人Carabin基因过表达的9型重组腺相关病毒的构建及功能鉴定  

Construction and Functional Identification of Recombinant Adeno-Associated Virus Type 9 Containing Human Carabin Gene

作　　者：周殿儒 程阔菊[1] 吴庆 冯胜红 罗健华[1] 黄河 罗云[1] ZHOU Dianru;CHENG Kuoju(Dazhou City Hospital of Integrated Traditionnal and Western Medicine,Sichuan Dazhou 635000,China)

机构地区：[1]四川省达州市中西医结合医院药学部,四川达州635000 [2]陆军军医大学第二附属医院麻醉科,重庆400037

出　　处：《河北医学》2021年第3期358-362,共5页Hebei Medicine

基　　金：国家自然科学基金,(编号:81370210);2015年达州市重点科技计划项目,(编号:达市财建[2015]61号)。

摘　　要：目的:构建人Carabin基因过表达的9型重组腺相关病毒,并对其进行结构和功能鉴定。方法:对目的基因质粒pUC57-carabin和载体质粒pAAV9-IRES-ZsGreen进行EcoRI+XhoI双酶切,在T4DNA Ligase作用下连接酶切产物,在JM109感受态细胞中筛选pAAV9-carabin-IRES-ZsGreen质粒,将pAAV9-carabin-IRES-ZsGreen质粒应用高效HET kit转染试剂盒转染至293AAV细胞,以包装rAAV9-carabin-IRES-ZsGreen腺相关病毒,利用Q-PCR检测病毒滴度。rAAV9-carabin-IRES-ZsGreen感染人心室肌细胞株AC16,western blot检测carabin蛋白表达。结果:pAAV9-carabin-IRES-ZsGreen过表达质粒构建成功,rAAV9-carabin-IRES-ZsGreen腺相关病毒包装顺利,滴度为5.09E+12 vg/mL,感染AC16后,carabin蛋白表达显著上调。结论:rAAV9-carabin-IRES-ZsGreen腺相关病毒构建成功,并在心肌细胞中具有高效表达carabin的效应。Objective:To construct recombinant adeno-associated virus type 9 containing human carabin gene and identify its function.Methods:PUC57-carabin and pAAV9-IRES-ZsGreen plasmids was digested by EcoRI+XhoI,then the enzymatic digestion product was ligated by T4DNA Ligase.The pAAV9-carabin-IRES-ZsGreen plasmid was screened in JM109 cells.The pAAV9-carabin-IRES-ZsGreen overexpression vector plasmid was transfected into 293AAV cells using high-efficiency transfection HET kit reagent to package rAAV9-carabin-IRES-ZsGreen.The viral titer was detected by Q-PCR.AC16 cells were infected with rAAV9-carabin-IRES-ZsGreen p,then the carabin protein expression was detected by western blot.Results:The pAAV9-carabin-IRES-ZsGreen overexpression plasmid was successfully constructed.RAAV9-carabin-IRES-ZsGreen was successfully packaged with a titer of 5.09E+12 vg/ml.The expression of carabin protein was significantly increased after AC16 cells were infected.Conclusion:RAAV9-carabin-IRES-ZsGreen has been successfully constructed and can express carabin efficiently in cardiomyocytes.

关 键 词：carabin 9型重组腺相关病毒 人心室肌细胞株AC16 

分 类 号：R373[医药卫生—病原生物学]