重组腺病毒载体pAdeasy-1／pAdtrack-CMV—GFP-β-NGF的构建和鉴定

Construction and identification of recombinant adenovirus vector pAdeasy-l/pAdtrack-CMV- GFP-β-NGF

目的构建携带绿色荧光蛋白（GFP）标记的小鼠β-NGF重组腺病毒载体。方法利用RT—PCR法从小鼠颌下腺总mRNA中扩增β-NGF全长cDNA片断，定向克隆于腺病毒穿梭质粒pAdtrack－CMV（已标记绿色荧光蛋白）中；在细菌中与缺陷型腺病毒基因组pAdeasy-1进行同源重组．构建Adeasy-1／pAdtrack—CMV－GFP－β－NGF载体，并进行酶切鉴定。结果成功获得小鼠β－NGF全长扩增片段；pAdTrack—CMV－β-NGF测序验证含有目的基因；双酶切鉴定成功构建重组pAdTrack－CMV－β-NGF质粒：限制性内切酶的酶切分析结果证明成功构建同源重组的Adeasy－1／pAdtrack－CMV－GFP－β-NGF载体。结论本实验所用方法可高效、安全构建腺病毒载体pAdeasy-1／pAdtrack—CMV－GFP－β-NGF。

Objective To construct the recombinant adenovirus vector carrying mouse β-nerve growth factor （β-NGF） labeled with green fluorescent protein （GFP）. Methods Adult healthy male Kunming mice, weighting （60-70） g, were chosen in our study. The full-length β-NGF eDNA was amplified from the total mRNA of mouse submandibular gland by RT-PCR, and then, this fragment was cloned into the adenovirus shuttle plasmid pAdtrack-CMV （marked with GFP）; and then, the pAdeasy-1/pAdtrack-CMV-β-NGF was constructed with defective adenovirus genome pAdeasy-1 by homologous recombination in bacteria; at last, restriction analysis was performed on this adenovirus vector pAdeasy-1/pAdtrack-CMV-GFP-β-NGF. Results Amplified full-length mouse β-NGF fragment was obtained by RT-PCR; the pAdTrack-CMV-β-NGF contained the target gene; double digestion indicated that the recombinant plasmid pAdTrack-CMV-β-NGF was successfully constructed; restriction enzyme digestion analysis indicated that pAdeasy-1/pAdtrack-CMV-βNGF adenovirus vector by homologous recombination was constructed. Conclusion The recombinant adenovirus vector pAdeasy- 1/pAdtrack-CMV-GFP-β-NGF is successfully constructed.