摘要
摘要：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pAdEasyl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）×10^11EFU／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。
Objective To construct a recombinant adenovirus encoding β-galactosidase gene （β-gal）, which may be used to treat the lactose intolerance caused by lactase deficiency, with modified AdEasy system. Methods The sequence fragment of β-gal was cloned into the shuttle plasmid pAdTrack-CMV, and the homologous recombination was completated with backbone plasmid pAdEasyl in the E. coli BJ5183 to construct the recombinant adenoviral plasmid. The adenoviruses were packaged and amplified in the HEK293 cells mediated by liposome. The viral titre was measured by limiting dilution assay and the transfection efficiency was observed by X-gal histochemical staining. Results The recombinant adenovirus with β-gal was constructed successfully and the viral titre was （2.5 -4.0） × 10^11EFU/ml. More than 70% HEK293 cells were transfected when the MOI was 10. Moreover, more than 65% cells were blue after X-gal histochemical staining. Conclusion Recombinant adenovirus carrying β-gal with high titre and efficient transfection is constructed successfully by AdEasy system, which supplies valuable experimental basis for the gene therapy to the lactose intolerance caused by lactase deficiency.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2009年第17期1649-1652,共4页
Journal of Third Military Medical University
