鹅星状病毒衣壳蛋白的表达及其免疫的小鼠B细胞受体组库特征分析  

Expression of goose astrovirus capsid protein and analysis of B cell receptor repertoire characteristics in mice immunized with the capsid protein

作　　者：赵冬敏[1,2] 刘青涛 韩凯凯[1,2] 黄欣梅 刘宇卓[1,2] 李银 ZHAO Dongmin;LIU Qingtao;HAN Kaikai;HUANG Xinmei;LIU Yuzhuo;LI Yin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing 210014,China;GuoTai(Taizhou)Center of Technology Innovation for Veterinary Biologicals,Taizhou 225300,China)

机构地区：[1]江苏省农业科学院兽医研究所/农业农村部兽用生物制品工程重点实验室,江苏南京210014 [2]兽用生物制品<泰州>国泰技术创新中心,江苏泰州225300

出　　处：《江苏农业学报》2025年第3期549-558,共10页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(23)3077]。

摘　　要：本研究根据鹅星状病毒(GoAstV)衣壳蛋白(Cap)基因序列,通过人工合成获得衣壳蛋白基因片段,并构建重组杆状病毒rBV-Cap。通过间接免疫荧光检测、Western Blot鉴定和透射电镜观察,发现rBV-Cap在sf9细胞中高效表达,衣壳蛋白可自组装形成与天然鹅星状病毒结构相似的病毒样颗粒。将纯化的衣壳蛋白免疫BALB/c小鼠,酶联免疫吸附试验(ELISA)和病毒噬斑减少中和试验结果表明,重组衣壳蛋白具有较好的免疫原性,可诱导小鼠产生高水平抗体。为进一步探究B细胞免疫应答特征,分别摘取对照小鼠和免疫后的小鼠脾脏组织,利用单细胞免疫谱测序技术对B细胞受体(BCR)组库进行测序分析。结果表明,对照小鼠BCR重链(H)-CDR3多肽链长度主要为13 aa,免疫后的小鼠BCR重链(H)-CDR3多肽链长度主要为14 aa。与对照相比,免疫后的小鼠免疫球蛋白G(IgG)数量和占比升高,表明Cap蛋白能够有效刺激小鼠产生IgG抗体。与对照相比,Cap蛋白免疫后的小鼠BCR组库多样性指数(D50值)降低,表明免疫后BCR多样性降低。但是免疫后的小鼠某些克隆型细胞数量增加,表明免疫会引起特定克隆型的扩增。对照小鼠对IGHV1、IGHV2、IGHV5基因的取用频率较高,而免疫后的小鼠对IGHV1、IGHV6、IGHV9基因的取用频率较高。对照小鼠和免疫后的小鼠对IGHJ基因的取用频率无显著差异。与对照相比,免疫后小鼠的IGHV7-3-IGHJ2配对和IGHV2-6-2-IGHJ2配对的频率增加。免疫后的小鼠体内存在IGHV2-2-IGHJ1和IGHV5-1-IGHJ1配对,而对照小鼠体内未检测到这2种类型的配对,表明Cap蛋白免疫导致小鼠V-J基因配对发生改变。本研究结果为基于B细胞表位的亚单位疫苗研发提供了理论支持。This study synthesized and obtained the capsid protein(Cap)gene fragment of goose astrovirus(GoAstV)based on its gene sequence and constructed the recombinant baculovirus rBV-Cap.Through indirect immunofluorescence detection,Western Blot identification,and transmission electron microscopy observation,it was found that rBV-Cap was efficiently expressed in sf9 cells,and the capsid protein(Cap)could self-assemble into virus-like particles similar in structure to the natural GoAstV.The purified capsid protein was used to immunize BALB/c mice.The results of enzyme-linked immunosorbent assay(ELISA)and plaque reduction neutralization tests indicated that the recombinant capsid protein had good immunogenicity and could induce high levels of antibodies in mice.To further explore the characteristics of B cell immune responses,spleen tissues were collected from control mice and immunized mice,and single-cell immune profile sequencing technology was used to sequence the B cell receptor(BCR)repertoire.The results showed that the length of the BCR heavy chain(H)-CDR3 polypeptide chain in control mice was mainly 13 aa,while in immunized mice,it was mainly 14 aa.Compared with the controls,the number and proportion of immunoglobulin G(IgG)in immunized mice were increased,indicating that Cap protein could effectively stimulate the production of IgG antibodies in mice.Compared with the controls,the diversity index for BCR repertoire(D50 value)of immunized mice decreased,indicating a reduction in BCR diversity.However,the cell number of certain clones increased in immunized mice,indicating that immunization could lead to the expansion of specific clones.The usage frequencies of IGHV1,IGHV2 and IGHV5 genes were higher in control mice,while those of IGHV1,IGHV6 and IGHV9 genes were higher in immunized mice.There was no significant difference in the usage frequency of IGHJ genes between control and immunized mice.Compared with the controls,the frequency of IGHV7-3-IGHJ2 and IGHV2-6-2-IGHJ2 pairings increased in immunized mice.The pairi

关 键 词：新型鹅星状病毒 衣壳蛋白 B细胞受体 互补决定区 

分 类 号：S858.33[农业科学—临床兽医学]