Preparation and identification of short peptides of rice Src homology-3 domain-containing protein 2 for polyclonal antibody production
文献类型： 外文期刊
作者： Wang, Yupeng 1 ; Xie, Yunjie 1 ; Yu, Xiangzhen 1 ; Lin, Yuelong 1 ; Luo, Xi 1 ; Xiao, Yanjia 1 ; Cai, Qiuhua 1 ; Xie, Hua'an 1 ; Zhang, Jianfu 1 ;
作者机构： 1.Fujian Acad Agr Sci, Rice Res Inst, Fuzhou 350019, Peoples R China
2.Fujian Agr & Forestry Univ, State Key Lab Ecol Control Crop Pests Fujian & Ta, Fuzhou 350002, Peoples R China
3.Natl Rice Improvement Ctr, Minist Agr & Rural Areas,State Key Lab Hybrid Ric, Fujian Engn Lab Crop Mol Breeding,State Key Lab C, Fujian Key Lab Rice Mol Breeding,Natl Engn Lab Ri, Fuzhou 350003, Peoples R China
关键词： short peptides; polyclonal antibody; OsSH3P2; peptide chain structure
期刊名称：CHINESE SCIENCE BULLETIN-CHINESE （ 影响因子：1.1； ）
ISSN： 0023-074X
年卷期： 2022 年 67 卷 13 期
页码：
收录情况： SCI
摘要： The successful preparation of specific antibodies is an important step in the study of the physiological and biochemical functions and molecular mechanisms of proteins in plants. In general, polyclonal antibodies are prepared using recombinant proteins expressed in prokaryotes as antigens. However, if the target protein has high sequence homology levels with other proteins, or is difficult to express or purify, specific antibodies cannot be obtained. To address these challenges, short peptide antibody preparation technology has emerged. This technology has gradually been adopted in animals, but it is rarely used in plants. In this study, we analyzed the antigenic epitopes and hydrophilic and homologous amino acid sequences of rice (Oryza sativa) SH3P2 and predicted the protein model using the RoseTTAFold system. Then, we selected three amino acid sequences, peptides 3, 4 and 5, having sequence specificity, high hydrophilicity, antigenic epitopes and different peptide chain structures as templates for the synthesis of short-peptide antigens. The short peptides were coupled with KLH and used independently as antigens to immunize New Zealand white rabbits to produce the antibodies Anti-OsSH3P2-1#, Anti-OsSH3P2-2# and Anti-OsSH3P2-3#, respectively. The serum titers of the polyclonal antibodies were detected using an ELISA, and the titers of the polyclonal antibodies were greater than 1.512000. Furthermore, we constructed the prokaryotic expression vector pMAL-MBP-OsSH3P2 and successfully induced protein production. To investigate the specificity of these three short-peptide polyclonal antibodies, we first detected their specificities to the recombinant protein pMAL-MBP-OsSH3P2. Anti-OsSH3P2-1# and Anti-OsSH3P2-2# successfully bound to the recombinant protein, with the former having the greatest specificity. Then, the plant endogenous OsSH3P2 protein was used to further detect the specificities of the three antibodies. Target bands were detected in OsSH3P2-over-expressing plants, which had significantly higher protein abundance levels, and wild-type plants. The bands indicated that Anti-OsSH3P2-1# specifically recognized endogenous OsSH3P2 without interference from other homologous proteins. In summary, the short peptide polyclonal antibodies of anti-OsSH3P2-1#, an antigen without alpha-helix and beta-fold structures, recognized OsSH3P2 effectively, and OsSH3P2 homologous proteins did not interfere with the binding, indicating that the antibodies had high immunological specificities. The successful production of polyclonal antibodies against OsSH3P2 short peptides lays a foundation for OsSH3P2-related biological functional studies and shed lights on the production of short-peptide antibodies in plants.
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