胞质蛋白NOD2重组腺病毒载体的构建

摘要

目的 构建人NOD2腺病毒表达载体.方法 采用分子克隆技术,从CoCa2细胞DNA中PCR扩增NOD2片段,经pcD N A 3.1克隆到穿梭质粒pShuttle-CMV,以Kpn Ⅰ和Not Ⅰ双酶切重组穿梭质粒,经电穿孔、抗性筛选,重组成pAdCMV-NOD2腺病毒质粒,经酶切鉴定正确后,在H EK 293细胞中包装成为重组rvAdCMV-NOD2腺病毒,并进行PCR鉴定测定.结果 成功扩增目的 基因并鉴定,重组穿梭质粒pShuttle-CMV-NOD2鉴定,经酶切鉴定证实重组腺病毒质粒构建成功.结论 腺病毒载体应用广泛,细菌体内同源重组腺病毒pAdCMV-NOD2合成效率高,为炎症的基因治疗研究奠定基础.%Objective To construct adenovirus vector containing the NOD2 gene for studying therapeutic inflammation disease. Methods NOD2 was obtained from human CoCa2 DNA based on Adopt molecule clone tech-nique, and then was cloned into plasmid pcDNA3.1 (+) and further cloned into plasmid pShutde-CMV ,pShuttleC-MV-NOD2 with double digestion of PI- Kpn Ⅰ and Ⅰ- Not Ⅰ. The recombinant adenoviral plasmid was identified and transferred into the adenoviral packaging cell HEK293 by lipofectamine 2000 mediated gent transfer method. The recombinant adenovius was confirmed by polymerase chain reaction (PCR). Results The recombinant pAdeno-NOD2 was correctly constructed and confirmed by restriction endonuclease analysis. The transferred HEK 293 cells were lysed by freezethawingto obtaining the recombinant adenovirus in the lysate. The PCR product of the lysate confirmed the presence ofrecombinant adenovirus. Conclusion The recombinant adenovims containing the NOD2 gene can be successfully constructed, which provides the further foundation of NOD2 gene therapy for inflammation disease.