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

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,.5P/N2.0为可疑,P/N1.5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。

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

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 greater than 0.5 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 specificity,which provided basis for identification of monoclonal antibody.