摘要
目的：构建重组质粒pcDNA3.1（＋）-增强型绿色荧光蛋白（EGFP）并观察其存大鼠体内的转染部位及表达时间。方法：构建重组质粒pcDNA3.1（＋）-增强型绿色荧光蛋白后直接注射到正常大鼠的蛛网膜下腔，观察其存大鼠体内转染后的时空效应。结果：双酶切结果符合真核表达载体pcDNA3.1（＋）约5.4kb，EGFP约750bp，另外测序分析与文献报道结果完全一致；动物实验结果表明，鞘内注射24h后存大鼠的脑脊膜和脊神经的外膜上有EGFP表达，在14d达到高峰，4周后明显下降，第7周消失；在实验组大鼠的心、肝、脾、肺、肾及股四头肌均没有绿色荧光。对照组所有取材部位均没有绿色荧光。结论：重组质粒pcDNA3.1（＋）-EGFP通过直接注射法被成功转染至大鼠的蛛网膜下腔，且外源基因得到了表达。
Objective： To construct pcDNA3.1（＋）-EGFP （Enhanced green fluorescence protein, EGFP） recombinant eu karyotic expression plasmid and to investigate the transfection location and duration of gene expression in normal rat.Methods： The recombinant plasmid pcDNA3.1（＋）- EGFP was constructed using gene recombination technology and transfected into the subarachnoid space of normal rat via direct intrathecal injection. Then, the feasibility of delivering exogenous genes was observed. Results： The recombinant pcDNA3.1（＋）-ECFP revealed a 5.4 kb and a 750 pb fragment by using restricted enzyme digestion, which was confirmed by DNA sequencing. After intrathecal injection of recombinant pcDNA3.1（＋）-EGFP, the expression of EGFP was detectable at 24 hours in meninges and lumbar dorsal roots dominantly, and peaked up at 14 days. The fluorescence of EGFP was attenuated since 4 weeks and disappeared at 7 weeks. No EGFP was detected in the other tissues. Conclusion： The recombinant pcDNA3.1（＋）-EGFP is constructed in success and can be transfected into me ninges and perilemma through the intrathecal injection to express EGFP.
出处
《解剖学杂志》
CAS
CSCD
北大核心
2009年第3期339-341,共3页
Chinese Journal of Anatomy
基金
河南省教育厅科技攻关项目（2007320040）
