大鼠瘦素基因重组腺相关病毒的构建及在原代神经元细胞中的过表达

Construction of rat leptin recombinant adeno-associated virus 2 vector and its overexpression in primary cultured neurons

目的构建携带大鼠瘦素(leptin)基因的重组腺相关病毒(adeno-associated virus,AAV),并鉴定其在原代鼠神经元细胞中介导的瘦素过表达,为肥胖症基因治疗研究奠定实验基础。方法提取大鼠脂肪组织总RNA,利用RT-PCR技术,获取目的基因瘦素cDNA,通过重组DNA技术,得到瘦素cDNA与pGEM-T载体的重组质粒,阳性重组子用PCR及测序分析鉴定。用Spe I和EcoR V双酶切将pGEM-Leptin中的瘦素基因片段切出,再克隆到AAV2表达质粒pTR-UF22中,构建瘦素重组AAV2载体pAAV2-CBA-leptin。以pDG作为辅助质粒用HEK293细胞包装AAV2-CBA-Leptin,并用一步重力流柱法纯化病毒,由荧光定量PCR测定病毒基因组DNA的拷贝数即为病毒滴度。然后将AAV2-CBA-Leptin及对照病毒AAV2-CBA-EGFP感染大鼠原代神经元细胞,分别用免疫染色和Western blotting鉴定外源基因在神经元的表达。结果测序证实瘦素基因与GenBank提供的原始序列完全一致。重组载体经酶切鉴定与预期结果完全一致,HEK293细胞包装病毒效果良好,得到滴度为1.5×1012vg/mL纯化的重组瘦素病毒AAV2-CBA-Leptin。Western blotting检测显示AAV2-CBA-Leptin能介导瘦素在大鼠神经元细胞中过表达,并随着病毒量的增加而增强。AAV2-CBA-EGFP感染鼠神经元细胞5d后95%左右的细胞有明显的绿色荧光,免疫染色和DAPI核酸染色显示荧光细胞均为神经元而神经胶质细胞无荧光。结论成功构建并包装了瘦素重组AAV2病毒并可介导瘦素在神经元细胞中高效、特异表达,从而为研究瘦素在中枢神经系统控制体重和糖尿病等方面的功能及基因治疗研究打下基础。

Objective The aim of this work was to construct a recombinant adeno-associated virus （rAAV） vector containing rat leptin gene, which mediates transduction of leptin into rat neurons in primary culture. Methods The leptin cDNA was obtained from rat adipose tissue by RT-PCR, cloned into pGEM-T vector, confirmed by sequencing, and then transferred into an AAV2 vector pTR-UF22 that carries CBA promoter. This vector, designated as pAAV2-CBA-Leptin, was packaged in human embryonic kidney （HEK） 293 ceils using pDG as helper plasmid and purified with a single-step gravity-flow column. Copies of viral genome DNA were determined by quantitative PCR. Vector doses were expressed as viral genome copies （ vg）. The viral vectors AAV2-CBA-Leptin and AAV2-CBA-EGFP were transduced into primary cultured cerebral cortical neurons, and leptin expression was determined using Western blotting. The cellular localization of EGFP in cultured neurons was determined by immunocytochemistry. Results Restriction enzyme digestion and sequencing results demonstrated successful cloning of the amplified leptin cDNA into pGEM-T vector and subsequent subcloning into pTR- UF22. Incubation of cerebral cortical neurons cultured with AAV2-CBA-Ieptin produced dose-dependent increases in leptin protein expression as compared with the background control with AAV2-CBA-EGFP. The AAV2-CBA-EGFP generated a high level of EGFP expression which colocalized with neuron-specific immunolabeling of NeuN, indicating a specificity of neuronal transduction by the vector. Conclusions We have developed a recombinant viral vector that efficiently transduces leptin into neurons. This vector offers a valuable bio-tool for studies of leptin in CNS function in the control of body weight and gene therapy of diabetes.