摘要
目的构建携带人碱性成纤维生长因子（bFGF）基因的重组慢病毒表达载体。方法采用聚合酶链反应（PCR）方法钓取人源性的bFGF和FGF4两个目的基因片段，将该基因克隆到慢病毒载体表达质粒pGC-FU[含绿色荧光蛋白（GFP）]中，得到pGCFU-FGF4-bFGF，通过PCR、酶切、测序和对比验证bFGF后，通过Lipofectamine2000的介导把pGCFU-FGF4-bFGF质粒和包装质粒pHelper1．0、pHelper2．0共转染至包装细胞293T，经同源重组产生重组慢病毒pGCFU-FGF4-bFGF，pGCFU-FGF4-bFGF在293T细胞内大量扩增，应用实时定量PCR法鉴定和测定滴度。结果克隆得到500bp目的bFGF全长基因，经过PCR扩增、酶切鉴定、序列测定证实，bFGF基因成功克隆到慢病毒载体中，可实现bFGF基因的表达，且病毒滴度为2．0×10^9TU／ml。结论成功构建表达人bFGF基因的慢病毒载体并能在293T细胞中扩增获得足够高的病毒滴度，可作为后续基因治疗研究工作的基因转染工具。
Objective To construct a recombinant lentiviral vector expressing human basic fibroblast growth factor （bFGF） gene. Methods Huamn hFGF and FGF4 genes were amplified by polymerase chain reaction （PCR） technique and then cloned into the pGC-FU vector which contained green fluorescent protein （GFP）. The resulting lentiviral vector containing FGF4 and hFGF was named the plasmid of pGC FU-FGF4-bFGF vector. The correct bFGF gene was confirmed by PCR, endoenzyme digestion, sequencing analysis and contrast. The plasmid of pGC FU-FGF4-bFGF vector was co-transfected together with lentivir- us-packaging plasmid pHelper 1.0 and pHelper 2. 0 into 293T packaging cells by lipofectamine 2000 medi- ation. The newly constructed recombinant lentivirus and the titer of virus were confirmed by real-time quan- titative PCR. Results The 500 bp DNA sequence showed that the cloned bFGF gene sequence was the same as that of the published sequence. The evidence of endonuclease digestion, DNA sequencing and PCR analysis confirmed that bFGF gene was correctly inserted into the lentiviral vector, and the titer of virus was 2. 0 ×10^9 TU/ml. Conclusion The recombinant lentivirus expressing human FGF4 and bFGF were successfully constructed and effectively expressed in 293T cells, which probably can provide a reliable tool for genetic transfection in further gene therapy researches.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2010年第5期580-582,共3页
Chinese Journal of Experimental Surgery
基金
福建省科技厅资助省属高校项目（2008F5022）
