Method for preparing cassava calmodulin monoclonal antibody, and antibody prepared with method

Abstract

The invention provides a method for preparing a manihot esculanta crantz calmodulin (CaM) monoclonal antibody, and the antibody prepared with the method. The method comprises the following steps: immunizing mice by purified fusion protein ACP-CaM and adjuvants; intraperitoneally impacting the mice with the fusion protein ACP-CaM diluted by normal saline; fusing spleen cells of the mice and myeloma cells; after fusion, performing cultivation by adopting HAT culture liquid and performing indirect ELISA detection screening to obtain positive hybridoma cells; cloning the positive hybridoma cells with a limiting dilution method until the positive monocloning rate is 100 percent; excluding the positive hybridoma cells, of which the antibodies crossly react with tag protein ACP; preparing a calmodulin monoclonal antibody with a mouse in vivo ascites induction method; and purifying the calmodulin monoclonal antibody. The calmodulin monoclonal antibody prepared with the method provided by the invention has high valence, high stability and high specificity.

Description

technical field [0001] The invention relates to the technical field of monoclonal antibodies, in particular to a method for preparing a cassava calmodulin monoclonal antibody and the antibody prepared by the method. Background technique [0002] The up-regulation of NADH-oxidase in cassava (Manihot esculanta Crantz) produces a large amount of ROS, induces the up-regulation of Calmodulin (CaM), thereby initiating a series of biological defense responses, increasing the expression of stress-resistant proteins and prolonging the storage time of roots. CaM plays an important regulatory role in postharvest cassava root decay (Jian Chunping. Postharvest cassava root storage capacity and proteomic analysis [D]. Haikou: Hainan University, 2013; Zhang Zhenwen. Protein regulation mechanism of cassava root postharvest physiology [D]. Haikou: Hainan University, 2012). Studies have shown that different biotic or abiotic stimuli, such as pests (Zhu Youlin, Wu Jiansheng, Wang Jinsheng. Tw...

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Problems solved by technology

Gel electrophoresis is rarely used because of its inaccurate detection; enzymatic method can detect active CaM, but it is easily disturbed during the determination process, resulting in low measured values; immunological methods mainly include radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA), these two methods are based on the specific combination of antigen and antibody, both need to prepare CaM antibody (Liang Jing, Zhang Xiaorong, Xu Yanhua. Research progress of calmodulin [J ].Journal of Clinical Stomatology, 2010,26(08):500-501)

The immunological detection method has strong specificity, relatively little interference from non-CaM components in the sample to be tested, and can detect the total amount of CaM, but cannot determine the active content of CaM (Wang Juan, Li Xiaojun, Pan Jiwen, etc. Calmodulin ELISA quantitative detection method Establishment and optimization of experimental conditions[J].Medical Graduate Journal,2003,16(6):401-403)

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