降钙素原的原核表达及单克隆抗体制备 |
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引用本文: | 郑姣,邱义兰,廖晶,李晓丹,张坚松,吴意,何赛,梁湘辉,郭向荣,刘如石. 降钙素原的原核表达及单克隆抗体制备[J]. 激光生物学报, 2016, 0(2): 147-155. DOI: 10.3969/j.issn.1007-7146.2016.02.009 |
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作者姓名: | 郑姣 邱义兰 廖晶 李晓丹 张坚松 吴意 何赛 梁湘辉 郭向荣 刘如石 |
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作者单位: | 1. 湖南师范大学 医学院,湖南长沙410013; 湖南师范大学 生命科学学院,湖南长沙410013;2. 湖南师范大学 生命科学学院,湖南长沙,410013;3. 湖南师范大学 医学院,湖南长沙,410013;4. 湖南师范大学 第一附属医院,湖南长沙,410013;5. 中南大学湘雅医院,湖南 长沙,410008 |
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基金项目: | 湖南省科技计划重点研发计划(2015SK20332,2015SK20665);湖南省教育厅高等学校科研项目(15C0829);湖南省自然科学基金项目(09JJ5027) |
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摘 要: | 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。
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关 键 词: | 降钙素原 原核表达 酶联免疫吸附 (ELISA) 纯化 单克隆抗体 (mAb) |
Prokaryotic Expression of Procalcitonin and Preparation of Anti-procalcitonin Monoclonal Antibody |
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Abstract: | 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. |
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Keywords: | procalcitonin Prokaryotic Expression enzyme-linked immunosorbent assay (ELISA ) purification monoclonal antibody (mAb) |
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