广东农业科学2026，Vol.53Issue(3)：65-75,11.DOI:10.16768/j.issn.1004-874X.2026.03.006
番茄SlHSP21-1/2基因原核表达及多克隆抗体制备与鉴定
Prokaryotic Expression of Tomato SlHSP21-1/2 and Preparation and Characterization of Its Polyclonal Antibody
摘要
Abstract
[Objective]To achieve prokaryotic expression of the tomato(Solanum lycopersicum)SlHSP21-1/2 gene and to prepare and characterize its polyclonal antibody,providing a foundation for further studies on its biological function.[Method]The coding sequences of SlHSP21-1/2 genes were cloned from tomato and subjected to bioinformatic analyses to predict their physicochemical properties and subcellular localization.Gene-specific primers were designed for amplification of the target fragments,which were then inserted into a prokaryotic expression vector.The recombinant plasmids were transformed into Escherichia coli BL21(DE3)expression strain,and recombinant protein expression was induced by IPTG.The expressed proteins were analyzed by SDS-PAGE and purified using Ni-NTA affinity chromatography.The purified proteins were used as antigens to immunize New Zealand rabbits.Polyclonal antibodies were obtained through multiple immunizations,and their specificity were evaluated.[Result]Bioinformatics analysis indicated that SlHSP21-1/2 proteins possess contain typical features of small heat shock protein,including a conserved α-crystallin domain and highly conserved N-and C-terminal regions.Subcellular localization predictions suggested that these proteins are localized in chloroplasts.SDS-PAGE analysis confirmed that the SlHSP21-1/2 fusion proteins were successfully expressed,predominantly as inclusion bodies.Following purification by Ni-NTA affinity chromatography,high-purity recombinant proteins were obtained,providing reliable antigens for immunization.Polyclonal antisera raised in New Zealand rabbits using the recombinant proteins specifically recognized the target protein bands in Western blot assays,demonstrating that the prepared antibodies possess strong specificity.[Conclusion]The SlHSP21-1/2 gene was successfully expressed in a prokaryotic system,and polyclonal antibody with good specificity was obtained.关键词
番茄/小热激蛋白/基因克隆/重组蛋白表达/叶绿体Key words
tomato/small heat shock protein/gene cloning/recombinant protein expression/chloroplast分类
农业科技引用本文复制引用
骆鑫灵,韩德阳,冼梓平,孟雪,李楠,张雪莲..番茄SlHSP21-1/2基因原核表达及多克隆抗体制备与鉴定[J].广东农业科学,2026,53(3):65-75,11.基金项目
国家自然科学基金(31972115) （31972115）
广东省自然科学基金(2025A1515012326) （2025A1515012326）
