ENC1的克隆,原核表达与数种细胞系表达谱分析

Cloning and Procaryotic Expression of ENC1 and Its Expression Status in Several Cell Lines

以人 3月胎脑总RNA为模板 ,用RT PCR的方法得到了ENC1(ectoderm neuralcortex 1)基因的cDNA ,经测序证实该cDNA的长度为 180 0bp ,包含ENC1的完整编码区 .将之克隆入pGEX 4T 1载体构建重组表达质粒 ,转化大肠杆菌BL2 1表达 ,经Sepharose 4B纯化得到目的蛋白 .通过Northern印迹和RT PCR检验了该基因在数种细胞系中的表达 ,结果表明其在神经胶质母细胞瘤细胞系U2 5 1中有较高的表达 ,而在包括神经母细胞瘤细胞系SH SY5Y的其他数种细胞系中无表达 .与在正常生理状态下神经系统中两种细胞的表达情况相反 .

Ectoderm-neural cortex-1(ENC1) was amplified by RT-PCR using the total RNA extracted from human three-month fetal brain as template. The sequencing shows that its 1.8 kb fragment includes the complete coding sequence of the gene. It was cloned into the expression vector pGEX-4T-1. The recombinant plasmid was induced to express after transformation into E. coli BL21. Then the expressed protein was purified by affinity chromatography. The expression of ENC1 in several cell lines were tested by Northern blot and RT-PCR, indicating that it is highly expressed in glioma U251, and has no expression in all the other cell lines including neuroblastoma SH-SY5Y. The expression pattern of the two cells,glioma U251 and neuroblastoma SH-SY5Y,were contrary to those in neurons under physiological state. This phenomena suggest that ENC1 has different roles in the neoplasis of the two different kinds of cells in neural system.