VCAM-1和EGFP双基因共表达重组腺病毒的构建

Construction of recombinant adenoviral vector co-expressing vascular cell adhesion molecules-1 and enhanced green fluorescent protein

目的构建VCAM-1和EGFP双基因共表达重组腺病毒载体。方法采用DNA重组技术,将目的基因VCAM-1克隆至含有报告基因EGFP的穿梭质粒;在BJ5183细胞中与pAdeasy-l质粒进行同源重组,产生重组腺病毒载体。结果(1)VCAM-1基因与pAdTrack-CMV载体成功连接,经NotⅠ/XhoⅠ双酶切后电泳可见2个大小约为9kb和2kb左右条带,经PCR法扩增后电泳可见一个600bp左右条带,证明pAdTrack-CMV-VCAM-1重组质粒构建成功;(2)pAdTrack-CMV-VCAM-1质粒与pAdeasy-l质粒同源重组后,产物经PacⅠ酶切后电泳可观察到2个大小约为31kb和4kb左右条带,与预期结果相符。结论Ad-VCAM-1-EGFP重组腺病毒载体构建成功。

Objective To construct the adenoviral vector co-expressing vascular cell adhesion molecules-1 （VCAM-1） and enhanced green fluorescent protein（EGFP）. Methods The target gene VCAM-1 was cloned into the shuttle plasmid expressed the report gene EGFP. Then the recombinant shuttle plasmid was transformed into BJ5183 bacteria to recombine with backbone vector pAdeasy-1. Results Identified by PCR and double digestion with restriction endonucleases Not Ⅰ/Xho Ⅰ, the VCA.M-1 fragment was successfully inserted into pAdTrack-CMV vector; after the recombination of pAdTrack-CMV-VCAM-1 and pAdeasy-1 in BJ5183 bacteria, the product was conformed by restriction endonuclease Pac I digestion. Conclusion The recombinant adenoviral vector Ad-VCAM-1-EGFP was successfully constructed.