非洲猪瘟病毒p30蛋白单克隆抗体制备和鉴定

doi:10.16431/j.cnki.1671-7236.2023.03.038

摘要/Abstract

摘要： 【目的】制备非洲猪瘟病毒(African swine fever virus,ASFV) p30蛋白的单克隆抗体,为非洲猪瘟快速诊断检测方法的建立和疫苗研究提供材料。【方法】构建p30原核表达质粒pET-30a(+)-p30,转化至大肠杆菌BL21(DE3)感受态细胞中进行诱导表达,通过镍柱亲和层析获得目的蛋白。p30复性后,每2周对BALB/c小鼠免疫3次,将免疫小鼠的脾脏与骨髓瘤细胞融合制备单克隆抗体,通过ELISA方法和亚克隆筛选分泌抗体的杂交瘤细胞。将筛选出的单克隆细胞注射进小鼠腹腔制备腹水,通过Western blotting检测、免疫荧光测定(IFA)和抗体亚型分析来鉴定单克隆抗体。【结果】试验成功构建了p30蛋白的原核表达质粒,并使用IPTG诱导、纯化获得了原核系统表达的重组p30蛋白;成功制备出4株能稳定分泌针对ASFV p30蛋白的单克隆抗体杂交瘤细胞株,分别命名为:1H3B4、3F2F5、6E3A1和9D6B9。经ELISA测定抗体腹水效价在1∶100000~1∶1000000之间。经Western blotting和IFA鉴定筛选得到的单克隆抗体与p30蛋白能发生特异性反应。经亚型鉴定,3F2F5和9D6B9 2株单克隆抗体的重链均为IgG2b,轻链均为Kappa;6E3A1株单克隆抗体的重链为IgM,轻链为Kappa;1H3B4株单克隆抗体的重链为IgG2b,轻链为Lambda。【结论】试验成功制备了4株针对ASFV p30蛋白的单克隆抗体,并分析了单克隆抗体的免疫学特性,为p30蛋白的生物学功能研究和ASFV血清学检测提供依据。

关键词: 非洲猪瘟病毒(ASFV); p30蛋白; 单克隆抗体; CP204L基因

Abstract: 【Objective】 The aim of this experiment was to prepare monoclonal antibody to African swine fever virus (ASFV) p30 protein,and lay the foundation for the establishment of a rapid diagnostic test for ASFV and the research of a vaccine.【Method】 The p30 prokaryotic expression plasmid pET-30a(+)-p30 was constructed,transformed into E.coli BL21 (DE3) competent cells for induced expression,and then the target protein was obtained by nickel column affinity chromatography.p30 was compounded and immunized to BALB/c mice for three times every two weeks,and the spleen of immunized mice was fused with myeloma cells to prepare monoclonal antibody by ELISA method and subclonal screening of antibody-secreting hybridoma cells.The screened monoclonal cells were injected into the peritoneal cavity of mice to prepare ascites.The monoclonal antibodies were identified by Western blotting detection,immunofluorescence assay (IFA) and antibody isotype analysis.【Result】 The p30 protein prokaryotic expression plasmid was successfully constructed,and the recombinant p30 protein expressed in the prokaryotic system was purified using IPTG to induce.Four monoclonal antibody hybridoma cell lines,named 1H3B4,3F2F5,6E3A1 and 9D6B9,which could stably secrete the ASFV p30 protein,were successfully prepared.The potencies of the antibodies were determined by ELISA in the range of 1∶100 000 to 1∶1 000 000.The screened monoclonal antibodies were identified by Western blotting and IFA and those antibodies were specifically reacted with p30 protein.The monoclonal antibodies were identified as IgG2b for the heavy chain and Kappa for the light chain of 3F2F5 and 9D6B9.6E3A1 had IgM for the heavy chain and Kappa for the light chain.1H3B4 had IgG2b for the heavy chain and Lambda for the light chain.【Conclusion】 Four monoclonal antibodies against ASFV p30 protein were successfully prepared in this experiment,and the immunological characteristics of the monoclonal antibodies were analyzed,the results provided basis for the biological function research of p30 protein and the serological detection of ASFV.

Key words: African swine fever virus (ASFV); p30 protein; monoclonal antibody; CP204L gene

中图分类号:

S852.65⁺1

侯浩宇, 郑南南, 吴宏举, 陈寅龙, 李潮, 张昂克, 姬鹏超, 万博, 杜永坤, 张改平. 非洲猪瘟病毒p30蛋白单克隆抗体制备和鉴定[J]. 中国畜牧兽医, 2023, 50(3): 1241-1249.

HOU Haoyu, ZHENG Nannan, WU Hongju, CHEN Yinlong, LI Chao, ZHANG Angke, JI Pengchao, WAN Bo, DU Yongkun, ZHANG Gaiping. Preparation and Characterization of Monoclonal Antibody Against p30 Protein of African Swine Fever Virus[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(3): 1241-1249.

参考文献

[1] 杨国淋,魏玲.非洲猪瘟的发生与防控[J].现代农业科技,2019,22:169-170. YANG G L,WEI L.Occurrence and control of African swine fever[J].Modern Agricultural Science and Technology,2019,22:169-170.(in Chinese)
[2] 魏园园.非洲猪瘟病毒的特点及预防措施[J].中国畜牧业,2021,23:42. WEI Y Y,Characteristics and preventive measures of African swine fever virus[J].China Animal Industry,2021,23:42.(in Chinese)
[3] 王岩,邴睿,张慧,等.OIE非洲猪瘟标准简介[J].中国动物检疫,2021,38(10):87-91. WANG Y,BING R,ZHANG H,et al.Introduction to OIE standard on African swine fever[J].China Animal Health Inspection,2021,38(10):87-91.(in Chinese)
[4] NAN W,DONGMING Z,JIALING W,et al.Architecture of African swine fever virus and implications for viral assembly[J].Science,2019,366(6465):640-644.
[5] 黄霞,康喜龙,孟闯,等.非洲猪瘟病毒及其疫苗研究进展[J].生物学杂志,2021,38(6):99-103. HUANG X,KANG X L,MENG C,et al.Research progress on African swine fever virus and its vaccines[J].Journal of Biology,2021,38(6):99-103.(in Chinese)
[6] SÁNCHEZ E G,QUINTAS A,NOGAL M,et al.African swine fever virus controls the host transcription and cellular machinery of protein synthesis[J].Virus Research,2013,173(1):58-75.
[7] LITHGOW P,TAKAMATSU H,WERLING D,et al.Correlation of cell surface marker expression with African swine fever virus infection[J].Veterinary Microbiology,2014,168(2-4):413-419.
[8] RAMIRO-IBÁÑEZ F,ESCRIBANO J M,ALONSO C.Application of a monoclonal antibody recognizing protein p30 to detect African swine fever virus-infected cells in peripheral blood[J].Journal of Virological Methods,1995,55(3):339-345.
[9] OH T,DO D T,LAI D C,et al.Chronological expression and distribution of African swine fever virus p30 and p72 proteins in experimentally infected pigs[J].Scientific Reports,2022,12(1):4151.
[10] HVBNER A,PETERSEN B,KEIL G M,et al.Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30 gene (CP204L)[J]. Scientific Reports,2018,8(1):1449.
[11] 崔钰晨,陈骐.中国非洲猪瘟疫情概况及其疫苗研究进展[J].福建农业科技,2021,52(4):66-71. CUI Y C,CHEN Q.Epidemic situation of African swine fever in China and the research progress of its vaccine[J].Fujian Agricultural Science and Technology,2021,52(4):66-71.(in Chinese)
[12] 欧动力.非洲猪瘟的症状及防控措施[J].中国动物保健,2022,24(6):91-92. OU D L.Symptoms and control measures of Africa swine fever[J].China Animal Health,2022,24(6):91-92.(in Chinese)
[13] GALLARDO M C,REOYO A D L T,FERNÁNDEZ-PINERO J,et al.African swine fever:A global view of the current challenge[J].Porcine Health Management,2015,1(1):21.
[14] VERGNE T,CHEN F C,LI S,et al.Pig empire under infectious threat:Risk of African swine fever introduction into the People’s Republic of China[J].Veterinary Record,2017,181(5):117.
[15] 张志,康京丽,李晓成.非洲猪瘟的流行特征和传播路线[J].中国动物检疫,2018,35(11):48-51. ZHANG Z,KANG J L,LI X C.The Epidemiological characteristics of African swine fever and its transmission routes[J].China Animal Health Inspection,2018,35(11):48-51.(in Chinese)
[16] ZHOU X,LI N,LUO Y,et al.Emergence of African swine fever in China,2018[J].Transboundary and Emerging Diseases,2018,65(6):1482-1484.
[17] SUBEDI D,SUBEDI S,KARKI S.First outbreak of African swine fever in Nepal[J].Transboundary and Emerging Diseases,2022,69(5):e3334-e3335.
[18] WANG F,ZHANG H,HOU L,et al.Advance of African swine fever virus in recent years[J].Research in Veterinary Science,2021,136:535-539.
[19] 杨佳萍,寇美玲,谢佳芮,等.非洲猪瘟疫苗和病原学诊断技术研究进展[J].中国动物检疫,2021,38(11):93-100. YANG J P,KOU M L,XIE J R,et al.Research progress on vaccines and etiological diagnostic technologies of ASF[J].China Animal Health Inspection,2021,38(11):93-100.(in Chinese)
[20] VLASOVA N N,VERKHOVSKY O A,ALIPER T I,et al.Problems of specific prevention of African swine fever[J].Problems of Virology,2022,67(3):206-216.
[21] URBANO A C,FERREIRA F.African swine fever control and prevention:An update on vaccine development[J].Emerging Microbes & Infections,2022,11(1):2021-2033.
[22] 杨继香,王长德,王长荣.非洲猪瘟的防控[J].畜禽业,2022,33(7):33-35. YANG J X,WANG C D,WANG C R.Prevention of African swine fever[J].Livestock and Poultry Industry,2022,33(7):33-35.(in Chinese)
[23] KUNO G,MACKENZIE J S,JUNGLEN S,et al.Vertebrate reservoirs of arboviruses:Myth,synonym of amplifier,or reality?[J].Viruses,2017,9(7):185.
[24] DIXON L K,CHAPMAN D A,NETHERTON C L,et al.African swine fever virus replication and genomics[J].Virus Research,2013,173(1):3-14.
[25] 周星怡,牛冬,李玉峰.非洲猪瘟病原学与传播特点及防控方法的研究进展[J].中国兽医科学,2022,52(9):1172-1180. ZHOU X Y,NIU D,LI Y F.Advancement in etiology,transmission and prevention and control of African swine fever[J].Chinese Veterinary Science,2022,52(9):1172-1180.(in Chinese)
[26] 刘厚荣,张强,钱明.非洲猪瘟检测方法研究进展[J].湖南畜牧兽医,2022,4:48-51. LIU H R,ZHANG Q,QIAN M.Rasearch progress on detection methods of African swine fever[J].Hunan Journal of Animal Science & Veterinary Medicine,2022,4:48-51.(in Chinese)
[27] OURA C A L,EDWARDS L,BATTEN C A.Virological diagnosis of African swine fever—Comparative study of available tests[J].Virus Research,2013,173(1):150-158.
[28] 刘靖,钟杰,陈伟业,等.非洲猪瘟病毒p30蛋白的真核表达纯化及单克隆抗体的制备[J].中国预防兽医学报,2021,43(10):1074-1079. LIU J,ZHONG J,CHEN W Y,et al.Eukaryotic expression of African swine fever virus p30 protein and preparation of monoclonal antibody[J].Chinese Journal of Preventive Veterinary Medicine,2021,43(10):1074-1079.(in Chinese)
[29] 李航,戚睿斌,陈宗艳,等.外源蛋白表达系统及其应用的研究进展[J].黑龙江畜牧兽医,2021,7:34-37. LI H,QI R B,CHEN Z Y,et al.Progress in research on foreign protein expression system and its application[J]. Heilongjiang Animal Science and Veterinary Medicine,2021,7:34-37. (in Chinese)