副猪嗜血杆菌5个重组蛋白的小鼠免疫保护效力比较  

Comparative analysis of protective efficacy of three antigenic proteins of Glaesserella parasuis on mice

作　　者：丁略烜 戚百川 史琳琪 杨金钱 詹鹏飞 关丽君 薛云[1] 赵战勤[1,2] DING Lue-xuan;QI Bai-chuan;SHI Lin-qi;YANG Jin-qian;ZHAN Peng-fei;GUAN Li-jun;XUE Yun;ZHAO Zhan-qin(Luoyang Key Laboratory of Prevention and Control Technology of Zoonotic Bacterial Infectious Diseases,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,China;Key-Disciplines Lab of safety of environment and Animal Product,College of Animal Science and Technology,Henan University of Science and Technology,Luoyang 471023,China)

机构地区：[1]河南科技大学动物科技学院/洛阳市动物细菌性传染病防控技术重点实验室,河南洛阳471023 [2]河南科技大学动物科技学院/河南省高等学校环境与畜产品安全重点学科开放实验室,河南洛阳471023

出　　处：《中国预防兽医学报》2023年第10期1074-1079,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(U1704117、31672530)。

摘　　要：为筛选副猪嗜血杆菌(GPS)亚单位疫苗的优势保护性抗原,本研究利用大肠杆菌BL21系统表达了GPS重要抗原r Hps06257、r D15和r Hps0675,及Hps0675 N端和C端的重组蛋白r Hps0675-N和r Hps0675-C,并通过SDS-PAGE检测各重组蛋白的表达情况。利用His标签蛋白纯化试剂盒纯化5个重组蛋白后,分别与Montanide^(TM)ISA 201 VG佐剂混合制成亚单位疫苗,对小鼠进行2次免疫,并分别在小鼠首免前、一免后21 d和二免后14 d(即首免后35 d)经尾根静脉采血分离血清,利用间接ELISA方法检测各组小鼠的抗体水平。二免后14 d分别利用5倍半数致死剂量(LD_(50))的5型GPS H5L3株和12型GPS H12L3株菌液对小鼠进行腹腔攻毒,攻毒后14 d内观察并记录各组小鼠的临床症状、发病及死亡情况,统计各组小鼠的免疫攻毒保护率。SDS-PAGE检测结果显示,5个重组蛋白均以包涵体形式表达,其表达量占菌体总蛋白的27.46%~42.37%。ELISA检测结果显示,各蛋白疫苗免疫组小鼠的Ig G抗体水平随免疫次数的增加而升高,与PBS对照组相比差异均极显著(P<0.01)。攻毒试验结果显示,分别利用5 LD_(50)GPS 5型H5L3株菌液和12型H12L3株菌液攻毒后,PBS和BL21菌体蛋白对照组小鼠攻毒后24 h内开始发病,3 d内全部死亡;发病小鼠主要症状为精神沉郁,食欲减退或废绝,被毛紊乱,对外界刺激无明显反应等;对死亡小鼠剖检可见其各组织器官的多发性浆膜炎和败血症性病理学变化。各蛋白疫苗免疫组小鼠中也出现了不同程度的发病和死亡情况,其中利用5 LD_(50)H5L3株攻毒后,r Hps06257、r D15、r Hps0675、r Hps0675-N和r Hps0675-C对免疫组小鼠的保护率分别为80%、0、0、40%和80%;利用5 LD_(50)H12L3株攻毒后,上述各组蛋白疫苗对小鼠的保护率分别为80%、20%、20%、10%和60%。本研究首次证实重组蛋白r Hps06257和r Hps0675-C能够诱导小鼠产生较强的免疫保护效力,具有作为亚单位疫苗靶抗原的潜力,�In order to screen for the dominant protective antigens of Glaesserella parasuis(GPS)subunit vaccine,the pET28a/BL21 system was used to express rHps06257,rD15 and rHps0675 proteins,as well as the N-terminal(rHps0675-N)and C-terminal(rHps0675-C)proteins of Hps0675.The expression of each recombinant protein was detected by SDS-PAGE.Five recombinant proteins were purified using His-tagged protein purification kit,and then mixed with Montanide^(TM) ISA 201 VG adjuvant to make subunit vaccine.Mice were immunized twice,and blood samples were collected and serum was isolated from tail root vein before the first immunization,21 days after the first immunization,and 14 days after the second immunization(i.e.35 d after the first immunization).The antibody levels in each group of mice were detected by indirect ELISA method.On 14 days after the second immunization,the mice were intraperitoneally challenged with 5 times the lethal dose(LD_(50))of type 5 GPS virulent strain H5L3 strain and type 12 GPS strain H12L3,respectively.The clinical symptoms,morbidity and mortality of the mice were observed and recorded for 14 days,and the challenge protection rate of each group was analyzed.SDS-PAGE showed that the five recombinant proteins were expressed in the form of inclusion bodies,accounting for 27.46%-42.37%of the total bacterial proteins.ELISA results showed that the IgG antibody level in each recombinant protein immune group increased with the increase of immunization times,and the difference was very significant compared with that in PBS control group(P<0.01).The challenge test results showed that the onset of disease began within 24 hours,and all died within 3 days of the PBS and BL21 protein control group after challenge with 5LD_(50) type 5 GPS strain H5L3 or 5LD_(50) type 12 strain H2L3.The main symptoms of the onset mice were depression,loss of appetite,disordered coat and no obvious response to external stimuli.Necropsy of dead mice mainly showed the pathological changes of multiple serositis and septicemia in various t

关 键 词：副猪嗜血杆菌 抗原 小鼠 保护效力 亚单位疫苗 

分 类 号：S852.61[农业科学—基础兽医学]