摘要
目的研究人激肽释放酶腺相关病毒载体的构建,观察重组病毒感染人脐静脉内皮细胞株后人激肽释放酶基因的表达。方法将人激肽释放酶基因定向克隆入AAV载体质粒pAAV-MCS中,并与两种辅助质粒pAAV-RC和pHelper共转染293细胞,包装成带有人激肽释放酶基因的重组腺相关病毒(rAAV);收集病毒颗粒并测定病毒滴度。以不同滴度的病毒分别感染人脐静脉内皮细胞,逆转录聚合酶链反应定量检测人激肽释放酶在该细胞中的表达。酶联免疫吸附法测定人脐静脉内皮细胞内人激肽释放酶的含量。结果成功获得了重组人激肽释放酶基因AAV载体,重组病毒滴度为6.2×1010个/L。以滴度分别为1×109个/L、1×108个/L和1×107个/L的病毒感染人脐静脉内皮细胞,与空白对照组比较,人激肽释放酶的表达均有增加(P<0.05),但以1×109个/L组升高最明显(P<0.001)。结论带有人激肽释放酶的重组腺相关病毒滴度可以稳定地达到1010个/L以上,感染人脐静脉内皮细胞后,人激肽释放酶基因在宿主细胞中的表达明显增强。
Aim To construct the recombinant adeno-associated virus vector （rAAV） expressing the human tissue kallikrein （HK） gene and detect the expression of interested gent in human umbilical vein endothelial cell （hUVEC） which were infected with different titer of rAAV. The feasibility of gene therapy for hypertension by rAAV mediated human tissue kallikrein gene transfection was discussed. Methods The HK gene was directionally cloned into the pAAV-MCS, and co-transfected AAV-293 cell with other two plasmids （the pAAV-RC, and pHelper） by lipofectamine. The recombinant AAV particles were harvested and the viral titer was measured. Then hUVEC were infected with different titer of rAAV particles. 72 hours later, the expression of human tissue kallikrein gene was detected by RT-PCR and ELISA. Results The AAV expressing system of human tissue kallikrein gene was successfully constructed with a titer of 6.2 × 10^10 pardcles/L measured with In Situ β-Galactosidase Staining Kit. Compared with control group, the expression of HK gene in groups infected with rAAV at different titer of 1×10^9, 1×10^8 1×10^7 increased significantly （P〈0.05）, especially in the group of 1×10^9 （P〈0.001）. Conclusions When co-transfecting AAV-293 cell with three plasmids（ the recombinant pAAV expression plasmid, pAAV-RC, and pHelper）, the titer of rAAV particles can reach 〉 10^10 particle/L stably. The interested gene can be expressed significantly and stably when using recombinant AAV viral to infect the cultured mammal cell.
出处
《中国动脉硬化杂志》
CAS
CSCD
2006年第4期321-324,共4页
Chinese Journal of Arteriosclerosis
