Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an 710062, PR China.
J Biotechnol. 2011 Mar 20;152(3):72-6. doi: 10.1016/j.jbiotec.2011.02.001. Epub 2011 Feb 12.
The generation of fiber-modified adenoviral vector has proven difficult. In the paper, we developed a new system for rapid construction of fiber-modified adenoviral vector containing foreign peptides in the HI loop or C-terminal of the fiber knob. The new system was established through the following processes. First, a unique BamHI mutation was made in the genome of Ad5 without causing amino acid change. Second, two unique restriction enzymes BamHI and SfuI, both with sticky end, were introduced in the HI loop or C-terminal of Ad5 fiber knob. Third, a lacza expression cassette was placed between BamHI and SfuI sites for a quick identification of positive cloning based on white-blue color screening. This system allows generation of recombinant adenoviral vector by a single step, in vitro ligation followed by quick white-color positive clone screening. To prove the principle of the method, Ad5HI-RGD by modifying HI-loop of the fiber knob with RGD motif and Ad5Cter-PK7 by modifying C-terminal of the knob with poly-lysine (pK7) were successfully generated in vitro. Ad5 with a knob modified in the HI loop of the fiber with Tat-PTD, NGR or SIKVAV peptide were also successfully developed. The transduction of the modified viruses for Hela, U87 MG and MDA-MB-231 cells was investigated in vitro compared with unmodified Ad5. In conclusion, the new vector system allows for a rapid generation of fiber-mutant adenovirus and provides useful tool for gene function analysis and cancer gene therapy.
纤维修饰型腺病毒载体的构建一直存在困难。在本研究中，我们开发了一种新的系统，可用于快速构建包含 HI 环或纤维蛋白 knob 末端的外源肽的纤维修饰型腺病毒载体。该新系统的建立经过以下步骤：首先，在不引起氨基酸改变的情况下，在 Ad5 基因组中引入独特的 BamHI 突变；其次，在 Ad5 纤维 knob 的 HI 环或末端引入两个独特的 BamHI 和 SfuI 内切酶位点，均具有粘性末端；最后，在 BamHI 和 SfuI 位点之间放置 lacza 表达盒，用于快速基于白色蓝色颜色筛选鉴定阳性克隆。该系统可通过一步法，体外连接，快速白色阳性克隆筛选，生成重组腺病毒载体。为了验证该方法的原理，我们通过修饰纤维 knob 的 HI 环，成功构建了携带 RGD 基序的 Ad5HI-RGD 和修饰 knob 末端的多聚赖氨酸（pK7）的 Ad5Cter-PK7。此外，我们还成功构建了纤维 knob 的 HI 环上修饰有 Tat-PTD、NGR 或 SIKVAV 肽的 Ad5。随后，我们研究了这些修饰病毒对 Hela、U87MG 和 MDA-MB-231 细胞的转导效率，并与未修饰的 Ad5 进行了比较。总之，该新型载体系统可快速生成纤维突变型腺病毒，为基因功能分析和癌症基因治疗提供了有用的工具。
