携强绿色荧光蛋白重组慢病毒的构建及其在原代培养SD大鼠皮层神经细胞中的表达

Construction of lentiviral vector containing EGFP and its expression in primary neocortical cells of SD rats

目的:构建携带强绿色荧光蛋白的重组慢病毒(Lent/EGFP),感染原代培养SD大鼠皮层神经细胞,观察其感染能力及报告基因的表达水平。方法:采用PCR方法克隆EGFP cDNA;将EGFP基因片段亚克隆到慢病毒穿梭质粒多克隆位点内;用4质粒共转染系统在293ET细胞中包装出携带EGFP基因的重组慢病毒Lent/EGFP;重组病毒感染NIH 3T3细胞,FACS检测重组病毒滴度;Lent/EGFP感染体外培养的原代SD大鼠皮层神经细胞,共聚焦荧光纤维镜观察原代神经细胞内绿色荧光蛋白的表达情况。结果:基因测序证明克隆的EGFP cDNA与GenBank提供的序列完全一致;琼脂糖凝胶电泳结果证实EGFP正确插入pLenti6/V5TOPO;FACS检测病毒滴度为2×106;在体外分离培养的原代SD大鼠皮层神经细胞中,重组病毒感染72 h后可见较强的绿色荧光蛋白表达。结论:采用改良的4质粒共转染方法成功构建了携EGFP的重组慢病毒载体;Lent/EGFP对非分裂的神经细胞具有较强的感染能力,且携带的EGFP基因可以在神经细胞内有效表达。

Objective To assess the efficacy of the improved lentiviral vector containing gene enhanced green flurosecent protein （EGFP） delivery pathway in nervous system disease. Methods The cDNA encoding EGFP was cloned by PCR with pIRES2-EGFP as the template. The resulting gene of EGFP was subcloned into the site BamH I and Xho I of pLenti6/V5 TOPO by using DNA recombinant technique. It was introduced into 293ET cells by Ca3 （PO4） 2 methods using four plasmids. The improved four-plasmid system was made up of the vector plasmids which consisted of EGFP, the packaging plasmid pLpl and pLp2, the envelope plasmid encoded the vesicular stomatitis virus-G glycoprotein （VSV-G）. 72 h after transfection, the viral supernatant on 293ET cells was collected. NIH 3T3 cells were infected with the rLent/EGFP and the fluorescence was detected. The titers of the lentiviral vector were determined by positive rate of EGFP cells by means of FACS. rLent/EGFP was transfected into the primary neocorticall cells of SD rats, the expression of EGFP was observed by confocal microscopy. Results The EGFP cDNA cloning and pLent/EGFP construction were confirmed by the evidences of DNA sequence analysis and restriction enzymes digestion. The transfected NIH 3T3 cells were found containing strong expression of EGFP, confirming that the four-plasmid system of the lentiviral vector and its packaging cell line as well, were successfully constructed. The titer of the rLent/EGFP was 2 × 10^6. 72 h after transfection, the higher fluorescent intensity of EGFP was found in the primary neocorticall cells under confocal microscopy. Oonclusion The recombinant Lent/EGFP is successfully constructed by the improved four-plasmid system. It could infect the non- dividing mammalian cells, as the primary neocorticall cells.