禽致病性大肠杆菌HlyE蛋白的免疫原性研究  

Studies on the immunogenicity of the HlyE protein of avian pathogenic Escherichia coli

作　　者：张春晓 王利丽 赵奇 孙欣艺 侯冠欣 刘畅[1] 史秋梅[1] 吴同垒[1] 张志强[1] ZHANG Chun-xiao;WANG Li-li;ZHAO Qi;SUN Xin-yi;HOU Guan-xin;LIU Chang;SHI Qiu-mei;WU Tong-lei;ZHANG Zhi-qiang(Key Laboratory of Preventive Veterinary Medicine in Hebei Province,Hebei Normal University of Science&Technology,Qinhuangdao 066600,China)

机构地区：[1]河北科技师范学院河北省预防兽医学重点实验室,河北秦皇岛066600

出　　处：《中国预防兽医学报》2024年第2期171-177,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：中央引导地方科技发展资金项目(236Z6604G);国家自然科学基金资助项目(31802186);河北省高等学校科学技术研究项目(BJ2021055);河北省重点研发计划项目(19226629D)。

摘　　要：为评估禽致病性大肠杆菌(APEC)溶血素HlyE蛋白的免疫原性,本研究对APEC溶血素HlyE蛋白进行原核表达和纯化,并对表达的重组蛋白进行SDS-PAGE和western blot分析,将纯化蛋白利用透析袋在4℃透析后,利用血琼脂平板对其溶血活性进行检测。SDS-PAGE和western blot结果显示,表达并纯化到分子量约为36 ku的重组HlyE蛋白(rHlyE),经ND-2000超微量核酸蛋白测定仪测定纯化后蛋白浓度为0.65 mg/mL;溶血活性检测结果显示,rHlyE具有溶血活性。以透析后的rHlyE作为抗原,按照50μg/只的剂量免疫小鼠,对照组于相同时间点注射等量PBS,共免疫3次间隔14 d,并于首免后不同时间采血,采用间接ELISA方法检测两组小鼠血清特异性抗体水平,并于三免后18 d以2 LD_(50)的APEC菌液攻毒小鼠,观察7 d内小鼠的死亡情况;于首免后28 d剖杀各组小鼠取其脾脏制备脾淋巴细胞,采用流式细胞术分别检测CD3^(+)、CD4^(+)、CD8^(+)T淋巴细胞亚型比率,对rHlyE的免疫原性进行评估。间接ELISA检测结果显示,该蛋白能够诱导机体产生体液免疫应答,分泌高表达量的IgG抗体,抗体水平于三免后15 d达到最高水平。rHlyE免疫攻毒保护试验结果显示,免疫组小鼠基本无明显临床症状,7 d内存活率达80%;而对照组小鼠表现明显临床症状,于攻毒后3 d内全部死亡。流式细胞术结果显示,与对照组相比,免疫组小鼠的CD3^(+)、CD4^(+)和CD8^(+)T淋巴细胞亚型比率均升高。上述结果表明,rHlyE在大肠杆菌BL21中为部分可溶性表达,将其免疫小鼠后可诱导小鼠产生较高水平的体液免疫应答,并且可对小鼠产生较好的免疫保护效果。本研究明确了APEC HlyE蛋白的免疫原性,为APEC免疫保护蛋白的筛选以及疫苗研发提供了借鉴与参考。To evaluate the immunogenicity of the hemolysin HlyE protein of avian pathogenic Escherichia coli(APEC),this study performed prokaryotic expression and purification of the APEC hemolysin HlyE protein.The expressed recombinant protein was analyzed by SDS-PAGE and western blot.After purification,the protein was dialyzed at 4℃ using a dialysis bag.The hemolytic activity of the dialyzed recombinant protein was then tested on blood agar plates.The results of SDS-PAGE and western blot analyses showed that the recombinant HlyE protein(rHlyE) with a molecular weight of approximately 36ku was successfully expressed and purified.The concentration of the purified protein was determined to be 0.65mg/m L using an ND-2000 ultra-micro nucleic acid and protein analyzer.The hemolytic activity test showed that rHlyE had hemolytic activity.The dialyzed rHlyE was used as an antigen to immunize mice at a dose of 50μg per mouse.The control group received an equal volume of PBS at the same time.An indirect ELISA method was used to detect the levels of specific antibodies in the serum of mice from both groups.On 18 days after the third immunization,the mice were challenged with APEC bacteria at 2LD_(50),and the survival of the mice was observed over 7 days.On 28 days after the first immunization,the mice from each group were sacrificed to obtain their spleens for preparing splenocytes.Flow cytometry was then used to detect the percentages of CD3~+,CD4~+,and CD8~+T lymphocytes,thereby assessing the immunogenicity of rHlyE.The results of the indirect ELISA showed that the protein was capable of inducing a humoral immune response in the organism,resulting in the secretion of high levels of Ig G antibodies.The antibody level peaked 15 days after the third immunization.The rHlyE immunoprotective experiment indicated that the immunized mice exhibited no significant clinical symptoms and had a survival rate of 80% within 7 days;whereas,the control group mice showed evident clinical symptoms and all died within 3 days after challenge.Flow cy

关 键 词：禽致病性大肠杆菌 重组HlyE蛋白 原核表达 溶血活性 免疫原性 流式细胞术 

分 类 号：S852.61[农业科学—基础兽医学]