摘要
目的构建小鼠T-bet基因重组腺病毒。方法采用RT-PCR方法从小鼠脾脏活化的T淋巴细胞中扩增的cD-NA序列,并将其克隆至穿梭质粒pShuttle-CMV中,形成转移质粒,酶切线性化后,与超螺旋腺病毒骨架质粒pAdEasy-l以电穿孔的方法共转化大肠杆菌BJ5183进行同源重组,经酶切鉴定筛选正确重组子,以线性化重组质粒转染293细胞,制备重组腺病毒小鼠T-bet重组腺病毒AdT-bet,最后以PCR及Western blot方法鉴定T-bet的表达。结果感受态大肠杆菌BJ5183细菌内同源重组24h后共获得35%阳性重组质粒克隆,经酶切获得一个30kb的大片段和一个4.5kb的小片断。该质粒转染293细胞后获得的重组腺病毒可高效表达T-bet,纯化后病毒滴度为2.0×1011PFU/mL。结论应用细菌内同源重组法成功快捷构建了含小鼠T-bet基因的重组腺病毒,所获得的AdT-bet可进一步应用支气管哮喘等疾病的基因治疗研究。
Objective To construct recombinant adenoviruse carrying the murine T-bet gene and amplify the adenovirus vector in HEK293 cells. Methods Mouse T-bet eDNA was amplified by RT-PCR from activated murine spleen T cells and subcloned into a shuttle vector pShuttle-CMV. Once construeted, the shuttle vector was linearized with Pine I and cotransformed into BJ5183 together with pAdEasy-1 and the supercoiled viral DNA plasmid. Transformants were selected for kanamyein resistance while recombinants were subsequently identified by restriction digestion. Then, the recombinant adenoviral construct was cleaved with Pac 1 and transfected into the packaging cell line HEK293. The adenoviral vector AdT-bet was propagated in 293 cells and purified by cesium chloride density centrifugation. PCR and Western blot analysis were performed to confirm T-bet expression. Results Total of 35 % positive recombinant plasmid clones were obtained 24 h after conclusion in BJ5183. Digestion of the recombinant Ad plasmid DNA with Pac I yielded a large fragment of 30 kb and a smaller fragment of 4.5 kb. Murine T-bet was expressed efficiently by AdT-bet transfection. The virus stock titer after CsCI banding was 2.0 × 10u PFU/mL. Conclusion The murine T-bet cDNA is cloned successfully and the murine T-bet recombinant adenovirus AdT-bet is constructed conveniently and efficiently by homologous recombination in bacteria with AdEasy system, which may be further used in gene therapy of asthma.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第1期84-87,共4页
Immunological Journal
基金
国家自然科学基金资助项目(30200116)
