摘要
为研究三叶青多糖(THP)作为佐剂对巴氏杆菌(Pm)灭活疫苗诱导小鼠免疫应答的影响,本研究将96只ICR雌性小鼠随机均分为4组:空白组(BC组)、Pm灭活疫苗组(Pm),含铝胶佐剂Pm疫苗组(Alum-Pm)、含THP佐剂的Pm疫苗组(THP-Pm),分别免疫相应疫苗两次,每次间隔两周。采用ELISA方法分别检测小鼠二免后7 d、14 d、21 d、28 d、35 d和42 d时血清IgG抗体水平,以及二免后28 d血清IgG1、IgG2a抗体亚类水平,以及Th1型(INF-γ)、Th2型(IL-4、IL-5)和Th17型(IL-17)细胞因子水平。结果显示,与Alum-Pm组相比,THP-Pm组小鼠血清中IgG抗体水平在二免后14 d和28 d显著升高(P<0.05),于28 d达到峰值,且THP-Pm组小鼠血清中IgG1和IgG2a抗体亚类水平显著高于其它各组(P<0.05)。与BC组相比,THP-Pm组小鼠血清IFN-γ和IL-17细胞因子分泌水平显著升高(P<0.05),IL-4和IL-5极显著升高(P<0.01、P<0.001);与Pm组相比,THP-Pm组小鼠血清IFN-γ和IL-5细胞因子的分泌水平显著升高(P<0.05),细胞因子IL-4的分泌水平极显著升高(P<0.01);与Alum-Pm组相比,仅IL-4的分泌水平显著升高(P<0.05)。二免后28 d取各组小鼠脾脏,制备脾淋巴细胞悬液,采用CCK-8法检测各组小鼠淋巴细胞增殖水平,采用荧光定量PCR(qPCR)检测淋巴细胞中T-bet和GATA-3基因的相对转录水平,采用流式细胞术检测淋巴细胞中CD4^(+)与CD8^(+)T淋巴细胞亚群的比值。CCK-8法检测结果显示,分别经灭活的Pm和LPS刺激后,THP-Pm组小鼠淋巴细胞的增殖水平极显著高于其余各组(P<0.01、P<0.001、P<0.0001),经ConA刺激后THP-Pm组小鼠淋巴细胞的增殖水平极显著高于BC组和Pm组(P<0.01)。qPCR检测结果显示,经Pm刺激后,THP-Pm组小鼠淋巴细胞T-bet mRNA的相对转录水平极显著高于BC组(P<0.0001)和Pm组(P<0.001),显著高于Alum-Pm组(P<0.01);THP-Pm组小鼠淋巴细胞GATA-3 mRNA的相对转录水平极显著高于BC组(P<0.001),显著高于Pm组和Alum-Pm组(P<0.05)。流式细胞术检测结果显示,与Pm组相比,THP-Pm组小鼠淋巴细胞CD4^(+)/CD8^(+)T淋巴细胞的比值显著升高(P<0.05)。二免后49 d通过攻毒试验检测免疫不同佐剂的Pm疫苗对小鼠的保护作用,结果显示,攻毒后10 d内BC组、Pm组、Alum-Pm组、THP-Pm组小鼠的存活率分别为0、33.3%、66.7%、83.3%,其中THP-Pm组小鼠的存活率极显著高于BC组和Pm组(P<0.01)。本研究首次证实THP佐剂可显著增强Pm灭活疫苗的免疫保护效果,为THP作为新型疫苗佐剂的开发与应用提供了重要参考依据。
To investigate the effect of Tetrastigma hemsleyanu polysaccharide(THP)as an adjuvant on the immune response induced by Pasteurella multocida(Pm)inactivated vaccine in mice,96 female ICR mice were randomly divided into four groups:blank control group(BC group),Pm vaccine group(Pm group),aluminum gel adjuvant Pm vaccine group(Alum-Pm group),and THP adjuvant Pm vaccine group(THP-Pm group).The mice were immunized twice with an interval of twoweeks.The serum IgG antibody levels were measured by ELISA at 7 days,14 days,21 days,28 days,35 days,and 42 days past the second immunization(dps).Additionally,serum IgG1 and IgG2a antibody subclasses and Th1-type(IFN-γ),Th2-type(IL-4,IL-5),and Th17-type(IL-17)cytokine levels were detected at 28dps after the second immunization.The results showed that compared with the Alum-Pm group,the THP-Pm group exhibited significantly higher IgG antibody levels at 14dps and 28dps(P<0.05),peaking at 28dps.The levels of antibody subclasses IgG1 and IgG2a in the THP-Pm group were significantly higher than those in the other groups(P<0.05)Compared with the BC group,the secretion levels of IFN-γand IL-17 cytokines in the serum of mice in the THP-Pm group were significantly increased(P<0.05),and the levels of IL-4 and IL-5 were extremely significantly increased(P<0.01,P<0.001).In comparison with the Pm group,the THP-Pm group exhibited significantly higher IFN-γand IL-5 levels(P<0.05),and a highly significant increase in IL-4(P<0.01).At 28 dps,splenic lymphocytes were isolated,and lymphocyte proliferation was assessed using the CCK-8 assay.The relative mRNA transcription levels of T-bet and GATA-3 transcription factors in lymphocytes were detected by fluoresecnce quantitative PCR(q-PCR),and the proportions of CD4^(+)and CD8^(+)T lymphocyte subsets were analyzed by flow cytometry.CCK-8 assay results showed that after stimulation with inactivated Pm and LPS respectivity,the lymphocyte proliferation level in the THP-Pm group was significantly higher than that in all other groups(P<0.01,P<0.001,P<0.0001).Following ConA stimulation,the lymphocyte proliferation level in the THP-Pm group was significantly higher than that in the BC and Pm groups(P<0.01).q-PCR results indicated that after Pm antigen stimulation,the relative transcription levels of T-bet mRNA in lymphocytes of the THP-Pm group was significantly higher than that in the BC group(P<0.0001)and the Pm group(P<0.001),and significantly higher than that in the Alum-Pm group(P<0.01).The relative transcription levels of GATA-3 mRNA in lymphocytes of mice in the THP-Pm group was significantly higher than that in the other groups(P<0.001).Flow cytometry results showed that the CD4^(+)/CD8^(+)ratio in the THP-Pm group was significantly higher than in the Pm group(P<0.05).At 49dpi,a challenge test was conducted to evaluate the protective efficacy of the different adjuvant-Pm vaccines.The results showed that within 10 days post-challenge,the survival rates of the BC,Pm,Alum-Pm,and THP-Pm groups were 0,33.3%,66.7%,and 83.3%,respectively,indicating that the survival rate in the THP-Pm group was significantly higher than in the BC and Pm groups(P<0.05).This study is the first to confirm that THP adjuvant can significantly enhance the immune protection effect of inactivated Pm vaccine,providing an important theoretical basis for the development and application of THP as a new vaccine adjuvant.
作者
向秋菊
崔雪梅
虞乐
罗艳芳
黄叶娥
季权安
石团员
胡子喆
宋厚辉
鲍国连
刘燕
XIANG Qiu-ju;CUI Xue-mei;YU-Le;LUO Yan-fang;HUANG Ye-e;JI Quan-an;SHI Tuan-yuan;HU Zi-zhe;SONG Hou-hui;BAO Guo-lian;LIU Yan(Institute of Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China;College of Animal Science and Technology,College of Veterinary Medicine,Zhejiang A&F University,Hangzhou 311300,China)
出处
《中国预防兽医学报》
北大核心
2025年第8期839-845,874,共8页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(32102714)
浙江省“尖兵”“领雁”研发攻关计划(2023C02047)
国家兔产业技术体系细菌病防控岗位专家项目(CARS-43-C-2)。
关键词
三叶青多糖
巴氏杆菌
灭活疫苗
佐剂
免疫保护
Tetrastigma hemsleyanum polysaccharides
Pasteurella multocida
inactivated vaccine
adjuvant
immune protection
