摘要
细胞自噬在植物生长发育和环境响应中扮演了重要角色,ATG5是参与自噬小体组装的核心因子之一。在前期工作中我们克隆了两个小麦ATG5基因TaATG5a和TaATG5b,并对其开展了初步的功能分析。鉴于后续基因功能研究中的免疫学方法涉及对特异性抗体的需求,通过载体构建、原核表达和亲和层析过程获得了TaATG5a的重组蛋白,经兔免疫过程制备了TaATG5a的抗血清,采用ELISA和Western杂交等方法鉴定了抗体的效价和特异性。结果表明,克隆于pET30a载体上的TaATG5a在大肠杆菌中能够被IPTG高效诱导表达,重组蛋白的表观分子量与理论值基本一致,表达量在诱导0-8h范围内逐渐增加。纯化的重组蛋白纯度较高,满足抗体制备要求。制备的抗血清能特异性识别原核表达和小麦内源的TaATG5a,效价达到1∶25600。此外,制备的抗血清通过Western杂交还能够识别共价连接的小麦ATG12-ATG5复合物,该复合物是小麦叶片中ATG5的主要存在形式。
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 andare under deep functional study in our lab. In order to meet the need of specific antibodies in immunological methods used in further functionalgene and protein study,a recombinant protein TaATG5a was acquired by vector construction,prokaryotic expression,and purification via theNi-column affinity chromatography method,then the antiserum was prepared in rabbit immunization. The titer and specificity of the preparedantiserum were tested by the ELISA and Western blot assays. The results showed that the expression of TaATG5a cloned in vector p ET30 a wasefficiently induced in Escherichia coli by IPTG,and gradually increased with the extension of IPTG induction time over 0-8 h. The apparentmolecular weight of recombinant protein TaATG5a was close to its theoretical value. The purified recombinant protein was in high-purity thatsatisfied the requirements for antibody preparation. The prepared antiserum had a high titer of 1∶25 600 and specifically recognized TaATG5ain 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 alsorecognized by the prepared antiserum through Western blot assay.
作者
张加姿
李开心
于宝佳
岳洁瑜
王华忠
ZHANG Jia-zi LI Kai-xin YU Bao-jia YUE Jie-yu WANG Hua-zhong(Tianjin Key Laboratory of Animal and Plant Resistance, School of Life Sciences, Tianfin Normal University, Tianfin 30038)
出处
《生物技术通报》
CAS
CSCD
北大核心
2017年第7期69-74,共6页
Biotechnology Bulletin
基金
天津市自然科学基金项目(17JCZDJC33800)
天津师范大学创新基金(52XC1604)
