马立克病毒pp38蛋白单克隆抗体的制备、抗原表位鉴定及应用

doi:10.3864/j.issn.0578-1752.2025.24.014

中国农业科学 ›› 2025, Vol. 58 ›› Issue (24): 5288-5298.doi: 10.3864/j.issn.0578-1752.2025.24.014

马立克病毒pp38蛋白单克隆抗体的制备、抗原表位鉴定及应用

伍楚妍¹(), 陆杭琼¹, 胡明雪¹, 林雨萌¹, 陈梦云¹, 刘长军¹, 刘永振¹, 崔红玉¹, 王素艳¹, 祁小乐¹, 陈运通¹, 段雨路¹, 高玉龙¹^,²^,³, 张艳萍¹^,^*()

¹ 中国农业科学院哈尔滨兽医研究所/动物疫病防控全国重点实验室，哈尔滨 150069
² 黑龙江省兽医免疫学重点实验室，哈尔滨 150069
³ 江苏高校动物重要疫病与人畜共患病协同创新中心，江苏扬州 225009

收稿日期:2025-04-02 接受日期:2025-07-01 出版日期:2025-12-22 发布日期:2025-12-22
通信作者:
张艳萍，E-mail：zhangyanping03@caas.cn
联系方式: 伍楚妍，E-mail：wuchuyan2021@163.com。
基金资助:
国家自然科学基金(32170170); 国家自然科学基金(U21A20260); 中国农业科学院科学中心项目(CAAS-CSLPDCP-202402); 现代农业产业体系肉鸡体系(CARS-41)

Preparation, Epitope Identification, and Preliminary Application of Monoclonal Antibodies Against Marek's Disease Virus pp38 Protein

WU ChuYan¹(), LU HangQiong¹, HU MingXue¹, LIN YuMeng¹, CHEN MengYun¹, LIU ChangJun¹, LIU YongZhen¹, CUI HongYu¹, WANG SuYan¹, QI XiaoLe¹, CHEN YunTong¹, DUAN YuLu¹, GAO YuLong¹^,²^,³, ZHANG YanPing¹^,^*()

¹ Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences/National Key Laboratory of Animal Disease Prevention and Control, Harbin 150069
² Heilongjiang Provincial Key Laboratory of Veterinary Immunology, Harbin 150069
³ Collaborative Innovation Center for Important Animal Diseases and Zoonoses in Jiangsu Higher Education Institutions, Yangzhou 225009, Jiangsu

Received:2025-04-02 Accepted:2025-07-01 Published:2025-12-22 Online:2025-12-22

摘要/Abstract

摘要：

【目的】通过制备马立克病毒（MDV）pp38单克隆抗体，鉴定其抗原表位，验证MDV应用价值。【方法】以MDV-1 GA株为模板扩增pp38，构建原核表达pColdⅠ-His-pp38重组质粒，转化大肠杆菌BL21，用IPTG诱导表达pp38-His重组蛋白并进行纯化，SDS-PAGE和Western blot进行蛋白鉴定。将该蛋白与弗氏佐剂乳化后免疫7周龄的BALB/c小鼠，每隔两周免疫一次，第5次免疫后采集小鼠尾尖血，间接ELISA检测血清抗体效价，选择效价最高的小鼠进行加强免疫。3d后，取小鼠脾细胞与SP2/0细胞进行细胞融合，通过间接ELISA筛选阳性杂交瘤细胞株，经两次流式分选亚克隆后扩大培养，连续传代20代，间接ELISA检测其抗体分泌的稳定性。通过抗体亚型检测试剂盒鉴定该抗体的重链与轻链类型，将pp38蛋白逐步截短并克隆至pCAGGS-GFP载体进行真核表达，Western blot鉴定该抗体的抗原识别表位。以pCAGGS-HA-pp38转染及MDV-1（GA株、Md5株、814株、CVI988株和LMS株）、MDV-2（SB-1株）和HVT（FC126株）感染的鸡胚成纤维细胞（CEF细胞）为抗原，该抗体作为一抗，进行间接免疫荧光试验（IFA），检测该抗体对外源性和内源性表达pp38蛋白的特异性。以MDV-1 GA株感染的CEF细胞为抗原，该抗体作为一抗，验证其在Western blot和流式细胞术方法检测MDV的应用。【结果】成功构建pColdⅠ-His-pp38重组质粒，获得高纯度的pp38-His重组蛋白。经小鼠免疫、血清抗体效价测定、细胞融合试验、细胞亚克隆试验，制备1株抗MDV pp38蛋白单克隆抗体2E8。2E8抗体能特异性识别MDV-1（GA株、Md5株、814株、CVI988株和LMS株），类型为κ轻链IgG1型，抗原识别位点位于pp38蛋白的第64—75个氨基酸区域内。初步验证该抗体适用于Western blot、IFA及流式细胞术等方法检测MDV-1。【结论】成功表达了MDV-1 pp38-His重组蛋白，制备了抗MDV-1 pp38蛋白的特异性单克隆抗体2E8，并鉴定其抗原表位，初步验证其应用范围，为MDV的流行病学调查、pp38蛋白的生物学功能及致病机制研究奠定基础。

关键词: 马立克病毒, pp38蛋白, 单克隆抗体, 抗原表位

Abstract:

【Objective】This study aimed to prepare monoclonal antibodies against the pp38 protein of Marek's disease virus (MDV), to identify their antigenic epitopes, and to verify their application value.【Method】The pp38 gene was amplified using the MDV-1 GA strain as a template, and a prokaryotic expression recombinant plasmid pColdⅠ-His-pp38 was constructed. The plasmid was transformed into E. coli BL21, and the expression of the pp38-His recombinant protein was induced by IPTG and purified. The purified protein was identified by SDS-PAGE and Western blot. The purified protein was emulsified with Freund's adjuvant and used to immunize 7-week-old BALB/c mice, with immunizations repeated every two weeks. After the fifth immunization, tail blood was collected from the mice, and the serum antibody titer was detected by indirect ELISA. The mouse with the highest antibody titer was selected for a booster immunization. Three days later, splenocytes from the mouse were fused with SP2/0 cells. Positive hybridoma cell lines were screened by indirect ELISA, and after two rounds of subcloning by flow cytometry, the cells were expanded and passaged continuously for 20 generations. The stability of antibody secretion was detected by indirect ELISA. The heavy and light chain types of the antibody were identified using an antibody isotyping kit. The pp38 protein was progressively truncated and cloned into the pCAGGS-GFP vector for eukaryotic expression. Western blot was used to identify the antigenic epitope recognized by the antibody. Indirect immunofluorescence assay (IFA) was performed using chicken embryo fibroblast (CEF) cells transfected with pCAGGS-HA-pp38 and infected with MDV-1 strains (GA, Md5, 814, CVI988, and LMS), MDV-2 strain (SB-1) and HVT strain (FC126) as antigens and the antibody as the primary antibody, to detect the specificity of the antibody against exogenously and endogenously expressed pp38 proteins. Using CEF cells infected with the MDV-1 GA strain as the antigen, the antibody was used as the primary antibody to validate its application in detecting MDV by Western blot and flow cytometry.【Result】The recombinant plasmid pColdⅠ-His-pp38 was successfully constructed, and a high-purity pp38-His recombinant protein was obtained. Through mouse immunization, serum antibody titer determination, cell fusion experiments, and subcloning of cells, a monoclonal antibody 2E8 against MDV pp38 protein was prepared. The 2E8 antibody specifically recognized MDV-1 strains (GA, Md5, 814, CVI988 and LMS), and was identified as a κ-light-chain IgG1 type. The antigen recognition site was located within the amino acids 64-75 of the pp38 protein. The antibody was preliminarily validated for use in Western blot, IFA, and flow cytometry for the detection of MDV-1.【Conclusion】This study successfully expressed the MDV-1 pp38-His recombinant protein and prepared the monoclonal antibody 2E8, which specifically targeted the pp38 protein of MDV-1. The antigenic epitope was identified, and the application scope was preliminarily verified, laying the foundation for the epidemiological investigation of MDV, the study of the biological functions of the pp38 protein, and the investigation of its pathogenic mechanisms.

Key words: Marek’s disease virus, pp38 protein, monoclonal antibody, antigenic epitope

伍楚妍, 陆杭琼, 胡明雪, 林雨萌, 陈梦云, 刘长军, 刘永振, 崔红玉, 王素艳, 祁小乐, 陈运通, 段雨路, 高玉龙, 张艳萍. 马立克病毒pp38蛋白单克隆抗体的制备、抗原表位鉴定及应用[J]. 中国农业科学, 2025, 58(24): 5288-5298.

WU ChuYan, LU HangQiong, HU MingXue, LIN YuMeng, CHEN MengYun, LIU ChangJun, LIU YongZhen, CUI HongYu, WANG SuYan, QI XiaoLe, CHEN YunTong, DUAN YuLu, GAO YuLong, ZHANG YanPing. Preparation, Epitope Identification, and Preliminary Application of Monoclonal Antibodies Against Marek's Disease Virus pp38 Protein[J]. Scientia Agricultura Sinica, 2025, 58(24): 5288-5298.

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图/表 10

表1

MDV pp38基因截短片段的PCR扩增引物"

引物 Primer	上游引物 Forward primer	下游引物 Reverse primer
pp38-A1	GAAGGATGCCCAGAAGGTACCGCCACCATGGAATTCGAAGCAGAACAC	CTTGCTCACGGTGGCCCCGGGATCCAAAGCGCTCATCTCGGCGTC
pp38-A2	GAAGGATGCCCAGAAGGTACCGCCACCATGGTCGCCGACGAGGCAGGGCA	CTTGCTCACGGTGGCCCCGGGCCCATCACCCTTTATTGGAATAGCC
pp38-A3	GAAGGATGCCCAGAAGGTACCGCCACCATGCGGGTCCAGAGGGACCGGTG	CTTGCTCACGGTGGCCCCGGGATTCCCTGACCTCCGGGGCTCAG
pp38-A4	GAAGGATGCCCAGAAGGTACCGCCACCATGAAGGCGATAGAATGCCAGGA	CTTGCTCACGGTGGCCCCGGGCCCCATCTGCTTCATACCATCTCCC
pp38-A5	GAAGGATGCCCAGAAGGTACCGCCACCATGGAACATCTTGACGAAAGTCG	CTTGCTCACGGTGGCCCCGGGCATTAATGCGCGAACGGAATGTACA
pp38-A6	GAAGGATGCCCAGAAGGTACCGCCACCATGGAGCTTGCCCAGCAGTGCGA	CTTGCTCACGGTGGCCCCGGGCACGGATAGAATAGTACGGGGTCGT
pp38-A7	GAAGGATGCCCAGAAGGTACCGCCACCATGCTGGCCGAAAGACAAAACCC	CTTGCTCACGGTGGCCCCGGGAAATGACATGCACGATCCTAGTAAT
pp38-A8	GAAGGATGCCCAGAAGGTACCGCCACCATGGAGTCAGAGAATGCAACAAT	CTTGCTCACGGTGGCCCCGGGAATGCCTGCACAGAAAGCCATTAAC
pp38-A9	GAAGGATGCCCAGAAGGTACCGCCACCATGTTCGCTGGTATGTTAGTCGG	CTTGCTCACGGTGGCCCCGGGATTTGATTCAGATTTTGTTTCTC
pp38-B1	GAAGGATGCCCAGAAGGTACCGCCACCATGGATCGGGTCCAGAGGG	CTTGCTCACGGTGGCCCCGGGGTGAGGGGGCGGAG
pp38-B2	GAAGGATGCCCAGAAGGTACCGCCACCATGTGGAGATTCAGTTCTCCGC	CTTGCTCACGGTGGCCCCGGGCCCCTTCCCCGTGAC
pp38-B3	GAAGGATGCCCAGAAGGTACCGCCACCATGTCTGGAGTCACGGG	CTTGCTCACGGTGGCCCCGGGCCCATCACCCTTTATTGG
pp38-C1	GAAGGATGCCCAGAAGGTACCGCCACCATGCGGGTCCAGAGGGA	CTTGCTCACGGTGGCCCCGGGAGAGTGAGGGGGCGG
pp38-C2	GAAGGATGCCCAGAAGGTACCGCCACCATGGTCCAGAGGGACCGG	CTTGCTCACGGTGGCCCCGGGTCCAGAGTGAGGGGGC
pp38-C3	GAAGGATGCCCAGAAGGTACCGCCACCATGCAGAGGGACCGGTGG	CTTGCTCACGGTGGCCCCGGGGACTCCAGAGTGAGGGG
pp38-C4	GAAGGATGCCCAGAAGGTACCGCCACCATGAGGGACCGGTGGAGAT	CTTGCTCACGGTGGCCCCGGGCGTGACTCCAGAGTGAGG
pp38-C5	GAAGGATGCCCAGAAGGTACCGCCACCATGGACCGGTGGAGATTCAGT	CTTGCTCACGGTGGCCCCGGGCCCCGTGACTCCAGAGTG
pp38-C6	GAAGGATGCCCAGAAGGTACCGCCACCATGCGGTGGAGATTCAGTTCTCC	CTTGCTCACGGTGGCCCCGGGCTTCCCCGTGACTCCAGA
pp38-D1	GAAGGATGCCCAGAAGGTACCGCCACCATGGATCGGGTCCAGAG	CTTGCTCACGGTGGCCCCGGGGTGAGGGGGCGGAGAA
pp38-D2	GAAGGATGCCCAGAAGGTACCGCCACCATGGTGAGGGGGCGGAGAA	CTTGCTCACGGTGGCCCCGGGAGGGGGCGGAGAACT

表1

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马立克病毒pp38蛋白单克隆抗体的制备、抗原表位鉴定及应用

Preparation, Epitope Identification, and Preliminary Application of Monoclonal Antibodies Against Marek's Disease Virus pp38 Protein

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