摘要
碱性螺旋-环-螺旋(basic helix-loop-helix,bHLH)转录因子在真核生物中广泛存在,是数量较多的转录因子家族之一。该文首次克隆并鉴定双孢蘑菇中的一个AbbHLH(Agaricus bisporus basic helix-loop-helix)转录因子,建立原核表达载体pET-28a(+)-AbbHLH并将其转至E. coli BL21(DE3)感受态细胞中诱导表达,得到主要以包涵体形式存在的6×His-AbbHLH重组蛋白,经纯化与复性后得到纯度超过80%的抗原蛋白。用制备的抗原蛋白免疫新西兰大白兔制备多克隆抗体,间接酶联免疫吸附测定法测定结果表明抗体血清的效价可达102 400。将抗体血清依次经Protein A及CNBr亲和纯化后,用Western Blotting检测抗体特异性,结果表明该抗体能够特异性识别双孢蘑菇中的AbbHLH蛋白。
The basic helix-loop-helix(bHLH) transcription factor family is one of the largest families of transcription factors in eukaryotes. In the present study,an Agaricus bisporus bHLH(AbbHLH)transcription factor was,for the first time,cloned in the A. bisporus,and the prokaryotic expression vector pET-28a(+)-AbbHLH was constructed and transferred into the E. coli BL21(DE3)competent cells to induce the expression of recombinant protein. The recombinant 6×His-AbbHLH protein was obtained mainly in the form of inclusion bodies,followed by purification and renaturation to produce the antigen protein with a purity higher than 80%.Polyclonal antibody was prepared by immunizing New Zealand white rabbits with the above prepared antigen protein,and the immunity serum titer reached 102 400 detected by indirect enzyme-linked immunosorbent assay(ELISA). After successive purification of the antibody serum by Protein A and CNBr,the antibody specificity was detected by Western blot,showing the specific recognition of the purified antibody to AbbHLH protein in A. bisporus.
作者
赵诗睿
奚志嫒
孟德梅
生吉萍
ZHAO Shi-rui;XI Zhi-ai;MENG De-mei;SHENG Ji-ping(College of Food Science and Engineering,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin Gasin-DH Preservation Technology Co.,Ltd.,Tianjin 300300,China;School of Agricultural Economics and Rural Development,Renmin University of China,Beijing 100872,China)
出处
《食品研究与开发》
CAS
北大核心
2022年第23期168-174,共7页
Food Research and Development
基金
国家自然科学基金(31501544)
中国博士后科学基金第69批面上资助项目(2021M692399)
国家级大学生创新创业训练计划资助项目(202210057023)。
关键词
双孢蘑菇
bHLH转录因子
原核表达
蛋白纯化
多克隆抗体
Agaricus bisporus
basic helix-loop-helix(bHLH)transcription factor
prokaryotic expression
protein purification
polyclonal antibody
