人BDNF和GFP基因共表达慢病毒载体的构建及神经干细胞转染  

Construction of bicistronic lentivirus vectors carrying hBDNF and GFP genes and transfection of neural stem cells

作　　者：刘勇[1] 陈二涛[1] 冯东福[1] 刘天津[2] 汪洋[3] 潘栋超[1] 

机构地区：[1]上海交通大学医学院第三人民医院神经外科,上海201900 [2]中国科学院上海细胞生物学研究所,上海200031 [3]复旦大学上海医学院解剖与组织胚胎学系,上海200032

出　　处：《上海交通大学学报（医学版）》2008年第10期1250-1253,1257,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：上海市教委自然科学基金(04BB23)~~

摘　　要：目的构建人脑源性神经营养因子（hBDNF）与绿色荧光蛋白（GFP）共表达的慢病毒载体并转染神经干细胞（NSCs）。方法运用基因重组技术，将hBDNF基因连接到带GFP的慢病毒表达载体pWPXL-MOD（pWPXL-GFP-IRES-GFP）中，构建慢病毒载体pWPXL-hBDNF-IRES-GFP，MluⅠ、EcoRⅠ双酶切反应及测序分析加以鉴定。将慢病毒载体主体质粒pWPXL-hBDNF-IRES-GFP、包装质粒HELPER和包膜质粒VSVG共转染293T细胞，包装慢病毒载体并测定滴度。将构建的pWPXL-hBDNF-IRES-GFP感染NSCs，并对感染细胞进行鉴定及分化活性检测。结果构建的慢病毒载体pWPXL-hBDNF-IRES-EGFP经MluⅠ和EcoRⅠ双酶切反应鉴定正确；测序分析证实与Genbank报道的hBDNF基因序列完全一致。三质粒共转染293T细胞后，荧光显微镜下可见大量绿色荧光。包装后慢病毒测定滴度为0．1×10^9～1×10^9 TU／mL。pWPXL-hBDNF-IRES-GFP感染后的NSCs表达绿色荧光，可在体外扩增，NSCs细胞标志物巢蛋白表达阳性；细胞贴壁后可分化为神经元和胶质细胞。结论成功构建hBDNF与GFP基因共表达的慢病毒载体并转染NSCs，转染后细胞仍能保持已知的原有生物学特性及良好的分化活性。Objective To construct human brain-derived neurotrophic factor （hBDNF） and green fluorescent protein （GFP） co-expressing lentiviral vector, and transfeet neural stem cells（NSCs）. Methods Gene recombinant technology was employed to clone hBDNF and GFP genes to lentivirus vector pWPXL-MOD （pWPXL-GFP-IRES-GFP） to construct a lentiviral vector pWPXL-hBDNF-IRES-GFP. Mlu Ⅰ and EcoR Ⅰ enzyme digestion and sequencing analysis were used for identification. Then, 293T cells were transfected with main vector pWPXL-hBDNF-IRES-GFP, packaging plasmid HELPER and coated plasmid VSVG. Lentiviral vectors were packaged and the titer was determined. The constructed pWPXL-hBDNF- IRES-GFP was used to infect NSCs, and the infected cells were identified and the differentiating activities were determined. Results The lentiviral vector plasmid pWPXL-hBDNF-IRES-EGFP was identified correct by Mlu Ⅰ and EcoR Ⅰ enzyme digestion, and DNA sequencing analysis confirmed that hBDNF gene sequencing was exactly the same with that reported by Genbank. After transfeetion, a large number of 293T cells with green fluorescence was observed by fluorescent microscopy. The concentration of the virus titer was 0.1×10^9 - 1 ×10^9 TU/mL. After infection with pWPXL-hBDNF-IRES-GFP, NSCs expressed green flourescence and nestin, and could differentiate into neurons and glial cells. Conclusion hBDNF with GFP gene expression lentiviral vectors can be successfully constructed. After infection, NSCs can maintain the intrinsic biological characteristics and differentiating activities.

关 键 词：脑源性神经营养因子 绿色荧光蛋白 转染 慢病毒 

分 类 号：Q78[生物学—分子生物学]