摘要
A method using immobilized affinity chromatography (IAC) was developed to screen for aflatoxin B1 (AFB1)-binding proteins. AFB1 and bovine serum albumin (BSA) coupled protein (BSA-AFB1) was prepared using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. The resulting coupled compound was immobilized onto PVDF transfer membranes, which were then incubated with total protein from mouse liver. AFB1-binding proteins were eluted, after non-specific washing, by specific elution, and the eluted proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two candidate AFB1-binding proteins were identified by liquid chromatography–tandem mass spectrometry as the 40S ribosomal protein SA (RPSA) and a putative uncharacterized protein. RPSA and AFB1 interactions were further analyzed by ELISA in vitro and laser confocal immunofluorescence analysis in vivo. The results from ELISA and immunofluorescence showed that RPSA efficiently bound AFB1 in vitro and in vivo. This study’s conclusion laid the foundation for further exploration of the role of AFB1-binding proteins in AFB1 toxicology towards hepatocytes and the entry pathway of AFB1 into hepatocytes.
摘要译文
使用固定亲和层析（IAC）的方法的开发是为了屏幕黄曲霉毒素B1（AFB1）结合蛋白AFB 1与牛血清白蛋白（BSA）偶联蛋白（BSA-AFB 1）中使用1-乙基-3-（3-二甲氨基丙基）碳二亚胺盐酸盐制备将所得偶联化合物被固定到PVDF转移膜，然后将其与总蛋白孵育从小鼠liverAFB1结合蛋白洗脱，后非特异性洗涤，通过具体的洗脱，并溶出蛋白质通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳分析两个候选AFB 1结合的蛋白质通过液相色谱鈥搕andem质谱作为40S核糖体蛋白的SA（RPSA）和推定的未表征的蛋白质鉴定RPSA和AFB1相互作用进行了进一步ELISA体内和体外激光共聚焦免疫荧光分析法分析从ELISA和免疫荧光结果表明RPSA有效的体外和体内结合AFB1这项研究鈥檚结论AFB1结合蛋白的毒理学AFB1的肝细胞对角色的进一步探索和AFB1的进入途径进入肝细胞奠定了基础。
Zhenhong Zhuang[1]; Yaling Huang[1]; Yanling Yang; Shihua Wang. Identification of AFB1-interacting proteins and interactions between RPSA and AFB1[J]. Journal of Hazardous Materials, 2016,301: 297–303