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目的构建含有神经特异性启动子PDGF的绿色荧光蛋白真核表达载体,并进行组织特异性鉴定。方法提取人外周血基因组DNA,采用PCR技术扩增PDGF-B链上游启动子序列,将其定向克隆至增强型绿色荧光蛋白报告基因表达质粒pEGFP-1中,构建真核表达载体pPDGF-EGFP。将重组载体转染至人神经母细胞瘤细胞SK-N-SH,以及4种其他组织来源细胞系,观察绿色荧光蛋白表达以鉴定其神经特异性。结果成功构建表达载体pPDGF-EGFP,经转染显示pPDGF-EGFP在人神经母细胞瘤细胞SK-N-SH中高表达,而在其它4种非神经组织来源细胞株中极少量表达或未见表达。结论重组真核表达载体pPDGF-EGFP有较高的神经组织特异性,为进一步研究神经系统疾病的靶向基因治疗奠定了基础。
Objective To construct a green fluorescent protein eukaryotic expression vector containing neuron specific promoter PDGF and to identify its tissue specificity. Methods The genomic DNA of human peripheral blood was extracted. The promoter sequence of PDGF-B chain was amplified by PCR and cloned into the enhanced green fluorescent protein reporter plasmid pEGFP-1. The eukaryotic expression vector pPDGF-EGFP was constructed. The recombinant vector was transfected into human neuroblastoma SK-N-SH cells and four other tissue-derived cell lines, and the expression of green fluorescent protein (GFP) was observed to identify its neural specificity. Results The expression vector pPDGF-EGFP was successfully constructed. The expression of pPDGF-EGFP was highly expressed in SK-N-SH cells of human neuroblastoma cells, but little or no expression in the other 4 non-neural cell lines . Conclusion The recombinant eukaryotic expression vector pPDGF-EGFP has high specificity of neural tissue, which lays the foundation for further research on targeted gene therapy of nervous system diseases.