人神经营养素3基因重组慢病毒载体的构建与鉴定

摘要

目的:构建携带人神经营养素3 (hNT3)基因的重组慢病毒表达载体.方法:通过双限制性内切酶消化和连接的方法构建pGC-E1-hNT3-EGFP质粒,将该质粒转化大肠杆菌DH5α,通过PCR、酶切、测序和对比验证hNT3,通过Lipofectamine 2000将pGC-E1-hNT3-EGFP、pHe1per 1.0和pHe1per 2.0三质粒系统共转染293T细胞包装病毒,经大量扩增后,应用实时定量PCR法鉴定和测定滴度.结果:克隆得到512 bp目的hNT3全长基因,经过PCR扩增、酶切鉴定、序列测定证实,hNT3基因成功克隆到慢病毒载体中,可实现hNT3基因的表达,且病毒滴度为5×107 TU/L.结论:成功构建表达人hNT3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,可作为基因转染的有效工具在将来神经损伤修复实验研究中得到应用.%Objective:To construct a recombinant lentiviral vector expressing human neurotrophin-3 (hNT3).Methods:pGC-E1-hNT3-EGFP plasmid was constructed by double restriction enzyme digestion and ligation,and then the plasmid was transformed into E.coli DH5 α.The correct hNT3 gene was confirmed with PCR,endoenzyme digestion,sequencing analysis and contrast.The plasmid of pGC-E1-hNT3-EGFP vector was cotransfected together with lentivirus-packaging plasmid pHelper 1.0 and pHelper 2.0 into 293T packaging cells by Lipofectamine 2000 mediation.The newly constructed recombinant lentivirus and the titer of virus were confirmed with real-time quantitative PCR.Results:The 512 bp DNA sequence showed that the cloned hNT3 gene sequence was the same as that of the published sequence in GeneBank.The evidence of endonuclease digestion,DNA sequencing and PCR analysis confirmed that hNT3 gene was correctly inserted into the Ientiviral vector.and the titer of virus was 5 × 107 TU/L.Conclusion:The recombinant lentivirus expressing neurotrophin-3 gene are successfully constructed and effectively expressed in 293T cells,which probably can provide a reliable tool for genetic transfection in further experimental researches of nerve injury repair.