pEGFP-C3-hNIS重组质粒的构建及其在人胃癌BGC-823细胞的表达

Construction of pEGFP-C3-hNIS and its expression in gastric carcinoma BGC-823 cells

目的构建人钠碘转运体(hNIS)基因真核绿色荧光蛋白融合表达载体pEGFP-C3-hNIS,并研究重组hNIS基因在人胃癌BGC-823细胞中的表达情况。方法采用Trizol一步法从甲状腺手术患者的甲状腺组织中提取总RNA,利用RT-PCR方法扩增得到hNIS的可编码区基因,并克隆到T载体上,测序后亚克隆到真核绿色荧光蛋白融合表达载体pEGFP-C3的多克隆位点,获得重组真核表达载体pEGFP-C3-hNIS。分别将pEGFP-C3-hNIS重组质粒(实验组)和pEGFP-C3(对照组)瞬时转染至人胃癌BGC-823细胞中,转染48 h后,于荧光显微镜下观察绿色荧光蛋白在细胞中的表达情况,并通过Western blot方法检测hNIS基因的蛋白表达水平。结果序列分析证实克隆片段与GenBank公布的hNIS基因序列(序列号:NM-000453)相似性达99.90%。转染48 h后,实验组与对照组细胞均可观察到较强绿色荧光信号,用hNIS基因特异性抗体检测表明实验组hNIS表达水平明显高于对照组。结论成功构建hNIS真核绿色荧光蛋白融合表达载体pEGFP-C3-hNIS,并能在人胃癌BGC-823细胞中高表达。

Objective To construct the eukaryotic green fluorescent protein fusion expression vector pEGFP-C3-human sodium iodide symporter（hNIS） and to observe the expression of recombinant hNIS in gastric carcinoma BGC-823 cells.Methods Total RNA was extracted from the thyroid tissue samples by Trizol reagent,the hNIS gene was amplified by RT-PCR,and then cloned into vector pGEM-T for sequencing.Then,the identified cDNA was subcloned into eukaryotic green fluorescent protein fusion expression vector pEGFP-C3 and transfected into gastric carcinoma BGC-823 cells.After recombinant plasmid transfection 48 hours,the green fluorescent signals were observed by fluorescence microscope,and the level of hNIS protein was detected by Western blot method.Results Sequence analysis indicated that the cloned fragment was 99.90% similarity to the released in GeneBank hNIS gene cDNA sequences.The level of hNIS protein was highly expressive in the cells transfected with pEGFP-C3-hNIS,whereas there was almost no expression in the cells transfected with pEGFP-C3,but the green fluorescent signals were obviously observed in all the cells after transfection 48 hours.Conclusion The construction and the expression of eukaryotic recombinant plasmid pEGFP-C3-hNIS have been achieved successfully.