安徽农业科学2024，Vol.52Issue(23)：79-84,6.DOI:10.3969/j.issn.0517-6611.2024.23.018
嗜水气单胞菌Hcp蛋白的原核表达·多克隆抗体制备及生物信息学分析
Prokaryotic Expression of Hcp Protein of Aeromonas hydrophila,Preparation of Polyclonal Antibody and Bioinformatics Analysis
摘要
Abstract
[Objective]In order to obtain a polyclonal antibody to the Hcp protein of Aeromonas hydrophila(AH)and its basic biological properties.[Method]The present study utilized RT-PCR to amplify the Hcp gene,constructed a recombinant expression plasmid and then trans-formed the BL21 receptor cells,which were induced to express the Hcp recombinant protein through IPTG(Isopropyl β-D-Thiogalactoside),purified and immunized rabbits,and then prepared a polyclonal antibody against the protein.Hcp recombinant protein was obtained by IPTG(Isopropyl β-D-Thiogalactoside)induced expression,purified and immunized rabbits,and polyclonal antibody against the protein was pre-pared,and the potency of the antibody was determined,identified by Western blotting,and its basic physicochemical properties,conserved structural domains,and phosphorylation sites were investigated using bioinformatics online tools,secondary and tertiary structure prediction.[Result]The results showed that the highest expression of the protein was obtained at the final concentration of IPTG of 0.6 mmol/L at 37℃for 6 h,and the protein mainly existed in the form of soluble protein;the results of Western blotting showed that the supernatant and precipitate of the culture of AH were able to specifically bind to the prepared rabbit antibody with good reactivity,and the potency of the prepared poly-clonal antibody reached 1.024×106.Bioinformatics analysis showed that the molecular formula of Aeromonas hydrophila Hcp protein was C846H1304N224O259S8,encoding a total of 172 amino acids,with a relative molecular mass of 19 013.49 u,an isoelectric point(pl)of 5.24,which is a stabilized protein;the secondary structure of the protein was 51.74％of the irregularly coiled,25.00％of the elongated chain,and 25.00％of the alpha-chain.25.00％,α-helix accounted for 19.19％,and β-turns accounted for 4.07％.[Conclusion]In this study,a poly-clonal antibody to Hcp protein was successfully prepared,which can provide technical support for the further study of Type Ⅵ secretion system(T6SS)of AH.关键词
嗜水气单胞菌/Hcp基因/原核表达/多克隆抗体制备/生物信息学Key words
Aeromonas hydrophila/Hcp gene/Prokaryotic expression/Preparation of polyclonal antibody/Bioinformatics分类
农业科技引用本文复制引用
许艺兰,陈忠伟,何颖,许佳乐,卢冰霞,陈婷婷,秦毅斌,刘思雨,全琛宇,许心婷,赵硕..嗜水气单胞菌Hcp蛋白的原核表达·多克隆抗体制备及生物信息学分析[J].安徽农业科学,2024,52(23):79-84,6.基金项目
广西重点研发计划项目(桂科AB21076008,桂科AB20297059) （桂科AB21076008,桂科AB20297059）
玉林市科学研究与技术开发计划项目(玉市科20220519). （玉市科20220519）
