IL-1ra-Fcε融合基因在大鼠体内表达条件优化及免疫原性分析

Optimization on expression condition of IL-1ra-Fcε fusion gene in rats and its immunogenicity analysis

目的探索基因治疗载体pCI-neo-IL-1ra-Fcε在大鼠体内的最适表达条件及免疫原性。方法采用气道滴注方式将pCI-neo-IL-1ra-Fcε载体滴注至大鼠肺部,利用RT-PCR及Westernblot方法检测不同剂量(0.25、0.5、2.5mg.kg-1)载体在不同时间(d4、d7、d20)的表达量,并采用免疫组化方法进行验证。间接ELISA法检测滴注后IL-1ra-Fcε蛋白IgG抗体变化,并对IgG抗体亚型IgG2a和IgG1进行分析。结果3种不同剂量载体在滴注后d20均可检测到目的蛋白表达,2.5mg·kg-1组蛋白表达量明显高于另外2组;以0.25mg·kg-1剂量滴注载体,目的基因d4时未见表达,d7时表达量增加,d20时达到最大值;免疫组化结果显示IL-1ra-Fcε基因在大鼠肺组织中得到有效表达。结论成功建立pCI-neo-Ira-Feε载体在大鼠体内最适表达条件,此载体可诱导机体产生Th1型免疫应答。

Aim To investigate the optimal condition for expression of pCI-neo-IL-1ra-Fcε plasmid for gene therapy and its immune response in rats.Methods Different doses（0.25,0.5,2.5 mg·kg^-1）pCI-neo-IL-1ra-Fcε plasmid were instilled into lungs of rats through tracheas,then expressions of IL-1ra-Fcε fusion protein were detected at different interval times（d 4,d 7,d 20）by RT-PCR and Western blot.Through immu-nohistochemistry analysis of rat lungs,those results of optimal condition were validated.Dynamic changes of rat serum IgG level,including IgG2a and IgG1 were detected by indirect ELISA.Results IL-1ra-Fcε could be detected at 20th day after instilling pCI-neo-IL-1ra-Fcε plasmid in all dose groups.The expression level in 2.5 mg·kg^-1 groups was higher than that in other two groups.IL-1ra-Fcε protein could not be expressed at 4th day,but increased at 7th day and reached maximum at 20th day after instillation.IL-1ra-Fcε gene could be efficiently expressed in rat lungs by immunohistochemistry analysis.Humoral immunity of rats was induced by pCI-neo-IL-1ra-Fcε plasmid.IgG2α expression was predominant in IgG subsets,showing that the humoral immunity was Th1-like immune response.Conclusion The optimal condition is established for gene therapy with pCI-neo-IL-1ra-Fcε plasmid which can induce Th1-like immune response in rats.