鸡白细胞介素18成熟蛋白的原核表达及抗体间接ELISA方法的建立  

Prokaryotic Expression of Chicken IL-18 Mature Protein Gene and Establishment of Indirect ELISA for Serum Antibody Detection

作　　者：苏倩倩[1] 李恩扩[1] 张海滨[1] 周霞[1] 胡敬东[1] 

机构地区：[1]山东农业大学动物医学院/山东省动物生物技术与疫病防治重点实验室,山东泰安271018

出　　处：《山东农业科学》2012年第4期5-9,共5页Shandong Agricultural Sciences

基　　金：山东省优秀中青年科学家科研奖励基金(2006BS06015);山东省自然科学基金(Y2008D12)资助

摘　　要：将鸡白细胞介素18的成熟蛋白(chicken interleukin 18 mature protein,mChIL-18)基因在原核系统表达,采用Ni-NTA亲和层析方法纯化得到该重组蛋白。纯化后重组蛋白包被反应板,通过方阵滴定确定最佳抗原包被浓度、血清稀释倍数、羊抗鼠酶标二抗稀释倍数,建立检测ChIL-18成熟蛋白抗体的间接ELISA方法。经方阵滴定结果确定最佳抗原包被质量浓度为4μg/ml,阳性血清稀释比例为1∶104,羊抗鼠IgG辣根过氧化物酶结合物最佳稀释比例为1∶12 000,抗原抗体最佳结合时间是1.5 h,血清与二抗最适反应时间为1 h。阴性、阳性血清的判定标准为:被检样本的OD值是一组阴性样本OD值的2或3倍且OD450≥0.5,即为阳性,比值以P/N表示1,.5<P/N<2.0为可疑,P/N<1.5为阴性。本试验结果证明该方法具有良好的稳定性、重复性和特异性,为下一步单抗的筛选鉴定奠定了必要的基础。The gene of chicken interleukin 18 mature protein was expressed in prokaryotic system. The recombinant mChIL - 18 protein was purified by Ni - NTA Resin affinity chromatography. The indirect ELISA for detecting mChIL - 18 protein antibody was established to determine the immunogenicity of the recombinant protein. The optimum working conditions for the ELISA were as follows： optimal concentration of mChIL- 18 for coating was 4 μg/ml; the dilution ratio of serum sample was 1 ： 104; the optimal coating condition of mChIL - 18 for ELISA was incubated at 37℃ for 2 hours and then at 4℃ over night; the best dilution ratio of HRP - labeled goat anti - mouse IgG was 1 ： 12000 ; the best incubation time for the response of serum sample with the mChIL - 18 protein and HRP - labeled goat anti - mouse IgG were 1.5 hours and 1 h respectively at 37℃. The assessment standard showed that the samples were positive when the OD value was equal and grea- ter than O. 5 and was 2 or 3 times of that of the negative samples, and were suspicious when 1.5 〈 P/N 〈 2, negative when P/N 〈 1.5. The results showed that this method possessed stability, reproducibility and speci- ficity, which provided basis for identification of monoclonal antibody.

关 键 词：鸡白细胞介素 18成熟蛋白(mChIL-18) 原核表达 蛋白纯化 间接ELISA 

分 类 号：Q503[生物学—生物化学]