降钙素原的原核表达及单克隆抗体制备

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

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。

Purpose: Recombinant human PCT was efficiently expressed in E. coli,and was purified,and then the highly sensitive and specific monoclonal antibodies( m Ab) 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 precipitation and affinity chromatography method,and then the recombinant protein was confirmed by mass spectrometry. Western blot and indirect ELISA assay was used to detect the immunoreactivity of the recombinant protein. The mice were immunized with the recombinant protein,and anti-PCT monoclonal antibodies( m Ab) were prepared by cell fusion and screening. Result: The recombinant protein was the human procalcitonin,which had good immunoreacitvity and immunogenicity,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.