降钙素原的原核表达及单克隆抗体制备

目的:高效表达与纯化可溶性重组人PCT蛋白,制备高灵敏度和高特异性的抗人PCT医用诊断单克隆抗体。方法:大肠杆菌表达重组人PCT蛋白后,利用饱和硫酸铵沉淀和亲和层析方法纯化PCT蛋白后,经质谱、Western blot和间接ELISA法进行性质鉴定和分析重组蛋白的表达与免疫反应性;重组蛋白免疫小鼠,经细胞融合及筛选制备抗PCT单克隆抗体(m Ab)。结果:在大肠杆菌中高效表达了人PCT蛋白;重组人PCT蛋白具有良好的免疫反应性与免疫原性;经筛选获得7株抗PCT单克隆抗体细胞株,经ELISA鉴定,筛选抗体可与PCT抗原有良好的特异性反应。结论:利用重组人PCT蛋白免疫制备了抗人PCT单克隆抗体,为进一步研发PCT快速诊断试剂提供了原料。

Prokaryotic Expression of Procalcitonin and Preparation of Anti-procalcitonin Monoclonal Antibody

Purpose:Recombinant human PCT was efficiently expressed in E.coli,and was purified,and then the high-ly sensitive and specific monoclonal antibodies (mAb)against PCT were prepared,which can be used as diagnostic raw materials for infectious diseases.Method:The recombinant PCT was purified with saturated ammonium sulfate precipita-tion and affinity chromatography method,and then the recombinant protein was confirmed by mass spectrometry.West-ern blot and indirect ELISA assay was used to detect the immunoreactivity of the recombinant protein.The mice were im-munized with the recombinant protein,and anti-PCT monoclonal antibodies (mAb)were prepared by cell fusion andscreening.Result:The recombinant protein was the human procalcitonin,which had good immunoreacitvity and immu-nogenicity,and 7 monoclonal antibodies against human PCT were obtained,and ELISA indicated the antibodies had a good specificity and sensitivity to PCT antigen.Conclusion:Highly sensitive and specific anti-PCT monoclonal antibody was prepared for recombinant human PCT,which will provide a raw material for the development of PCT rapid diagnostic reagent.