Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus

doi:10.15933/j.cnki.1004-3268.2021.12.017

Journal of Henan Agricultural Sciences ›› 2021, Vol. 50 ›› Issue (12): 149-154.DOI: 10.15933/j.cnki.1004-3268.2021.12.017

Special Issue: 非洲猪瘟专题

• Animal Science Veterinary Medicine • Previous Articles Next Articles

Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus

YAN Shijun^1,2，HAO Liying³，BAI Lulu^1,2，ZHENG Dingding³，SONG Huanhuan^1,2，LI Shengqiang^1,2，WANG Tongyan^1,2，TAN Feifei^1,2，DENG Junhua³，TIAN Kegong^1,2,3

（1.National Research Center for Veterinary Medicine，Luoyang 471000，China；2.Publike Bioengineering Co.，Ltd.，Luoyang 471000，China；3.Luoyang Putai Biotechnology Co.，Ltd.，Luoyang 471000，China）

Received:2021-05-07 Published:2021-12-15 Online:2022-01-28

非洲猪瘟病毒p22 蛋白的表达及单克隆抗体制备

颜世君^1,2，郝丽影³，白露露^1,2，郑丁丁³，宋欢欢^1,2，李胜强^1,2，王同燕^1,2，谭菲菲^1,2，邓均华³，田克恭^1,2,3

（1.国家兽用药品工程技术研究中心，河南洛阳471000；2.普莱柯生物工程股份有限公司，河南洛阳471000；3.洛阳普泰生物技术有限公司，河南洛阳471000）

通讯作者: 邓均华（1978-），女，四川阆中人，兽医师，硕士，主要从事动物疫病诊断与防控技术研究。E-mail：dengbetter88@163.com 田克恭（1964-），男，山西万荣人，研究员，博士，主要从事动物疫病诊断与防控技术研究。E-mail：vetvac@126.com
作者简介:颜世君（1986-），女，山东淄博人，工程师，硕士，主要从事基因工程疫苗研究。E-mail：xiaohuolong_001@163.com 郝丽影为同等贡献作者
基金资助:
洛阳市重大科技专项（1901029A）

Abstract

Abstract: In order to develop an immunodiagnostic reagent for African swine fever virus，the Bac‐to‐Bac baculovirus expression system was used to express the p22 protein of African swine fever virus，the anti‐p22 protein monoclonal antibody was prepared and its reactogenicity with African swine fever virus was identified. The results showed that the recombinant baculovirus AcNPV‐p22 was successfully obtained，and the soluble recombinant protein of p22 with the size of about 22 ku was expressed.Western blot analysis confirmed that the protein had a good reactivity with African swine fever virus positive serum.Positive hybridoma cells（3D2，4A7）stably secreting monoclonal antibodies against African swine fever virus p22 protein were screened. The ascites ELISA titers of the two hybridoma cells were higher than 1∶128 000.Monoclonal antibodies isotype assay showed that the heavy chain was IgG1，and the light chain was κ.The IFA results showed that the prepared monoclonal antibody could specifically react with African swine fever virus.In summary，this study successfully expressed African swine fever virus p22 protein by baculovirus‐insect expression system and the monoclonal antibodies against African swine fever virus p22 protein were successfully prepared.

Key words: African swine fever virus, p22 protein, Baculovirus expression system, Monoclonal antibody, Indirect immunofluorescence assay

摘要： 为研发非洲猪瘟病毒的免疫诊断学试剂，利用Bac-to-Bac杆状病毒表达系统表达非洲猪瘟病毒p22蛋白，制备抗p22蛋白单克隆抗体并鉴定其与非洲猪瘟病毒的反应性。结果显示，成功制备重组杆状病毒AcNPV-p22，表达得到大小约22 ku可溶性的重组p22蛋白。Western blot鉴定结果表明，重组p22蛋白可以与非洲猪瘟病毒阳性血清发生特异性反应。筛选到能够稳定分泌抗非洲猪瘟病毒p22蛋白单克隆抗体的杂交瘤细胞株（3D2、4A7），腹水ELISA效价均高于1∶128 000；单克隆抗体亚类分型鉴定结果表明，重链均为IgG1，轻链均为κ；间接免疫荧光试验（IFA）结果表明，制备的单克隆抗体能与非洲猪瘟病毒发生特异性反应。综上，利用杆状病毒-昆虫表达系统成功表达了可溶性的非洲猪瘟病毒重组p22蛋白，并制备抗p22蛋白单克隆抗体。

关键词: 非洲猪瘟病毒, p22蛋白, 杆状病毒表达系统, 单克隆抗体, 间接免疫荧光试验

CLC Number:

S855.3

YAN Shijun, HAO Liying, BAI Lulu, ZHENG Dingding, SONG Huanhuan, LI Shengqiang, WANG Tongyan, TAN Feifei, DENG Junhua, TIAN Kegong. Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus[J]. Journal of Henan Agricultural Sciences, 2021, 50(12): 149-154.

颜世君, 郝丽影, 白露露, 郑丁丁, 宋欢欢, 李胜强, 王同燕, 谭菲菲, 邓均华, 田克恭. 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.

Figures/Tables 5

References

［1］GALINDO I，ALONSO C.African swine fever virus：Areview［J］.Viruses，2017，9（5）：103.
［2］CISEK A A，DABROWSKA I，GREGORCZYK K P，et al.African swine fever virus：A new old enemy of Europe［J］.Ann Parasitol，2016，62（3）：161‐167.
［3］HESS W R. African swine fever：A reassessment［J］.Adv Vet Sci Comp Med，1981，25：39‐69.

［4］焦福林，韩一超，李文刚，等.非洲猪瘟的检测与防控［J］.山西农业科学，2017，45（12）：2006‐2008.

JIAO F L，HAN Y C，LIU W G，et al.Detection and control of African swine fever［J］.Journal of Shanxi Agricultural Sciences，2017，45（12）：2006‐2008.
［5］OURA C A L，POWELL P P，ANDERSON E，et al.The pathogenesis of African swine fever in the resistant bush
pig［J］.Journal of General Virology，1998，79：1439‐1443.
［6］陈腾，张守峰，周鑫韬，等.我国首次非洲猪瘟疫情的发现和流行分析［J］.中国兽医学报，2018，38（9）：1831‐1832.
CHEN T，ZHANG S F，ZHOU X T，et al.The discovery and epidemic analysis of the first African swine fever epidemic in China［J］.Chinese Journal of Veterinary Medicine，2018，38（9）：1831‐1832.
［7］耿笑林，孙杰，王彦伟，等.非洲猪瘟病毒K145R蛋白的原核表达及其单克隆抗体制备［J］.河南农业科学，2021，50（8）：154‐159.
GENG X L，SUN J，WANG Y W，et al.Prokaryotic expression of African swine fever virus K145R protein and preparation of monoclonal antibody［J］.Journal of Henan Agricultural Sciences，2021，50（8）：154‐159.
［8］祁元明，陈玉梅，王爱萍.非洲猪瘟病毒特异性抗体检测研究进展［J］.河南农业大学学报，2021，55（5）：807‐814.
QI Y M，CHEN Y M，WANG A Q.Advances in detection of specific antibodies against African swine fever virus［J］.Journal of Henan Agricultural University，2021，55（5）：807‐814.
［9］YANEZ R J，RODRIGUEZ J M，NOGAL M L，et al.Analysis of the complete nucleotide sequence of African swine fever virus［J］.Virology，1995，208（1）：249‐278.
［10］TAKAMATSU H H，DENYER M S，LACASTA A，et al.Cellular immunity in ASFV responses［J］.Virus Research，2013，173（1）：110‐121.
［11］SALAS M L，ANDRES G.African swine fever virus morphogenesis［J］.Virus Research，2013，173（1）：29‐41.
［12］DE VILLIERS E P，GALLARDO C，Arias M，et al.Phylogenomic analysis of 11 complete African swine fever virus genome sequences［J］.Virology，2010，400（1）：128‐136.
［13］ALEJO A，MATAMOROS T，GUERRA M，et al.A proteomic atlas of the African swine fever virus particle［J］.Journal of Virology，2018，12，92（23）：1293‐1310.
［14］李杰，张星星，郭晶，等.非洲猪瘟病毒p30-54融合蛋白基因的构建、表达及其多克隆抗体制备［J］.河南农业科学，2018，47（9）：132‐136.
LI J，ZHANG X X，GUO J，et al.Construction，expression of p30-54 fusion protein gene of African swine fever virus and preparation of polyclonal antibody［J］.Journal of Henan Agricultural Sciences，2018，47（9）：132‐136.
［15］CAMACHO A，VINUELA E.Protein p22 of African swine fever virus：An early structural protein that is incorporated into the membrane of infected cells［J］.Virology，1991，181（1）：251‐257.
［16］GOMEZ P P，RODRIGUEZ F，OVIEDO J M，et al.The African swine fever virus proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody‐mediated protective immune response［J］.Virology，1998，243（2）：461‐471.
［17］BARDERAS M G，RODRIGUEZ F，GOMEZ P P，et al.Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins［J］.Arch Virol，2001，146（9）：1681‐1691.
［18］NEILAN J G，ZSAK L，LU Z，et al.Neutralizing antibodies to African swine fever virus proteins p30，p54，and p72 are not sufficient for antibody‐mediated protection［J］.Virology，2004，319（2）：337‐342.
［19］白晶晶，宋欢欢，白晨雨，等.非洲猪瘟病毒p17蛋白单克隆抗体的制备与鉴定［J］.河南农业科学，2020，49（12）：137‐143.
BAI J J，SONG H H，BAI C Y，et al.Preparation and identification of monoclonal antibodies against African swine fever virus p17 protein［J］.Journal of Henan Agricultural Sciences，2020，49（12）：137‐143.
［20］郝丽影，王彦伟，孙玉洁，等.非洲猪瘟病毒P30蛋白单克隆抗体的制备与应用［J］.河南农业科学，2020，49（10）：124‐129.
HAO L Y，WANG Y W，SUN Y J，et al.Preparation and application of monoclonal antibodies against P30 protein
of African swine fever virus［J］.Journal of Henan Agricultural Sciences，2020，49（10）：124‐129.
［21］贾云飞，赵福杰，朱静静，等.非洲猪瘟病毒SYBR Green Ⅰ 实时荧光定量PCR检测方法的建立［J］.河南农业大学学报，2020，54（1）：69‐73.
JIA Y F，ZHAO F J，ZHU J J，et al.Development of a SYBR Green Ⅰ based real‐time quantitative PCR method for the detection of African swine fever virus［J］.Journal of Henan Agricultural University，2020，54（1）：69‐73.
［22］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.
［23］JANCOVICH J K，CHAPMAN D，HANSEN D T，et al.Immunization of pigs by DNA prime and recombinant vaccinia virus boost to identify and rank African swine Fever virus immunogenic and protective proteins［J］.Virology，2018，92（8）：e02219‐17.
［24］NATASHA N，GAUDREAULT，JUERGEN A，et al.Subunit vaccine approaches for African swine fever virus［J］.Vaccines，2019，7（2）：56.

[1]	XU Jia, SUN Huaichang. Prokaryotic Expression of Vsp Protein and Preparation of Monoclonal Antibody against Mycoplasma bovis [J]. Journal of Henan Agricultural Sciences, 2023, 52(12): 142-148.
[2]	LUO Jun, DANG Lu, ZHENG Luping, LIU Jinling, CHAI Shujun, ZHAO Dong, YANG Yanyan, TENG Man. Preparation and Identification of Specific Monoclonal Antibody against Chicken hnRNPAB Protein [J]. Journal of Henan Agricultural Sciences, 2022, 51(2): 127-132.
[3]	WANG Xun, LI Ge, LI Qingmei, LÜ Rongzhou, MENG Zekun, CHAI Shujun, YANG Jifei, GUO Junqing, ZHANG Gaiping. Preparation and Identification of Monoclonal Antibody Broadly Recognizing HA Protein of H7 Subtype Avian Influenza Virus [J]. Journal of Henan Agricultural Sciences, 2022, 51(11): 119-126.
[4]	GENG Xiaolin, SUN Jie, WANG Yanwei, HUANG Tian, LIU Peng, CAO Hongmei, PANG Wenqiang, HAO Liying, DENG Junhua, HUANG Yuxin, TIAN Kegong. Prokaryotic Expression of African Swine Fever Virus K145R Protein and Preparation of Monoclonal Antibody [J]. Journal of Henan Agricultural Sciences, 2021, 50(8): 154-159.
[5]	WANG Zhaoqin, ZHANG Fusheng, DENG Jiajun, MA Yuhua, CUI Tingting, CUI Na, WAN Yuping. Rapid Detection of Profenofos Using Colloidal Gold Immunoassay Based on Monoclonal Antibody [J]. Journal of Henan Agricultural Sciences, 2020, 49(12): 77-82.
[6]	BAI Jingjing, SONG Huanhuan, BAI Chenyu, HAO Liying, YAN Shijun, DU Mengmeng, LI Xiangdong, DENG Junhua, TIAN Kegong. Preparation and Identification of Monoclonal Antibodies against African Swine Fever Virus p17 Protein [J]. Journal of Henan Agricultural Sciences, 2020, 49(12): 137-143.
[7]	TENG Man, CHI Jiaqi, CHAI Shujun, LUO Jun. Development of Monoclonal Antibodies against Chicken LTBP1 Protein [J]. Journal of Henan Agricultural Sciences, 2020, 49(12): 144-150.
[8]	HAO Liying, WANG Yanwei, SUN Yujie, CAO Hongmei, HUANG Tian, PANG Wenqiang, LI Xiangdong, DENG Junhua, TIAN Kegong. Preparation and Application of Monoclonal Antibodies against P30 Protein of African Swine Fever Virus [J]. Journal of Henan Agricultural Sciences, 2020, 49(10): 124-129.
[9]	CHENG Xuan, FU Pengfei, DU Yongkun, CHU Beibei, YANG Guoyu, WANG Jiang. Preparation and Identification of Monoclonal Antibody against gB Protein of Porcine Pseudorabies Virus Variant Strain HN1201 [J]. Journal of Henan Agricultural Sciences, 2020, 49(10): 137-142.
[10]	. Expression of Pseudorabies Virus gE Protein in Pichia pastoris and Screening of Its Monoclonal Antibodies#br# [J]. Journal of Henan Agricultural Sciences, 2019, 48(10): 133-139.

Expression and Monoclonal Antibody Preparation of the p22 Protein of African Swine Fever Virus

非洲猪瘟病毒p22 蛋白的表达及单克隆抗体制备

PDF

Knowledge

Abstract

Cite this article

share this article

Figures/Tables 5

References

Related Articles 10

Recommended Articles

Metrics