应用AdEasy腺病毒改良载体系统构建携带β-半乳糖苷酶基因重组腺.DOCVIP

应用AdEasy腺病毒改良载体系统构建携带β-半乳糖苷酶基因重组腺病毒的实验研究 洪思琦 王佚 毕杨 郭振华（重庆医科大学附属儿童医院，重庆，400014） 提要： 目的 应用AdEasy腺病毒改良载体系统构建携带β-半乳糖苷酶(β-galactosidase，β-gal)基因重组腺病毒。方法 将β-gal基因克隆到腺病毒载体质粒pAdTrack-CMV上，在BJ5183大肠杆菌内与pAdEasy1骨架质粒完成同源重组；经脂质体介导转入HEK293细胞包装、扩增并经反复乒乓感染获得携带β-gal基因的重组腺病毒Ad-β-gal；有限稀释法检测病毒滴度；X-gal组织化学染色检测重组腺病毒Ad-β-gal在HEK293细胞中的功能。结果 成功构建携带β-gal基因的重组腺病毒Ad-β-gal，病毒滴度达2.5~4.0×1011efu/ml；感染复数（multiplicity of infection，MOI）为10时对HEK293细胞48h转染率达70%，并在X-gal试剂作用下，细胞呈现蓝色。结论 成功构建了携带β-gal基因的重组腺病毒，能高效感染293细胞，并功能性表达β-半乳糖苷酶，为进一步研究应用β-gal基因治疗乳糖酶缺乏所致乳糖不耐受症提供实验基础。 关键词 腺病毒；β-半乳糖苷酶；基因治疗；转染；乳糖不耐受症 中图法分类号 文献标识码：A Research on the construction of the recombinant adenovirus vector with β-galactosidase gene by modified AdEasy system HONG Si-qi，WANG Yi，BI Yang，GUO Zhenhua（Chongqing Children’s Hospital, Chongqing Medical University, Chongqing 400014, China） Abstract: Objective The recombinant adenovirus with β-galactosidase gene, which may be used to treat the lactose intolerance caused by lactase deficiency, was constructed by modified AdEasy system. Methods The fragment of β-gal gene was cloned into the shuttle plasmid pAdTrack-CMV, and the homologous recombination was completated with backbone plasmid pAdEasy1 in the E.coli BJ5183 to constructed the recombinant adenoviral plasmid. The adenoviruses were packaged and amplified in the HEK293 cells mediated by liposome. The viral titre was checked by limiting dilution assay and the transfection efficiency was observed by X-gal histochemical stain. Results The recombinant adenovirus with β-gal gene was constructed successfully and the viral titre was 2.5~4.0×1011efu/ml. More than 70% HEK293 cells could be transfected when the MOI was 10. Moreover, more than 65% cells were blue after X-gal histochemical stain. Conclusions Recombinant adenovirus carring β-gal gene with high titre and efficient transfrection could be constructed by AdEasy system, which would supply valuable experimental basis for the gene therapy to the lactose intolerance caused by la