CAJ | 학술논문

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-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。
목적구건휴대록색형광단백(GFP)표기적소서β-NGF중조선병독재체。 방법이용RT-PCR법종소서합하선총mRNA중확증β-NGF전장cDNA편단，정향극륭우선병독천사질립pAdtrack-CMV(이표기록색형광단백)중；재세균중여결함형선병독기인조pAdeasy-1진행동원중조，구건Adeasy- 1/pAdtrack-CMV-GFP-β-NGF재체，병진행매절감정。 결과성공획득소서-NGF전장확증편단；pAdTrack-CMV-β-NGF측서험증함유목적기인；쌍매절감정성공구건중조pAdTrack-CMV-β-NGF질립；한제성내절매적매절분석결과증명성공구건동원중조적Adeasy-1/pAdtrack-CMV-GFP-β-NGF재체。 결론본실험소용방법가고효、안전구건선병독재체pAdeasy- 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 cDNA 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-l/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-l/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.