牦牛源大肠杆菌OmpA外膜蛋白的原核表达及免疫效果评价  

Prokaryotic expression and preliminary analysis of immunogenicity of outer membrane protein of yak-derived Escherichia coli OmpA

作　　者：章石楠 韩生义 石田 李淑萍 胡国元 高瑞[1,2,3] 田嘉琪 周雯雯 李生庆 ZHANG Shinan;HAN Shengyi;SHI Tian;LI Shuping;HU Guoyuan;GAO Rui;TIAN Jiaqi;ZHOU Wenwen;LI Shengqing(College of Agriculture and Animal Husbandry,Qinghai University,Xining 810016,China;Qinghai Province Academy of Animal Husbandry and Veterinary Medicine,Xining 810016,China;Qinghai Province Key Laboratory of Pathogen Diagnosis and Green Prevention and Control Technology Research of Animal Diseases,Xining 810016,China)

机构地区：[1]青海大学农牧学院,青海西宁810016 [2]青海省畜牧兽医科学院,青海西宁810016 [3]青海省动物疫病病原诊断与绿色防控技术研究重点实验室,青海西宁810016

出　　处：《中国兽医学报》2025年第3期458-465,472,共9页Chinese Journal of Veterinary Science

基　　金：青海省科学技术厅科技成果转化专项资助项目(2022-NK-118)。

摘　　要：用生物信息学软件将本实验室分离的Escherichia coli QML2206-1(E.coli QML2206-1)株与不同菌种的外膜蛋白A(OmpA)蛋白氨基酸序列进行同源性分析,并对OmpA蛋白进行理化性质分析、跨膜结构和信号肽预测、B细胞抗原表位预测、二级和三级结构预测;将OmpA基因片段与pET-32a载体连接,构建原核表达载体,在BL21(DE3)进行原核表达并优化表达条件后用镍柱亲和纯化系统进行纯化;将纯化后的重组蛋白与弗氏佐剂充分混匀后免疫小鼠,利用ELISA方法检测小鼠特异性IgG抗体水平及小鼠血清中细胞因子CD4、CD8、IL-4的表达水平,并通过小鼠攻毒保护试验评价其免疫保护效果。结果显示,OmpA蛋白为亲水性蛋白,无跨膜结构域,二级结构主要由无规则卷曲(47.98%)和α-螺旋(29.77%)构成,有12个能与B细胞产生的抗体结合的抗原表位。经原核表达成功获得相对分子质量约为55 kDa的重组蛋白OmpA,且在37℃、IPTG浓度0.0004 mol/L诱导6 h的表达量最高。重组蛋白免疫小鼠后血清特异性IgG抗体效价最高可达1∶32000;与对照组相比,免疫小鼠血清中CD4、CD8及IL-4的表达水平均有升高。用最小致死量(minimum lethal dose,MLD)、2倍最小致死量(2MLD)攻毒后小鼠存活率分别为80%、40%。结果表明,OmpA重组蛋白具有良好的抗原性和一定的免疫保护作用。该研究为下一步基于OmpA蛋白的牦牛适用性E.coli基因工程亚单位疫苗的研制提供了技术依据。The amino acid sequences of the OmpA protein isolated from Escherichia coli QML2206-1(E.coli QML2206-1)in our laboratory were analyzed for homology with different strains of OmpA proteins using bioinformatics software,and the OmpA protein was analyzed for its physicochemical properties,transmembrane structure and signal peptide prediction,B-cell antigenic epitope prediction,secondary and tertiary structure prediction.The OmpA gene fragment was ligated with pET-32a vector to construct a prokaryotic expression vector,which was purified by a nickel column affinity purification system after prokaryotic expression and optimization of expression conditions in BL21(DE3).The purified recombinant protein was fully mixed with Freund's adjuvant to immunize mice,and the levels of mouse-specific IgG antibody and the expression levels of cytokines CD4,CD8 and IL-4 in mouse serum were detected by ELISA,and the immunoprotective effect was evaluated by mouse attack protection test.OmpA protein is a hydrophilic protein with no transmembrane structural domains and a secondary structure consisting mainly of irregular coils(47.98%)and a-helices(29.77%),with 12 antigenic epitopes that can bind to antibodies produced by B cells.The recombinant protein OmpA with a relative molecular mass of about 55 kDa was successfully obtained by prokaryotic expression,and the highest expression was induced by IPTG concentration of 0.0004 mmol/L for 6 h at 37℃.The serum-specific IgG antibody potency of recombinant protein immunized mice was up to 1:32000;the expression levels of CD4,CD8 and IL-4 in the serum of immunized mice were elevated compared with those of the control group.The survival rate of mice was 80%and 40%after attack with minimum lethal dose(MLD)and 2 times minimum lethal dose(2MLD),respectively.OmpA recombinant protein has good antigenicity and certain immunoprotective effects,and this study provides a technical basis for the next step in the development of a genetically engineered subunit vaccine against yak-applicable E.coli based

关 键 词：牦牛源大肠杆菌 OmpA蛋白 原核表达 生物信息学分析 免疫保护效果 

分 类 号：S852.4[农业科学—基础兽医学] S852.61[农业科学—兽医学]