腺病毒介导CTLA4Ig和Iκ-Bα双基因在ECV304细胞中的表达

目的: 通过制备细胞毒性T淋巴细胞相关抗原 4融合蛋白(CTLA4Ig)和核因子的抑制蛋白 (Iκ- Bα)双基因共表达腺病毒载体, 检测重组腺病毒在ECV304细胞中的表达。方法: 将Iκ- Bα和CTLA4Ig通过内部核糖体进入部位(IRES2 )连接, 并以基因重组的形式插入腺病毒表达载体, 构建重组腺病毒, 检测Iκ- Bα和CTLA4Ig在ECV304细胞中的蛋白表达情况及其对炎症介质TNF的调控效应。结果: ( 1 )构建pAdTrack -CMV- CTLA4Ig IRES2- Iκ-Bα, 与pAdEasy在BJ5183重组后转染 293, 获得重组腺病毒。(2)PCR鉴定重组腺病毒正确。(3)用重组腺病毒感染体外培养的ECV304后,该腺病毒可表达Iκ- Bα蛋白及CTLA4Ig蛋白, 且可以下调LPS攻击后TNF α的产生。结论: 构建人CTLA4Ig, Iκ- Bα双基因共表达腺病毒载体, 可以有效的抑制炎症因子的表达,为进一步的CTLA4Ig免疫耐受基因治疗中的抑制, 因NF- κB的过度激活而产生的炎性反应提供研究基础。

Construction and identification of recombinant adenovirus vector harboring CTLA4Ig-IRES2-Iκ-B α gene in ECV-304 cell

AIM: To construct adenovirus vector harboring CTLA4Ig-IRES 2-IkappaBalpha gene,and investigate its expression in ECV304 cells. METHODS: Recombinant adenovirus vector harboring CTLA4Ig-IRES 2-IkappaBalpha was constructed by homologous recombination in E.coli BJ5183. Then recombinant vector was packaged and propagated in 293 cells. ECV304 cells were infected with the recombinant adenovirus, and Western Blot was used to detect IkappaBalpha and CTLA4Ig protein expression in infected ECV304 cells. The effect of expressed IkappaBalpha and CTLA4Ig on the expression of TNF-alpha of ECV304 cells stimulated with LPS was also investigated. RESULTS: The recombinant CTLA4Ig-IRES 2-IkappaBalpha adenovirus was generated by homologous recombination and identified by PCR methods. Ikappa-Balpha and CTLA4Ig were expressed in ECV304 cells infected with the recombinant adenovirus. The production of TNF-alpha induced by the LPS was inhibited in infected ECV304 cells. CONCLUSION: The recombinant CTLA4Ig-IRES 2-IkappaBalpha adenovirus may offer a method to down regulate the inflammatory cytokines in the inflammatory reaction which may lay the foundation for genetherapy of immunosuppression.