免疫增强剂CVC1302调控体细胞高频突变的机制研究  

Mechanism of immunopotentiator CVC1302 regulating somatic hypermutation

作　　者：杜露平[1,2] 鲁海燕 侯立婷 于晓明[1,2] 程海卫 张元鹏[1,2] 陈瑾 郑其升[1,2] 侯继波 DU Luping;LU Haiyan;HOU Liting;YU Xiaoming;CHENG Haiwei;ZHANG Yuanpeng;CHEN Jin;ZHENG Qisheng;HOU Jibo(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,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

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

出　　处：《华中农业大学学报》2025年第1期211-216,共6页Journal of Huazhong 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CVC1302调控生发中心B细胞发生体细胞高频突变的免疫机制,本研究将4-羟基-3-硝基苯乙酰基耦联鸡卵白蛋白(NP-OVA)混合免疫增强剂CVC1302后,利用ISA206乳化获得疫苗。BALB/c小鼠分为2组,分别后腿肌肉注射NP-ISA206和NP-CVC1302-ISA206,每只50μg NP-OVA,免疫后14 d,利用流式分选获得生发中心B细胞,利用巢氏PCR扩增B细胞免疫球蛋白序列可变区VH_(186.2),Western blot检测诱导活化的胞苷脱氨酶(AID)、Pax5表达水平,β-actin作为内参,比较组间AID、Pax5表达差异;利用荧光定量PCR检测AID、Pax5基因转录水平。试验结果显示:CVC1302显著诱导生发中心B细胞免疫球蛋白序列VH186.2突变频率,试验组W33L突变频率为62.2%,而对照组仅为20.25%;CVC1302可提升AID蛋白、Pax5蛋白表达水平,其中试验组AID蛋白相对表达水平为0.72,对照组仅为0.16,试验组Pax5蛋白相对表达水平为0.62,对照组仅为0.26;CVC1302增强AID基因、Pax5基因转录水平,相较于对照组分别提高2.36、4.13倍。推断CVC1302依赖Pax5介导AID表达,调控生发中心B细胞发生体细胞高频突变。ISA206 was used to obtain the vaccine by emulsifying 4-hydroxy-3-nitrophenylacetyl con-jugated chicken egg white protein(NP-OVA)with immunopotentiator CVC1302 to study the immune mechanism of CVC1302 regulating the occurrence of somatic hypermutation in germinal center B(GC B)cells.Six-week-old BALB/c female mice were divided into 2 groups and immunized with NP-CVC1302-ISA206,NP-ISA206 in the hind leg muscles,with each mouse receiving 100μL vaccine and 50μg NP-OVA.Germinal center B cells were obtained with flow cytometry after14 days of immunization,and the variable region VH_(186.2) gene sequence of immunoglobulin in GC B cells was amplified with nested-PCR.Western blot was used to detect the expression level of cytidine deaminase(AID)and Pax5 induced activa-tion,and the differences in the expression level of AID and Pax5 between groups were compared withβ-ac-tin as an internal reference.The transcription level of AID and Pax5 in GC B cells was analyzed with fluo-rescence quantitative PCR.The results showed that immunopotentiator CVC1302 significantly induced the mutation frequency of VH186.2 in the immunoglobulin sequence of germinal center B cells,with a W33L mu-tation frequency of 62.2%in the NP-CVC1302 immunized group and only 20.25%in the NP immunized group.CVC1302 increased the expression level of AID protein and Pax5 protein in GC B cells.The rela-tive expression level of AID protein in the NP-CVC1302 immunized group was 0.72,while that in the NP immunized group was only 0.16.The relative expression level of Pax5 protein in the NP-CVC1302 immu-nized group was 0.62,while that in the NP immunized group was only 0.26.CVC1302 enhanced the tran-scription level of AID gene and Pax5 gene in the NP-CVC1302 immunized group,which was increased by 2.36 and 4.13 times compared to that in the NP immunized group.It is indicated that CVC1302 relies on Pax5 mediated AID expression to regulate the occurrence of somatic hypermutation in germinal center B cells.

关 键 词：免疫增强剂CVC1302 体细胞高频突变 免疫机制 生发中心B细胞 胞苷脱氨酶 PAX5 

分 类 号：S852.4[农业科学—基础兽医学]