摘要
为嗜水气单胞菌外膜蛋白AH6169亚单位疫苗的研究奠定理论基础,采用生物信息学方法分析了AH6169的理化性质、结构与功能;分子克隆构建AH6169表达菌株,正交试验筛选其最佳诱导表达条件;通过包涵体洗涤和SDS-PAGE电泳切胶纯化AH6169蛋白;小鼠免疫蛋白制备多克隆抗血清;Western blotting鉴定抗血清特异性与效价;酶联免疫法体外模拟AH6169抗血清与重要水产病原菌的相互识别作用。结果表明,AH6169属跨膜蛋白,无规则卷曲占全序列46.79%,延伸25.49%,α-螺旋22.69%;不同种类的气单胞菌AH6169蛋白保守性较高;成功构建AH6169表达菌株,最佳表达条件为诱导时菌液浓度OD600=0.8,IPTG浓度0.1 mmol·L^(-1),32℃诱导8 h;纯化获得AH6169蛋白,制备的AH6169小鼠抗血清具有较好的特异性,且效价达到1∶1 600;AH6169蛋白抗血清与嗜水气单胞菌、荧光假单胞菌和溶藻弧菌存在相互识别作用。说明AH6169蛋白有较好的抗原性,AH6169可为不同种类的气单胞菌提供交叉免疫保护作用,并且其细胞表位较为丰富,可能是一种潜在的疫苗候选成分。
To lay a theoretical foundation for the study on the subunit vaccine of outer membrane protein AH6169 of Aeromonas hydrophila,bioinformatics methods were used to analyze the physical and chemical properties,structure and function of AH6169;Molecular cloning was used to construct AH6169 expression strains,and orthogonal experiments were used to screen the optimal expression conditions;The AH6169 protein purified by washing the inclusion bodies and cutting the gel by SDS-PAGE electrophoresis;polyclonal antiserum from mouse immune proteins was prepared;Western blotting was used to identify the specificity and titer of antiserum;enzyme-linked immunoassay was used to simulate the mutual recognition of AH6169 antiserum and important aquatic pathogens in vitro.The results show that AH6169 is a transmembrane protein,with random coils accounting for 46.79%of the total sequence,25.49%extension,and 22.69%α-helix;Different species of Aeromonas AH6169 have higher protein conservation;The AH6169 expression strain was successfully constructed.The optimal expression conditions were as follows:the concentration of the bacterial solution during induction was OD 600=0.8,the concentration of IPTG was 0.1 mmol·L-1,and the induction was 8 h at 32℃;The AH6169 protein was purified.The prepared AH6169 mouse antiserum has good specificity and the titer is 1∶1600;AH6169 protein antiserum has mutual recognition effect with Aeromonas hydrophila,Pseudomonas fluorescens and Vibrio alginolyticus.It shows that AH6169 protein has good antigenicity.AH6169 can provide cross-immunity protection for different kinds of Aeromonas,and its cell epitopes are relatively abundant,which may be a potential vaccine candidate component.
作者
康超
荣娜
简思杰
孙薇
刘祥
陈琛
陈春琳
KANG Chao;RONG Na;JIAN Sijie;SUN Wei;LIU Xiang;CHEN Chen;CHEN Chunlin(Chinese-German Joint Institute for Naturel Product Research,College of Biological Sciences and Engineering,Shaanxi University of Technology,Hanzhong 723001,China)
出处
《河南农业大学学报》
CAS
CSCD
2021年第1期80-88,共9页
Journal of Henan Agricultural University
基金
国家外国专家局项目(GDT20186100426)
陕西省科技厅重点研发计划项目(2020LSFP2-38)
陕西理工大学人才项目(SLGQD1803)。
