摘要
目的建立布鲁氏菌外膜蛋白OMP10间接ELISA检测方法,评价OMP10在小鼠体内的免疫效果。方法本研究表达和纯化了布鲁氏菌外膜蛋白OMP10,通过Western Blot进行验证,建立了OMP10间接ELISA检测方法,然后将OMP10与弗氏佐剂和LDH佐剂配伍成2种亚单位疫苗,免疫小鼠后检测抗体水平、脾脏淋巴细胞的增殖水平、CD4^(+)和CD8^(+)T细胞比值以及细胞因子分泌,最后通过攻毒试验评估OMP10的免疫保护效果。结果OMP10作为诊断抗原的特异性和符合率均高于70%;免疫小鼠后2种亚单位疫苗均能产生高滴度的IgG抗体,CD4^(+)/CD8^(+)T的比值均高于PBS对照组,IFN-γ高于PBS对照组,差异具有统计学意义(P<0.01),但IL-4未发生明显变化,表明OMP10能够诱导很好的体液免疫及Th1型免疫应答。免疫保护结果显示,2种亚单位疫苗免疫组小鼠脾脏指数和脾脏载菌量低于布鲁氏菌M5感染组,差异具有统计学意义(P<0.01),表明OMP10蛋白配伍的亚单位疫苗对布鲁氏菌M5感染小鼠具有高度的保护作用。结论外膜蛋白OMP10在布鲁氏菌诊断和疫苗方面具有一定的应用价值。
This study was aimed at establishing an indirect ELISA assay based on the Brucella outer membrane protein OMP10,and preliminarily evaluating the immunological effect of OMP10 in mice.We expressed and purified the Brucella outer membrane protein OMP10,as validated by western blotting,then established an indirect ELISA assay based on OMP10.We subsequently formulated OMP10 with Freund’s adjuvant and LDH adjuvant to form two subunit vaccines,and immunized mice to detect antibody levels,proliferation levels of splenic lymphocytes,CD4^(+)and CD8^(+)T cell ratios Finally,we evaluated the immunoprotective effect of OMP10 with attack tests.The specificity and compliance rate of OMP10 as a diagnostic antigen exceeded 70%,and both subunit vaccines produced high titers of IgG antibodies after immunization of mice.The CD4^(+)/CD8^(+)T ratios were significantly higher than those in the control group,and IFN-γwas significantly higher than that in the control group(P<0.01),but IL-4 did not significantly differ,thus indicating that OMP10 induced strong humoral immune and Th1-type immune responses.The attack tests demonstrated that the spleen index and spleen load in mice in the two subunit vaccine immunized groups were significantly lower than those in the Brucella M5-infected group(P<0.01),thus indicating that the OMP10 protein-matched subunit vaccine was highly protective in Brucella M5-infected mice.In conclusion,these results suggested that the outer membrane protein OMP10 has value in Brucella diagnostics and vaccine development.
作者
邓肖玉
徐锦凤
何金科
谢珊珊
王勇
王震
陈创夫
DENG Xiao-yu;XU Jin-feng;HE Jin-ke;XIE Shan-shan;WANG Yong;WANG Zhen;CHEN Chuang-fu(School of Animal Science and Technology of Shihezi University,Shihezi 832000,China;Collaborative Innovation Center for the Prevention and Treatment of Infectious Diseases Involving Humans and Animals,Shihezi 832000,China;Wuwei Vocational College,Wuwei 733000,China;Xinjiang Second Medical College,Karamay 834000,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2023年第2期107-114,共8页
Chinese Journal of Zoonoses
基金
兵团重大科技项目(No.2017AA003)
国家自然基金项目(No.U1803236)
河北省重点科技项目(No.21322912D)联合资助。
作者简介
通讯作者:王震,Email:wzhen2018@shzu.edu.cn,ORCID:0000-0003-0427-0078;陈创夫,Email:ccf-xb@163.com,ORCID:0000-0001-5181-6518。
