Cx43和GFP双基因共表达重组腺病毒载体的构建和鉴定

Construction and identification of recombinant adenoviral vector co-expressing connexon 43 and green fluorescent protein

目的构建间隙连接蛋白43（Cx43）和绿色荧光蛋白（GFP）双基因共表达重组腺病毒载体。方法利用PCR方法从真核表达载体pcDNA3.0-Cx43扩增Cx43片断,利用DNA重组技术,将目的基因Cx43克隆至含有报告基因GFP的穿梭质粒pAdTrack-CMV,然后在BJ5183细胞中将重组穿梭质粒pAdTrack-Cx43与pAdeasy-l质粒进行同源重组,产生重组腺病毒载体pAd-Cx43-GFP,用PacⅠ单酶切鉴定重组载体;将pAd-Cx43-GFP转入293细胞中包装,荧光显微镜观察报告基因GFP的表达,Western blot方法检测293细胞中Cx43表达。结果（1）通过PCR方法从载体pcDNA3.0-Cx43扩增出单一条带,与Cx43片断大小相符;（2）重组穿梭质粒pAdTrack-Cx43经NotⅠ/XhoⅠ双酶切后,电泳可见2个大小约为9、1kb条带,证明pAdTrack-Cx43构建成功;（3）同源重组产物pAd-Cx43-GFP经PacⅠ酶切后,电泳可观察到2个大小约为31、4kb左右条带,与预期结果相符,提示同源重组成功;（4）荧光显微镜下观察可见转染pAd-Cx43-GFP后293细胞在培养1-2d即有绿色荧光表达;（5）利用WesternBlot法检测到转染pAd-Cx43-GFP后293细胞中Cx43的表达较未转染组增强。结论Ad-Cx43-GFP重组腺病毒载体构建成功。

Objective To construct and identify the recombinant adenoviral vector co-expressing connexon 43 and green fluores- cent protein. Methods The Cx43 gene was amplified by PCR from eukaryotic expression vector pcDNA3.0-Cx43 and cloned into the shuttle plasmid pAdTrack-CMV which expressed the report gene GFP. Then the recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-1. The recombinant plasmid pAd-Cx43-GFP was confirmed with single digestion by Pac Ⅰ . Further, the plasmid pAd-Cx43-GFP was transfeeted into 293 cells, and the expression of Cx43 and GFP in 293 cells were respectively detected by fluorescent microscopy and Western blot. Results （1）The single fragment which length approached to Cx43 was obtained using PCR; （2）Double digested with restriction endonucleases Not Ⅰ/Xho Ⅰ , the recombinant shuttle plasmid pAdTrack-Cx43 was confirmed by two products which length were respectively about 9kb and lkb; （3）Two fragments of about 31kb and 4kb were obtained, after the recombinant plasmid pAd-Cx43-GFP was digested with Pac Ⅰ ,which suggested a successful homologous recombination; （4）After transfected by pAd-Cx43-GFP, the expression of GFP was monitored by fluorescent microscopy in 293 cells; （5）In contrast to no-transfection group, higher expression of Cx43 in 293 cells transfected by pAd- Cx43-GFP was detected by Western blot. Conclusion The recombinant adenoviral vector Ad-Cx43-GFP was successfully constructed.