非洲猪瘟病毒p22蛋白单克隆抗体制备及其抗原表位鉴定

doi:10.16431/j.cnki.1671-7236.2025.02.028

摘要/Abstract

摘要： 【目的】采用原核表达系统表达并获得非洲猪瘟病毒(ASFV)p22重组蛋白，并进一步制备、鉴定p22重组蛋白的单克隆抗体。【方法】采用PCR方法扩增ASFV p22蛋白编码基因，构建重组表达质粒pET-28a(+)-p22，并通过IPTG诱导表达p22蛋白。将纯化后重组蛋白免疫小鼠，取免疫小鼠脾细胞与SP2/0骨髓瘤细胞进行融合制备单克隆抗体。通过Western blotting鉴定单克隆抗体特异性，通过ELISA方法测定腹水效价；利用在线软件预测p22蛋白抗原表位分布情况并合成重叠多肽，通过Dot blotting鉴定抗体识别的抗原表位。【结果】本研究成功构建了p22重组蛋白的原核表达质粒，获得原核系统表达的p22重组蛋白，分子质量约为22 ku。用纯化后p22蛋白作为免疫原免疫小鼠，成功筛选到4株阳性单克隆杂交瘤细胞株，分别为1G3D7G11、2G5H6H8、6D10G7D6和8F6F8B9，其腹水效价均达到1∶500 000。Western blotting结果显示，4株单克隆抗体均能与p22蛋白发生特异性反应。单克隆亚型鉴定结果显示，1G3D7G11、2G5H6H8和6D10G7D6株单克隆抗体的重链均为IgG1类，8F6F8B9株单克隆抗体的重链为IgG2a类，4株单克隆抗体轻链均为Kappa型。Dot blotting检测结果显示，1G3D7G11、2G5H6H8株单克隆抗体识别抗原表位位于第58—89位氨基酸处；8F6F8B9株单克隆抗体识别抗原表位位于第126—150位氨基酸处。【结论】本研究成功制备了4株ASFV p22蛋白的单克隆抗体，并初步鉴定了单克隆抗体所识别的B细胞表位区间。研究结果为p22蛋白的生物学功能研究和ASFV血清学检测提供了参考依据。

关键词: 非洲猪瘟病毒; p22蛋白; 单克隆抗体; 抗原表位

Abstract: 【Objective】 The purpose of this study was to express and obtain recombinant protein p22 of African swine fever virus (ASFV) using the prokaryotic expression system,and further prepare and identify the monoclonal antibody against recombinant protein p22. 【Method】 PCR was used to amplify the ASFV p22 protein coding gene and construct the recombinant expression plasmid pET-28a(+)-p22，and induced to express p22 protein by IPTG.The purified recombinant protein was immunized to mice,and the spleen cells of immunized mice were fused with SP2/0 myeloma cells to prepare monoclonal antibody.The specificity of monoclonal antibody was determined by Western blotting,and the titer of ascites was determined by ELISA. Online software was used to predict the distribution of p22 protein epitopes and synthesize overlapping peptides,and the epitopes recognized by antibodies were identified by Dot blotting. 【Result】 In this study,the prokaryotic expression plasmid of p22 recombinant protein was successfully constructed,and the recombinant protein p22 expressed in the prokaryotic system was obtained with a molecular mass of about 22 ku.After immunizing mice with purified p22 protein as immunogen,4 monoclonal hybridoma cell lines were successfully screened，which was 1G3D7G11,2G5H6H8,6D10G7D6 and 8F6F8B9,and the ascites titer reached 1∶500 000.Western blotting results showed that 4 monoclonal antibodies could react specifically with p22 protein.The identification results of monoclonal subtypes showed that the heavy chains of monoclonal antibodies of 1G3D7G11,2G5H6H8 and 6D10G7D6 were IgG1,the heavy chain of monoclonal antibody of 8F6F8B9 was IgG2a,and the light chains of the 4 monoclonal antibodies were Kappa.Dot blotting test results showed that the epitopes of the recognition antigen of monoclonal antibodies 1G3D7G11 and 2G5H6H8 were located at amino acid 58-89.The epitope of monoclonal antibody 8F6F8B9 was located at amino acid 126-150. 【Conclusion】 In this study,4 monoclonal antibodies against ASFV p22 protein were successfully prepared,and the B cell epitope interval recognized by the monoclonal antibody was preliminarily identified.The results provided a reference for the study of biological function of p22 protein and serological detection of ASFV.

Key words: African swine fever virus; p22 protein; monoclonal antibody; epitope

中图分类号:

S852.65⁺1

王小格, 司煊瑛, 闫志伟, 王飞, 尤龙琪, 刘梗, 蔡茂, 梁珺珹, 梁玉秀, 杜永坤, 张改平. 非洲猪瘟病毒p22蛋白单克隆抗体制备及其抗原表位鉴定[J]. 中国畜牧兽医, 2025, 52(2): 781-791.

WANG Xiaoge, SI Xuanying, YAN Zhiwei, WANG Fei, YOU Longqi, LIU Geng, CAI Mao, LIANG Juncheng, LIANG Yuxiu, DU Yongkun, ZHANG Gaiping. Preparation of Monoclonal Antibody Against African Swine Fever Virus p22 Protein and Identification of the Antigenic Epitope[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(2): 781-791.

参考文献

[1] OH S I,LEE H S,BUI V N,et al.Dynamic variations in infrared skin temperature of weaned pigs experimentally inoculated with the African swine fever virus:A pilot study[J].Veterinary Sciences,2021,8(10):223.
[2] DODANTENNA N,CHA J W,CHATHURANGA K,et al.The African swine fever virus virulence determinant DP96R suppresses type Ⅰ，IFN production targeting IRF3[J]. International Journal of Molecular Sciences,2024,25(4):23-29.
[3] EDWARD S,VIVIAN O,ELIZABETH V,et al.Full genome sequence for the African swine fever virus outbreak in the dominican republic in 1980[J].Scientific Reports,2023,13(1):1024.
[4] CLAUDIA T,MARIANGELA F,LUIGI B,et al.The evolution of African swine fever virus in Sardinia (1978-2014) as revealed by whole-genome sequencing and comparative analysis[J]. Transboundary and Emerging Diseases,2020,67(5):1971-1980.
[5] CHUCHARD P,PRATHUMWAN D,TRACHOO K,et al.The SLI-SC mathematical model of African swine fever transmission among swine farms:The effect of contaminated human vector[J]. Axioms,2022,11(7):329.
[6] YAO H,ZANG C,ZUO X,et al.Tradeoff analysis of the pork supply and food security under the influence of African swine fever and the COVID-19 outbreak in China[J].Geography and Sustainability,2022,3(1):32-43.
[7] 戈胜强,沙洲,左媛媛,等.越南非洲猪瘟疫苗AVAC ASF LIVE(ASFV-G-ΔMGF)研究工作分析与思考[J].中国动物检疫,2024,41(2):36-41.GE S Q,SHA Z,ZUO Y Y,et al.Analysis and reflection on the research work of Viet Nam African swine fever vaccine AVAC ASF LIVE (ASFV-G-ΔMGF)[J]. China Animal Health Inspection,2024,41(2):36-41.(in Chinese)
[8] KIRAMAGE C,JONGSOO L.African swine fever virus (ASFV):Immunity and vaccine development[J]. Vaccines,2023,11(2):199.
[9] WANG G,XIE M,WU W,et al.Structures and functional diversities of ASFV proteins[J].Viruses,2021,13(11):2124.
[10] LOPERA-MADRID J,MEDINA-MAGVES L G,GLADUE D P,et al.Optimization in the expression of ASFV proteins for the development of subunit vaccines using Poxviruses as delivery vectors[J].Scientific Reports,2021,11(1):23476.
[11] CARLOS D,JIRI S,DAGMAR K B,et al.Examination of immunogenic properties of recombinant antigens based on p22 protein from African swine fever virus[J].Journal of Veterinary Research,2022,66(3):297-304.
[12] VUONO E A,RAMIREZ-MEDINA E,PRUITT S,et al.Evaluation of the function of the ASFV KP177R gene,encoding for structural protein p22,in the process of virus replication and in swine virulence[J]. Viruses,2021,13(6):986.
[13] 陈艳,祝贺,童明龙,等.非洲猪瘟病毒CD2v蛋白单克隆抗体的制备与鉴定[J].畜牧与兽医,2024,56(3):86-93.CHEN Y,ZHU H,TONG M L,et al.Preparation and identification of monoclonal antibody to African swine fever virus CD2v protein[J]. Animal Husbandry & Veterinary Medicine,2018,56(3):86-93.(in Chinese)
[14] 陈艳,龙云凤,姜焱,等.非洲猪瘟病毒p54蛋白单抗制备及B细胞线性抗原表位鉴定[J].畜牧与兽医,2024,56(1):115-122.CHEN Y,LONG Y F,JIANG Y,et al.Preparation of African swine fever virus p54 protein monoclonal antibody and identification of linear epitopes in B cells[J].Animal Husbandry & Veterinary Medicine,2024,56(1):115-122.(in Chinese)
[15] DIXON K L,STAHL K,JORI F,et al.African swine fever epidemiology and control[J].Annual Review of Animal Biosciences,2020,8(1):221-246.
[16] GAO Z,SHAO J J,ZHAGN G L,et al.Development of an indirect ELISA to specifically detect antibodies against African swine fever virus:Bioinformatics approaches[J].Virology Journal,2021,18(1):97.
[17] GALLARDO C,NIETO R,SOLER A,et al.Assessment of African swine fever diagnostic techniques as a response to the epidemic outbreaks in Eastern European Union countries:How to improve surveillance and control programs[J].Journal of Clinical Microbiology,2015,53(8):2555-2565.
[18] SONG J,WANG M,ZHOU L,et al.A candidate nanoparticle vaccine comprised of multiple epitopes of the African swine fever virus elicits a robust immune response[J].Journal of Nanobiotechnology,2023,21(1):424.
[19] JIN J,BAI Y,ZHANG Y,et al.Establishment and characterization of a novel indirect ELISA method based on ASFV antigenic epitope-associated recombinant protein[J].International Journal of Biological Macromolecules,2023,253(7):127311.
[20] NGUYEN T L,SAMUEL LEON MAGDALENO J,TAJJAK SHAIKH A,et al.Designing a multi-epitope candidate vaccine by employing immunoinformatics approaches to control African swine fever spread[J].Journal of Biomolecular Structure Dynamics,2023,41(19):10214-10229.
[21] ZHANG S,WANG R,ZHU X J,et al.Identification and characterization of a novel epitope of ASFV-encoded dUTPase by monoclonal antibodies[J]. Viruses,2021,13(11):2175.
[22] 石正发,马源,袁洪,等.非洲猪瘟病毒p72蛋白单克隆抗体的制备及阻断ELISA方法的建立[J].中国预防兽医学报,2023,45(5):488-493.SHI Z F,MA Y,YUAN H,et al.Preparation of monoclonal antibody to African swine fever virus p72 protein and establishment of blocking ELISA method[J].Chinese Journal of Preventive Veterinary Medicine,2023,45(5):488-493.(in Chinese)
[23] GENG R,SUN Y,LI R,et al.Development of a p72 trimer-based colloidal gold strip for detection of antibodies against African swine fever virus[J]. Applied Microbiology and Biotechnology,2022,106(7):2703-2714.
[24] 卢丽飞,党佳佳,吴青萍,等.非洲猪瘟EP364R蛋白的原核表达及间接ELISA方法的建立[J].中国动物传染病学报,2023,31(5):79-86.LU L F,DANG J J,WU Q P,et al.Prokaryotic expression of EP364R protein in African swine fever and the establishment of indirect ELISA method[J].Chinese Journal of Animal Infectious Diseases,2023,31(5):79-86.(in Chinese)
[25] 王兆贵,赵亚茹,杨吉飞,等.基于非洲猪瘟病毒pA104R蛋白的间接ELISA检测方法的建立与应用[J].中国兽医科学,2021,51(4):403-411.WANG Z G,ZHAO Y R,YANG J F,et al.Establishment and application of an indirect ELISA method for the detection of African swine fever virus pA104R protein[J]. Veterinary Science in China,2021,51(4):403-411.(in Chinese)
[26] 颜世君,郝丽影,白露露,等.非洲猪瘟病毒p22蛋白的表达及单克隆抗体制备[J].河南农业科学,2021,50(12):149-154.YAN S J,HAO L Y,BAI L L,et al.Expression of African swine fever virus p22 protein and preparation of monoclonal antibody[J].Journal of Henan Agricultural Sciences,2021,50(12):149-154.(in Chinese)
[27] SHI L F,REN H,ZHANG B,et al.Preparation and epitope mapping of monoclonal antibodies against African swine fever virus p22 protein[J]. International Journal of Biological Macromolecules,2023,255:128111.