人MxA基因重组腺病毒载体的构建及鉴定  

Construction and identification of the recombinant adenovirus AdMxA

作　　者：毛国强[1] 彭明利[2] 黄琴[1] 赵瑞秋[1] 朱朝敏[1] 任红[2] 许红梅[1] 

机构地区：[1]重庆医科大学附属儿童医院感染消化科 [2]重庆医科大学感染性疾病分子生物学教育部重点实验室重庆医科大学病毒性肝炎研究所,重庆400010

出　　处：《重庆医科大学学报》2006年第4期498-501,508,共5页Journal of Chongqing Medical University

基　　金：重庆市卫生局科技基金资助项目(03125)

摘　　要：目的:克隆中国人MxA基因并构建含有MxA基因的重组腺病毒载体,为下一步应用其进行抗乙型肝炎基因治疗研究奠定基础。方法:从健康中国人外周血中分离单个核细胞,用Trizol试剂提取总RNA。应用逆转录PCR(RT-PCR)方法,以自行设计的引物扩增全序列MxA基因,应用基因克隆技术将MxA基因克隆到腺病毒穿梭质粒pAdTrack(携带有绿色荧光蛋白基因)中,将线性化的pAdTrack-MxA和腺病毒骨架质粒pAdEasy-1共同电转化入新鲜感受态大肠杆菌BJ5183中,两者在其中完成同源重组,MxA重组腺病毒质粒经卡那霉素抗性鉴别和PacI酶切确证,验证正确的MxA重组腺病毒线性化转染入293细胞中包装成完整病毒颗粒,观察绿色荧光蛋白(GFP)表达并检测重组腺病毒的病毒滴度。结果:重组腺病毒穿梭质粒pAd-Track-MxA连接成功,测序结果表明克隆的MxA基因序列与GenBank中人MxA基因序列仅有5个核苷酸不同但所编码氨基酸仅1个发生改变,且此氨基酸位于非功能区。MxA重组腺病毒经PacI酶切后产生30kb和4.5kb大小2个片段表明同源重组成功。重组腺病毒经293细胞包装后可观察到绿色荧光,收获病毒并测感染滴度为1.59×108(pfu/ml),具有较高感染效率。结论:我们已成功克隆了中国人的MxA基因,并成功构建重组腺病毒质粒,为进一步应用此基因进行抗乙型肝炎基因治疗研究奠定了基础。Objective： To clone the Chinese MxA gene and to construct the recombinant adenovirus plasmid carring human MxA gene for further study. Methods： The peripheral blood mononuclear cells（PBMCs） were isolated by gradient fractionation,then total RNA was extracted from the PBMCs.The MxA gene was amplified by RT-PCR using the primers based on the published sequence of MxA.The PCR product, double-digested with restrictive endonucleases NotⅠ and XbaⅠ, was cloned into pAdTrack-CMV by molecular cloning technique. The plasimid pAdTrack-MxA was linearized with PmeⅠ ,and subsequently co-transformed into electrocompetent BJ5183 cells with an adenoviral backbone plasmid （pAdEasy-1）. Recombinants were selected for kanamycin resistence and confirmed by PacI.Finally,the linearized recombinant plasmid was transfected into HEK 293 cells.Recombinant adenoviruses were generated within 7 to 12 days,then viral tite was checked by GFP. Results：Sequencing analysis revealed that the amino acid sequence of MxA gene had only one amino acid residue displace from isoleucine to valine（compared with that of published sequence） although there were five variations in nucleotide sequence.The restrictive endonuclease analysis confirmed that correct recombinant adenovirus plasmid was constructed.24 hours after transfection,the fluorescence was observed in 293 cells, and viral title checked by GFP was 1.59 × 10^8（ pfu/mL ）. Conclusion： The MxA gene is cloned from a Chinese donor and a recombinant adenovirus vector containing human MxA gene is constructed successfully.It lays the foundation for further study of gene therapy for HBV.

关 键 词：MxA基因 逆转录 基因克隆 腺病毒 

分 类 号：R512.6[医药卫生—内科学]