构建睫状神经营养因子和绿色荧光蛋白基因转导的重组腺病毒载体

Construction of ciliary neurotrophic factor and green fluorescence protein-transduced recombinant adenovirus

目的以重组腺病毒（rAd）为载体构建腺病毒-睫状神经营养因子-内部核糖体进入位点-绿色荧光蛋白（Ad—CNTF-IRES-GFP）。方法先构建Psp-CNTF-IRES-GFP质粒，再制备PDC316-CNTF-IRES—GFP质粒。然后在脂质体的作用下，用构建好的PDC316-CNTF-IRES—GFP质粒与骨架质粒PBHG在293-LP细胞中构建Ad—CNTF-IRES—GFP腺病毒，并扩增、纯化，鉴定病毒活性。最后，将Ad-CNTF-IRES-GFP转染人源性骨髓间充质细胞（MSCs），观察MSCs的CNTF表达情况。结果成功扩增CNTF基因，扩增后的CNTF基因与基因文库序列完全相符；成功制备PDC316-CNTF-IRES-GFP质粒及Ad-CNTF-IRES-GFP腺病毒．测得Ad—CNTF-IRES—GFP腺病毒的病毒活性单位（pfu）为2．3×10^11；构建好的Ad-CNTF-IRES-GFP成功转染MSCs，而且转染后的MSCs表达CNTF的量为未转染MSCs表达量的20倍。结论本方法能够成功构建Ad-CNTF-IRES—GFP腺病毒载体，而且转染后的MSCs高度表达CNTF。

Objective To construct an adenoviral vector carrying the gene encoding ciliary neurotrophic factor （CNTF）. Methods The gene fragment encoding CNTF was amplified from pMEG-CNTF plasmid by PCR and the Psp-CNTF-IRES-GFP and PDC316-CNTF-IRES-GFP plasmids were constructed. Using PDC316-CNTF-IRES-GFP and PBHG plasmids, the Ad-CNTF-IRES-GFP vector was constructed, and the constructed vector was amplified, purified and identified in 293-LP cells. Ectopic overexpression of CNTF was induced using the constructed vector in human bone marrow-derived mesenchymal stem cells （MSCs） to investigate the role of CNTF in promoting remyelination. Results The Ad-CNTF-IRES-GFP vector was successfully constructed with a pfu of 2.3×10^11. CNTF concentration in the MSCs transfected with Ad-CNTF-IRES-GFP vector was 20-fold higher than that in either non-transfected or Ad-EGFP-transfected MSCs. Conclusion The constructed Ad-CNTF-IRES-GFP vector allows CNTF overexpression in human MSCs by 20 folds, which provides a strategy for gene therapy targeting CNTF.