摘要
特异性抗体的成功制备是研究蛋白功能的重要环节,目前短肽抗体制备技术在动物中的应用日益成熟,然而该项技术在植物中的应用甚少.本研究通过分析水稻OsSH3P2蛋白的抗原表位、亲水性以及同源蛋白氨基酸序列,同时利用RoseTTAFold系统进行蛋白模型预测.选取了3条序列特异、亲水性高、具有抗原表位和不同肽链结构的氨基酸序列,通过人工合成短肽抗原、偶联KLH载体蛋白后,免疫新西兰大白兔制备得到相应多克隆抗体.经酶联免疫吸附测定检测获得了3份Anti-OsSH3P2-1#、Anti-OsSH3P2-2#、Anti-OsSH3P2-3#高效价短肽多克隆抗体血清.用免疫印迹(Western blotting)方法对OsSH3P2原核重组蛋白和植株内源的OsSH3P2蛋白进行检测,以进一步确定短肽抗体的特异性.结果显示,由不含α-螺旋和β-折叠结构的肽段作为抗原,免疫制得的Anti-OsSH3P2-1#短肽多克隆抗体能有效识别OsSH3P2蛋白,且不受同源蛋白干扰,说明该抗体具有较高的免疫特异性.OsSH3P2短肽多克隆抗体的成功制备,为进一步挖掘OsSH3P2生物学功能奠定基础,且为植物短肽抗体制备提供新的思路.
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 Rose TTAFold 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 p MAL-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 p MAL-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-overexpressing 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 withoutα-helix andβ-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.
作者
王昱澎
谢云杰
余想珍
林悦龙
罗曦
肖晏嘉
蔡秋华
谢华安
张建福
Yupeng Wang;Yunjie Xie;Xiangzhen Yu;Yuelong Lin;Xi Luo;Yanjia Xiao;Qiuhua Cai;Hua’an Xie;Jianfu Zhang(Rice Research Institute,Fujian Academy of Agricultural Sciences,Fuzhou 350019,China;State Key Laboratory for Ecological Control of Crop Pests between Fujian and Taiwan,Fujian Agriculture and Forestry University,Fuzhou 350002,China;National Engineering Laboratory of Rice/South China Research Base of State Key Laboratory of Hybrid Rice/Incubating base of State Key Laboratory of Crop Germplasm Innovation and Molecular Breeding between Fujian Province and Ministry of Science and Technology,Fuzhou Branch of National Rice Improvement Center,South China Key Laboratory of Hybrid Rice Germplasm Innovation and Molecular Breeding of Ministry of Agriculture and Rural Areas/Fujian Engineering Laboratory of Crop Molecular Breeding/Fujian Key Laboratory of Rice Molecular Breeding,Fuzhou 350003,China)
出处
《科学通报》
EI
CAS
CSCD
北大核心
2022年第13期1449-1458,共10页
Chinese Science Bulletin
基金
国家水稻产业技术体系项目(CARS-01)
福建省人民政府-中国农业科学院农业高质量发展超越“5511”协同创新工程项目(XTCXGC2021001)
福建省科技重大专项(2020NZ08016)
福建省省属公益类科研专项(2020R1023008)资助。
关键词
短肽
多克隆抗体
OsSH3P2
肽链结构
short peptides
polyclonal antibody
OsSH3P2
peptide chain structure
