A rapid generation of adenovirus vector with a genetic modification in hexon protein
A rapid generation of adenovirus vector with a genetic modification in hexon protein
复制标题
快速生成六邻体蛋白基因修饰的腺病毒载体。
DOI：
10.1016/j.jbiotec.2011.12.022
复制
发表时间：
2012-02-10
影响因子：
4.1
通讯作者：
Xia, Haibin
中科院分区：
文献类型：
--
作者：
Di, Bingyan;Mao, Qinwen;Xia, Haibin
六邻体修饰的腺病毒载体的产生已被证明是困难的。本研究建立了一种快速制备六邻体修饰的腺病毒载体的新方法，即体外一步连接，然后快速进行白色/蓝色筛选。新系统具有以下特点。首先，在E1区由CMV启动子驱动的eGFP表达充当报告基因以评估六邻体修饰的腺病毒的体外嗜性。第二，它有两个独特的限制性内切酶位点，粘性末端位于六邻体HVR 5区域。第三，将plac启动子控制下的lacZ表达盒置于两个限制酶位点之间，这允许使用蓝色/白色筛选来选择重组体。为了证明该方法的原理，通过将NGR、RGD或达特PTD肽插入六邻体HVR 5中成功地产生了遗传修饰的腺病毒。此外，达特PTD修饰的病毒的转导效率在A172和CHO-K1细胞中显示出显著增强。总之，新的系统使真正的重靶向载体的生产更有前途，这将是癌症基因治疗的实质性好处。(C)2012年爱思唯尔B。V.保留所有权利。
The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy. (C) 2012 Elsevier B. V. All rights reserved.