[1]冯健,李运明,刘毓刚,等.空肠弯曲菌PEB1的B细胞免疫优势表位的鉴定及保护效果的评价[J].第三军医大学学报,2019,41(04):302-307.
 FENG Jian,LI Yunming,LIU Yugang,et al.Finemapping of immunodominant linear Bcell epitopes of C.jejuni PEB1 antigen using short overlapping peptides [J].J Third Mil Med Univ,2019,41(04):302-307.
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空肠弯曲菌PEB1的B细胞免疫优势表位的鉴定及保护效果的评价
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第04期
页码:
302-307
栏目:
基础医学
出版日期:
2019-02-28

文章信息/Info

Title:
Finemapping of immunodominant linear Bcell epitopes of C.jejuni PEB1 antigen using short overlapping peptides
 
作者:
冯健李运明刘毓刚王艳艳胡宗海熊杰张睿
西部战区总医院:老年医学科,信息科,检验科
Author(s):
FENG Jian LI Yunming LIU Yugang WANG Yanyan HU Zonghai XIONG Jie ZHANG Rui

Department of Geriatrics, Department of Information, Department of Clinical Laboratory, General Hospital of Western Theater Command, Chengdu, Sichuan Province, 610083, China

关键词:
 
Keywords:
 
分类号:
R378.99; R392-33; R392.13
文献标志码:
A
摘要:

目的 利用表位预测分析技术筛选空肠弯曲菌(C.jejuni) 黏附蛋白PEB1的B细胞免疫优势表位,并评价其免疫保护效果。方法 采用氨基酸步移策略,合成18 mer氨基酸重叠肽段。ELISA系统筛选鉴定PEB1的B细胞免疫优势表位。采用KLH偶联免疫优势表位肽免疫BALB/c小鼠,ELISA测定优势表位肽诱导的IgG抗体效价。末次免疫后7 d,经口灌胃感染C.jejuni 11168,在感染后的28 d,定量检测感染攻毒后各组小鼠的空肠组织C.jejuni的定植量以及qRT-PCR技术检测炎性细胞因子TNF-α的相对表达水平。通过HL-60细胞调理吞噬实验测定表位肽诱导产生抗体所介导的调理吞噬杀伤功能。免疫B细胞缺失小鼠,感染攻毒后检测肠组织C.jejuni的定植量。结果 PEB155-72aa、PEB197-114aa、PEB1211-228aa均能与PEB1抗血清产生强烈IgG抗体反应。抗PEB155-72aa、抗PEB197-114aa和 PEB1211-228aa血清均能与重组的PEB1产生较强的抗原抗体反应。与CFA/IFA组相比,免疫PEB155-72aa、PEB197-114aa、PEB1211-228aa后血清中的抗体能够显著增强抗体介导的HL-60细胞的调理吞噬作用(P<0.01),均显著降低C.jejuni在空肠组织中的定植量,同时也均显著降低炎性细胞因子TNF-α的相对表达水平(P<0.01)。PEB155-72aa-KLH+CFA/IFA组,PEB197-114aa-KLH+CFA/IFA组,PEB1211-228aa-KLH+CFA/IFA组中,免疫B cell Knock out(B细胞缺失)小鼠攻毒后,C.jejuni在空肠组织中的定植量均显著高于WT(野生型)小鼠的定植量(P<0.01)。结论 成功鉴定出3个具有良好免疫原性和免疫保护作用的优势B细胞抗原表位(PEB155-72aa、PEB197-114aa、PEB1211-228aa),可用于C.jejuni疫苗的后续开发研究。

Abstract:

Objective To map the immunodominant linear B-cell epitopes of Campylobacter jejuni (C.jejuni) PEB1 antigen and evaluate the protective immune responses elicited by these epitopes in a mouse model of oral infection with C.jejuni.  Methods The B-cell immunodominant PEB1 epitopes were identified using synthetic overlapping peptide ELISA. BALB/c mice were immunized with the immunodominant PEB1 peptides conjugated with KLH plus CFA/IFA, and the IgG titers against these peptides were detected using ELISA. Seven days after the last immunization, the mice were orally infected with C.jejuni 11168, and the bacterial burden and expression of tumor necrosis factor-α (TNF-α) mRNA in the jejunum were analyzed using qRT-PCR in 28 d after the challenge. We assessed the effect of the antibodies against the immunodominant PEB1 epitopes in mediating the opsonophagocytic killing of C.jejuni by HL-60 cells. We also assessed the bacterial burden in the jejunum following C.jejuni challenge in a B cell-knockout mice immunized with the peptides.  Results The immunodominant peptides PEB155-72aa, PEB197-114aa, and PEB1211-228aa all induced strong IgG responses to PEB1 antiserum, and the antisera of these immunodominant peptides also showed strong IgG responses to recombinant PEB1. Compared with the antiserum of CFA/IFA, the antisera of these immunodominant peptides induced significantly enhanced opsonophagocytic activity of HL-60 cells (P<0.01). Both the bacterial burdens and TNF-α mRNA expression level in the jejunum were significantly lowered in the mice immunized with the 3 immunodominant peptides in comparison with the control mice immunized with CFA/IFA  (P<0.01). In B cellknockout mice, immunization with the 3 peptides did not provide immune protection against C.jejuni, and the  bacterial burden in the jejunum after C.jejuni challenge was significantly greater than that in immunized wildtype mice (P<0.01).  Conclusion We successfully identified 3 linear B-cell epitopes of C.jejuni PEB1 (PEB155-72aa, PEB197-114aa, and PEB1211-228aa), which exhibit good immunogenicity and immunoprotective activities and may facilitate the future development of vaccines against C.jejuni infection.

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更新日期/Last Update: 2019-02-21