摘要
[目的]对鲍曼不动杆菌潜在生物标志物蛋白Dcap进行生物信息学分析,并制备其多克隆抗体。[方法]采用生物信息学对Dcap蛋白进行初步分析,通过PCR扩增Dcap基因片段,并克隆入p ET-28a载体中构建重组表达载体。将重组载体p ET-28a-Dcap转入大肠杆菌BL21(DE3)中诱导表达,用纯化后的蛋白免疫小鼠制备多克隆抗体,Western blot实验分析抗体特异性。[结果] Dcap基因全长1 305个碱基对,编码434个氨基酸,在鲍曼不动杆菌中高度保守,是一种含有信号肽的稳定亲水性跨膜蛋白,二级结构主要由无规则卷曲组成,占比达到51.15%,三维模型质量评估系数(GMQE)为0.76,具有较高的可靠性,含有15个潜在的线性B细胞抗原表位。免疫实验结果显示,纯化的Dcap蛋白能够有效刺激小鼠产生多克隆抗体,其效价高达1:256 000,能特异性识别Dcap蛋白。[结论]生物信息学分析表明Dcap蛋白具备稳定的理化性质,位于细胞外膜,展现出良好的免疫原性。通过基因克隆、蛋白质纯化以及免疫小鼠实验,成功制备了Dcap蛋白的多克隆抗体,表明Dcap蛋白有望成为鲍曼不动杆菌检测的新靶标。
[Objective]Bioinformatics analysis of Dcap,a potential biomarker protein of Acinetobacter baumannii,and prepara-tion of its polyclonal antibody.[Method]Preliminary analysis of Dcap protein was carried out using bioinformatics,and the Dcap gene fragment was amplified by PCR and cloned into pET-28a vector to construct a recombinant expression vector.The recombinant vector pET-28a-Dcap was transferred into the expression bacterium Escherichia coli BL21(DE3)to induce ex-pression,and the Dcap protein was purified by using Ni-NTA,and the purified protein was immunized to mice to prepare poly-clonal antibodies.The specificity of the prepared polyclonal antibodies was identified by Western blot analysis.[Result]The Dcap gene is 1305 base pairs in length,encodes 434 amino acids,is highly conserved in Acinetobacter baumannii,is a stable hydrophilic transmembrane protein containing a signal peptide,has a secondary structure consisting mainly of random coil with a percentage of 51.15%,shows high reliability with a three-dimensional model quality assessment coefficient(GMQE)of 0.76,and contains 15 potentially linear B cell antigenic epitopes.The results of immunological experiments showed that the pu-rified Dcap protein could effectively stimulate mice to produce polyclonal antibodies with a titer as high as 1∶256000,which could specifically recognize Dcap protein.[Conclusion]Bioinformatics analysis showed that the Dcap protein has stable physi-cochemical properties,is located in the extracellular membrane,and exhibits good immunogenicity.Through gene cloning,pro-tein purification and immunization of mice,a polyclonal antibody against Dcap protein was successfully prepared,indicating that Dcap protein is a new target for A.baumannii detection.
作者
华明明
李增辉
王毅
孔萌萌
胡征
杨波
HUA Mingming;LI Zenghui;WANG Yi;KONG Mengmeng;HU Zheng;YANG Bo(School of Life and Health Engineering,Hubei University of Technology,Wuhan 430068;Key Laboratory of Fermentation Engineering(Ministry of Education),Hubei University of Technology,Wuhan 430068;Co-construction Cooperative Innovation Center for Industrial Fermentation(Ministry of Education&Hubei Province),Hubei University of Technology,Wuhan 430068,China)
出处
《生物技术》
2025年第6期677-683,676,共8页
Biotechnology
关键词
鲍曼不动杆菌
Dcap蛋白
生物信息学
蛋白结构
抗原表位
包涵体复性
蛋白纯化
多克隆抗体
Acinetobacter baumannii
Dcap protein
bioinformatics
protein structure
antigenic epitope
inclusion body re-folding
protein purification
polyclonal antibody
