免疫增强剂CVC1302诱导高亲和力抗体机制分析  

Analysis of the mechanism of immunopotentiator CVC1302 inducing high affinity antibody

作　　者：杜露平 鲁海燕[1,2,3] 侯立婷 于晓明[2,3] 程海卫 张元鹏[2,3] 陈瑾 郑其升[1,2,3] 甘芳 DU Luping;LU Haiyan;HOU Liting;YU Xiaoming;CHENG Haiwei;ZHANG Yuanpeng;CHEN Jin;ZHENG Qisheng;GAN Fang(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China;Institute of Veterinary Immunology&Engineering,Jiangsu Academy of Agricultural Sciences/National Research Center of Engineering and Technology for Veterinary Biologicals/Jiangsu Key Laboratory for Food Quality and Safety-State Key Laboratory Cultivation Base,Ministry of Science and Technology,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

机构地区：[1]南京农业大学动物医学院,南京210095 [2]江苏省农业科学院动物免疫工程研究所/国家兽用生物制品工程中心/江苏省食品质量安全重点实验室,南京210014 [3]兽用生物制品(泰州)国泰技术创新中心,江苏泰州225300

出　　处：《中国农业大学学报》2023年第10期166-172,共7页Journal of China Agricultural University

基　　金：十四五重点研发专项(2022YFD1800800);国家自然科学基金项目(32102690);江苏省农业自主创新专项(CX(21)3135)。

摘　　要：为探究免疫增强剂CVC1302诱导机体产生高亲和力抗体的免疫机制,本研究利用4-羟基-3-硝基苯乙酰基耦联鸡卵白蛋白(NP-OVA)与免疫增强剂CVC1302配伍后,与ISA206佐剂乳化制备疫苗。将33只6周龄BALB/c雌性小鼠随机分为3组,分别后腿肌肉注射NP、NP-ISA206和NP-CVC1302-ISA206,每只100μL疫苗,50μg NP-OVA/只,利用ELISA检测高亲和力NP特异性抗体水平;利用流式细胞仪分析生发中心B细胞增殖水平、生发中心B细胞诱导活化的胞苷脱氨酶(AID)表达水平;利用荧光定量PCR检测AID基因和HoxC4基因转录水平。结果表明:1)免疫增强剂CVC1302可诱导小鼠产生高亲和力的NP特异性抗体。2)免疫增强剂CVC1302可显著提升抗原特异性生发中心B细胞数量占总B细胞数量比例。3)免疫增强剂CVC1302可显著增加生发中心B细胞HoxC4基因转录水平。4)免疫增强剂CVC1302可显著增强生发中心B细胞AID蛋白表达水平。生发中心B细胞表达的AID可针对B细胞免疫球蛋白序列可变区进行点突变,发生体细胞高频突变,提升B细胞BCR针对抗原的亲和力,进而在辅助性T细胞的阳性选择下分化为长寿浆细胞,持续性分泌高亲和力抗体,因此本研究推断免疫增强剂CVC1302依赖于正向调控AID诱导机体产生高亲和力抗体,为机体提供免疫保护力,以抵抗病原体的感染,为后续新型免疫增强剂的研制提供思路和理论基础。In order to elucidate the immune-mechanism of immunopotentiator CVC1302inducing high affinity antibody,NP-OVA,as model antigen,mixed with CVC1302,then emulsified with ISA206,was prepared for vaccine.33Six week-old BALB/c female mice were divided into 3groups and immunized with NP-CVC1302-ISA206,NP-ISA206or NP,each mouse immunized with 100μL vaccine,as well as 50μg NP-OVA.The levels of high-affinity NP-specific antibody were detected by ELISA,the percentages of NP-specific germinal center(GC)B cells in B cells and expression levels of AID in GC B cells were analyzed by flow cytometry.GC B cells were harvested by FACS sorting.The transcriptional levels of AID and HoxC4 in GC B cells were analyzed by real-time PCR.The results showed that:1)Immunopotentiator CVC1302induced high affinity NP-specific antibodies.2)CVC1302induced higher percentage of NP-specific GC B cells in B cells.3)CVC1302improved transcriptional levels of HoxC4 in GC B cells.4)CVC1302enhanced the expression levels of AID in GC B cells.Considering AID in GC B cells inducing high affinity antibodies through point mutation,then GC B cells differentiated into long-term plasma cells under the positive selection of follicular helper T cells,we concluded that CVC1302induced high affinity antibody dependent on positive control of AID,which shed light on the development of new immunopotentiators.

关 键 词：免疫增强剂CVC1302 高亲和力抗体 AID 生发中心B细胞 HoxC4 

分 类 号：S852.4[农业科学—基础兽医学]