生物技术通报2017，Vol.33Issue(7)：69-74,6.DOI:10.13560/j.cnki.biotech.bull.1985.2017-0020
小麦自噬相关因子ATG5的原核表达和抗血清制备
Prokaryotic Expression of Wheat Autophagy-related Factor ATG5 and Preparation of Its Antiserum in Rabbits
摘要
Abstract
Autophagy is closely implicated in plant growth and development,as well as responses to environmental stimuli. ATG5 is one of the core factors involved in autophagosome assembly. Two wheat ATG5-encoding genes,TaATG5a and TaATG5b were cloned previously and are under deep functional study in our lab. In order to meet the need of specific antibodies in immunological methods used in further functional gene and protein study,a recombinant protein TaATG5a was acquired by vector construction,prokaryotic expression,and purification via the Ni-column affinity chromatography method,then the antiserum was prepared in rabbit immunization. The titer and specificity of the prepared antiserum were tested by the ELISA and Western blot assays. The results showed that the expression of TaATG5a cloned in vector pET30a was efficiently induced in Escherichia coli by IPTG,and gradually increased with the extension of IPTG induction time over 0 - 8 h. The apparent molecular weight of recombinant protein TaATG5a was close to its theoretical value. The purified recombinant protein was in high-purity that satisfied the requirements for antibody preparation. The prepared antiserum had a high titer of 1:25600 and specifically recognized TaATG5a in E. coli or wheat total protein. In addition,the ATG12-ATG5 in conjugated form,i.e.,the main form of wheat ATG5 in leaves,was also recognized by the prepared antiserum through Western blot assay.关键词
小麦/自噬相关因子ATG5/原核表达/抗血清Key words
wheat/ATG5/prokaryotic expression/antiserum引用本文复制引用
张加姿,李开心,于宝佳,岳洁瑜,王华忠..小麦自噬相关因子ATG5的原核表达和抗血清制备[J].生物技术通报,2017,33(7):69-74,6.基金项目
天津市自然科学基金项目(17JCZDJC33800),天津师范大学创新基金(52XC1604) （17JCZDJC33800）
