摘要
目的:构建并制备血管生成素-1和EGFP双基因共表达重组腺病毒载体.方法:采用基因克隆技术克隆目的基因ANG-1,并将得到的基因亚克隆至含有报告基因EGFP的穿梭载体pAdTrack-CMV;使用Padeasy-1腺病毒系统将含有目的基因的穿梭载体与腺病毒骨架质粒在细菌BJ5183中同源重组,产生重组腺病毒载体;线性化重组的腺病毒转染入QBI-293A细胞中对重组的腺病毒进行包装并扩增.结果:ANG-1基因与腺病毒穿梭载体连接成功,经Xho I/EcoR V双酶切后琼脂糖电泳可见在1.5kb处出现特异性条带,证明pAdTrack-CMV-ANG-1重组质粒构建成功;重组的穿梭载体与pAdeasy-1质粒重组后,产物经PacI酶切后琼脂糖电泳可见一大一小两个片段.大小约为30kb和4.5kb,与预期结果相符,证明重组腺病毒pAd-ANG-1-EGFP构建成功;线性化重组的腺病毒转染入QBI-293A细胞后包装成功.扩增后病毒滴度为2×1014v.g./L,EGFP活性检测腺病毒感染细胞阳性率在30%以上.结论:成功制备了pAd-ANG-1-EGFP腺病毒.为骨组织工程血管化的研究打下基础.
AIM： To construct and prepare the recombinant adenovirus vector co-expressing angiopoietin-1 （ ANG-1 ） and report gene EGFP. METHODS： The ANG-1 gene was cloned by PCR and then subcloned into vector pAdTrack-CMV which carries the report gene EGFP. The pAdTrack-CMV-ANG-1 and Padeasy-1 were recombined in BJ5183. After identity, the precombinated adenovirus vector of plasmid pAd-ANG-1-EGFP was used to package adenovirus in 293A cells. The identity, titer and EGFP activity of adenovirus were analyzed by methods of PCR, dot-blot and virus infection, respectively. RESULTS： The enzyme digestion of the products with Xho I/EcoR V displayed a fragment at 1. 5 kb, suggesting that the recombinant plasmid, pAdTrack-CMV- ANG-1 was constructed successfully. The enzyme digestion of the products with Pac I showed 2 gene fragments： one was about 30 kb, the other was about 4.5 kb, suggesting that the recombinant adenovirus vector, pAd-ANG-1-EGFP, was successfully reconstruced. In 293A cells the recombinant adenovirus vector was successfully packaged. The titer was 2 × 10^14 v. g./L, the positivity rate of adenovirus infected cells was over 30%. CONCLUSION ： The recombinant adenovirus vector expressing angiopoietin-1 （ANG-1） is successfully prepared. The virus serves to explore the vascularization in bone tissue engineering.
出处
《第四军医大学学报》
北大核心
2008年第11期1002-1004,共3页
Journal of the Fourth Military Medical University
基金
国家自然科学基金(30672142)
