Prokaryotic Expression of Wheat Autophagy-related Factor ATG5 and Preparation of Its Antiserum in Rabbits

doi:10.13560/j.cnki.biotech.bull.1985.2017-0020

Abstract

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∶25 600 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.

Key words: wheat, ATG5, prokaryotic expression, antiserum

ZHANG Jia-zi ,LI Kai-xin ,YU Bao-jia ,YUE Jie-yu, WANG Hua-zhong. Prokaryotic Expression of Wheat Autophagy-related Factor ATG5 and Preparation of Its Antiserum in Rabbits[J]. Biotechnology Bulletin, 2017, 33(7): 69-74.

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