摘要
为筛选可用于副猪格拉瑟菌(GPS)亚单位疫苗的优势保护性抗原,本研究利用原核表达系统表达GPS的CyaY、SmpA、AfuA和OppA14个抗原的重组蛋白,采用His标签蛋白纯化试剂盒纯化这4个重组蛋白后,通过SDS-PAGE检测各重组蛋白的表达及纯化效果,采用western blot检测各重组蛋白的反应原性。SDS-PAGE检测结果显示,4个重组蛋白r CyaY、r SmpA、rAfuA和rOppA1的分子量分别约为12 ku、16 ku、41 ku和62 ku,均主要以可溶性形式表达,其表达量占菌体总蛋白的16.50%~26.50%。4个重组蛋白的纯化效果均较好,其浓度介于1.00 mg/mL~4.00 mg/mL。Western blot结果显示,rOppA1和rAfuA与GPS猪阳性血清的反应原性最强,rCyaY的反应原性中等,rSmpA的反应原性最弱。采用超微量核酸蛋白浓度测定仪测定4个重组蛋白的浓度后分别与ISA 201佐剂混合制成疫苗,对小鼠经皮下注射方式免疫2次,分别在小鼠免疫前、首免后21 d和二免后14 d采血,采用间接ELISA方法检测各组小鼠血清中的抗体水平;二免后14 d分别以5 LD_(50)(8.40×10^(8)cfu)和10 LD_(50)(1.68×10^(9)cfu)的5型GPS H5L3株对小鼠经腹腔攻毒,统计各重组蛋白疫苗对小鼠的免疫攻毒保护率。ELISA结果显示,各重组蛋白疫苗免疫组小鼠血清中IgG抗体水平随免疫次数的增加而升高,且均与PBS对照组抗体水平差异极显著(P<0.01)。小鼠攻毒试验结果显示,采用5 LD_(50)H5L3株攻毒后,rCyaY、rSmpA、rAfuA和rOppA1蛋白疫苗对小鼠的免疫保护率分别为83.3%(5/6)、83.3%(5/6)、33.3%(2/6)和100%(6/6);采用10 LD_(50)H5L3株攻毒后,rCyaY、r SmpA和rOppA1蛋白疫苗仍能对小鼠提供33.3%(2/6)、16.7%(1/6)和50%(3/6)的免疫保护率,但rAfuA蛋白疫苗不能为小鼠提供有效的免疫保护力(0/6)。本研究首次在相同条件下表达了GPS的4个重要保护性重组抗原蛋白rCyaY、rSmpA、r AfuA和r OppA1,并评价了它们对小鼠的免疫保护效力,证实rOppA1具有成为亚单位疫苗候�
To screen the superior protective antigens of the Glasserella parasuis(GPS)subunit vaccine,the recombinant proteins of four antigens,GPS CyaY,SmpA,AfuA and OppA1,were successfully expressed by prokaryotic expression system and purified using a His-tagged protein purification kit,and their expression and purification were monitored through SDS-PAGE analysis,and the reactogenicity of each recombinant protein was detected by western blot.The SDS-PAGE results showed that the four recombinant proteins,rCyaY,rSmpA,rAfuA,and rOppA1,exhibited molecular weights approximately 12ku,16ku,41ku,and 62ku,respectively.All proteins were primarily expressed in a soluble form,accounting for 16.50%to 26.50%of the total bacterial protein.The four recombinant proteins were effectively purified,with concentrations ranging from 1.00mg/mL to 4.00mg/mL.Western blot analysis showed that the reactivity of rOppA1 and rAfuA with positive GPS porcine sera was strongest,and it's moderate and weakest for rCyaY and rSmpA,respectively.Subsequently,each of the purified recombinant proteins was mixed with ISA 201 VG adjuvant to prepare vaccines after determination of concentration using an ultra-micro nucleic acid protein concentration analyzer.Mice were immunized twice with these vaccines via subcutaneous injection.Blood samples were collected from the mice prior to immunization,21 days after the first immunization,and 14 days after the second immunization.The antibody levels in mice sera from different groups were detected using an indirect ELISA method.Fourteen days after the second immunization,the mice were challenged with approximately 5 LD_(50)(8.40×10^(8)cfu)and 10 LD_(50)(1.68×10^(9)cfu)of the serotype 5 GPS strain H5L3 via intraperitoneal injection.The protective rates of the mice vaccinated with the prepared vaccines were then statistically analyzed to determine their efficacy on Glässer's disease.ELISA results indicated that the levels of IgG antibodies in the sera of mice immunized with the corresponding recombinant protein vaccines
作者
董玉岗
杨金钱
丁略烜
史琳琪
杨梦娟
张俊峰
关丽君
司丽芳
薛云
赵战勤
DONG Yu-gang;YANG Jin-qian;DING Lue-xuan;SHI Lin-qi;YANG Meng-juan;ZHANG Jun-feng;GUAN Li-jun;SI Li-fang;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 471000,China;Laboratory of Microbial Molecular Testing Technology,College of Medical Technology and Engineering,Henan University of Science and Technology,Luoyang 471000,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第10期1057-1064,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(31672530、U1704117)。
关键词
副猪格拉瑟菌
抗原
小鼠
保护效力
亚单位疫苗
Glasserella parasuis
antigen
mouse
protective efficacy
subunit vaccine
