified from human embryonic kidney 293FT cells and cloned into the pEGFP-N1 vector containing the reporter gene of green fluorescence protein.The recombinant pEGFP-WTX expression vector,after digestion by restriction enzyme to identify the size of target gene fragment,was transfected into 293FT cells and the expression of fluorescent reporter gene was observed under fluorescence microscope.pEGFP-WTX vector was transfected into human gastric cancer BGC-823 cell line to establish BGC-823/WTX-EGFP cell line stably expressing WTX.Quantitative RT-PCR and immunocytochemical staining were used to detect the expression of WTX in both BGC-823/WTX-EGFP and control BGC-823 cells.Results The recombinant pEGFP-WTX plasmid was successfully constructed and verified by PCR and sequencing.The mRNA and protein expressions of MTX were significantly increased in BGC-823/WTX-EGFP cells as compared with those in the control cells.Conclusion The full-length WTX expression vector（pEGFP-WTX） and BGC-823/WTX-EGFP gastric cancer cell line have been successfully established to facilitate further functional study of WTX gene.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2011年第3期392-396,共5页
Journal of Southern Medical University
基金
国家自然科学基金(81071989)
南方医科大学基础医学院院长基金(B1100364)
广东省自然基金(8451051501000555)~~
关键词
WTX
293FT细胞
载体构建
Wilms tumor gene on X chromosome
293FT cells
vector construction
