神经特异性启动子PDGF-EGFP载体的构建及组织表达特异性鉴定  

Construction of nervous specific promoter PDGF-EGFP vector and identification of tissue specificity

作　　者：任凯[1] 丁岩[2] 崔云燕[3] 杨玲玲[4] 张静[4] 崔行[4] 

机构地区：[1]西安医学院生物化学教研室,西安710021 [2]西安交通大学生命科学与技术学院生物工程系,西安710049 [3]济南市中心医院,济南250013 [4]山东大学医学院生物化学与分子生物学研究所,济南250012

出　　处：《山东大学学报（医学版）》2009年第5期50-53,共4页Journal of Shandong University:Health Sciences

基　　金：高等学校博士学科点专项科研基金(No.20050422045)

摘　　要：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 an expression vector of enhanced green fluorescent protein （EGFP） containing nervous tissue- specific promoter-PDGF. Methods The PDGF-promoter sequence was amplified by PCR and the template was the genome of human blood cells. The PCR products were cloned into the pEGFP-1 vector and then pPDGF-EGFP vector was constructed. The vector was transfected into human neuroblastoma cells, SK-N-SH and four kinds of non-nervous recourse cells by lipofectamine. Expression of EGFP of transfected cells and tissue specificity of the vector were determined by transfection. Results The vector of pPDGF-EGFP was successfully constructed. Mter a transfection experiment, the EGFP was highly expressed in SK-N-SH cells and scarcely expressed in the other four cells. Conclusion The vector of pPDGF-EGFP has a high nervous tissue specificity. It lays a foundation for studying target gene therapy of nervous system diseases.

关 键 词：血小板源性生长因子 神经特异性启动子 细胞株 

分 类 号：R741.02[医药卫生—神经病学与精神病学]