摘要
目的构建肌凝蛋白轻链蛋白-2(myosin light chain-2,MLC-2p)启动子调控的钠/碘转运体(sodium/iodide symporter,NIS)的腺相关病毒载体,探讨外源基因在心肌细胞中特异性表达的可行性。方法构建rAAV9-rMLC-2p-rNIS及rAAV9-CMV-rNIS重组腺相关病毒,经酶切鉴定及效价测定。rAAV9-rMLC-2p-rNIS及rAAV9-CMV-rNIS体外感染心肌细胞和非心肌细胞,行动态摄碘及NaClO4抑制实验观察NIS蛋白的功能和特性。结果成功构建rAAV9-rMLC-2p-rNIS及rAAV9-CMV-rNIS,测得效价为rAAV9-rMLC-2p-rNIS 2.46×1012μg/ml,rAAV9-CMV-rNIS 4.21×1012μg/ml。动态摄碘实验表明感染rAAV9-rMLC-2p-rNIS的心肌细胞的Na125I摄取明显高于非心肌细胞,且能被NaClO4特异性抑制,证实了rMLC-2p启动子的心肌特异性及转染细胞表达的rNIS蛋白有功能。结论成功构建了MLC-2p启动子调控NIS的腺相关病毒载体,证实了外源基因在心肌细胞中的特异性表达,为进一步以该报告基因系统的体内动物实验打下基础。
Objective To construct a recombinant adenoassociated vector containing the rat NIS gene with the myosin light chain - 2 （MLC -2p） gene as the promoter and evaluate its specific expression and feasibility as a reporter gene in cardiomyoeytes. Methods MLC -2p promoter and NIS were subcloned into an adenoassoeiated shuttle vector. Positive recombinant adenoassociated vector was se- lected, packaged and amplified in the 293AAV cells to obtain recombinant adenoassociated virus rAAV9 - rMLC - 2p - rNIS. rAAV9 - cytomegaoviyns （CMV） - NIS with cytomegalovirus as the promoter. Cardiomyocytes and non - cardiomyocytes were infected by the ade- noassociated virus. The function and features of NIS protein were evaluated by dynamic iodide uptake and NaC104 iodine uptake inhibition test in vitro. Results Recombinant NIS adenoassoeiated virus was successfully constructed. Dynamic iodide uptake tests showed that the peaks of iodide uptake of the cardiomyocytes were higher than non - eardiomyocytes transfected with rAAV9 - rMLC - 2p - rNIS. The iodide uptake function of cardiomyocytes were inhibited by NaC104. Conclusion The MLC - 2p promoter - driven rNIS protein was established successfully, showing a strong capability of iodide uptake. It will be the basement for the further research of the stem cells treatment of heart diseases.
出处
《医学研究杂志》
2014年第5期82-86,共5页
Journal of Medical Research
基金
广州市医药卫生科技项目(201102A213021)
广州市属高校科研项目(10A226)
广州医学院博士启动基金(2009)
广州医学院科研项目(2011C49)
