摘要
目的提供一种制备精氨酸酶特异性多克隆抗体的方法。方法首先对双孢蘑菇中编码精氨酸酶的基因(AbArg)进行聚合酶链反应(polymerase chain reaction,PCR)扩增,构建重组质粒pET-28a(+)-Arg,再将质粒转入感受态细胞E.coli BL 21中诱导表达,获得含有6×His标签的融合蛋白,最后用纯化后的蛋白免疫新西兰大白兔,获得抗血清。结果构建了重组质粒pET-28a(+)-Arg,聚丙烯酰胺凝胶电泳(SDS-PAGE)分析表明诱导出的融合蛋白以包涵体形式存在,经纯化后得到的融合蛋白纯度>90%、浓度>0.5 mg/ml。用间接ELISA法测定抗体的效价达51200。Western印迹实验表明制备的多克隆抗体特异性良好,可成功识别双孢蘑菇中的精氨酸酶蛋白。结论成功制备了精氨酸酶的多克隆抗体,为进一步研究精氨酸代谢的机制奠定了基础。
Objective To provide a method for preparing a specific polyclonal antibody of ARG.Methods Firstly,the tech⁃nology of polymerase chain reaction(PCR)was adopted to amplify the AbArg gene encoding arginase in Agaricus bisporus.Then the re⁃combinant plasmid pET-28a(+)-Arg was constructed and transformed into E.coli BL21 for the expression of fusion protein.After⁃wards,the purified protein was used to immunize New Zealand white rabbits,and the serum samples were collected.Results The prokaryotic expression plasmid pET-28a(+)-Arg was constructed and the results of SDS-PAGE analysis showed that the fusion protein existing in form of inclusion body was successfully induced and expressed.After purification,the fusion protein with high purity(above 90%)was obtained.The immunity serum titer was 51200 and Western blotting results showed that the polyclonal antibody could specifically recognize ARG protein in A.bisporus.Conclusion Polyclonal antibody anti-ARG was prepared successfully,which lays a foundation for the further study on the mechanism of arginine metabolism.
作者
奚志嫒
杨可心
孟德梅
XI Zhi-ai;YANG Ke-xin;MENG De-mei(State Key Laboratory of Food Nutrition and Safety,Technology Cooperation Institute for Health Biotechnology,College of Food Science and Engineering,Tianjin University of Science&Technology,Tianjin 300457,China)
出处
《国际药学研究杂志》
CAS
北大核心
2020年第8期632-637,共6页
Journal of International Pharmaceutical Research
基金
国家自然科学基金资助项目(31501544)
天津市京津冀三地联合攻关项目(19YFSLQY00100)。
关键词
精氨酸酶
融合蛋白
多克隆抗体
免疫印迹
arginase
fusion protein
polyclonal antibody
Western blotting
