非洲猪瘟病毒K205R蛋白单克隆抗体的制备与鉴定
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摘要
将合成的非洲猪瘟病毒K205R基因序列插入p ET-28a(+)表达载体,经IPTG诱导表达p ET-28aK205R重组蛋白。用制备的重组抗原免疫BALB/c小鼠,采用化学融合方法融合、筛选,获得了6A4、7F5和7G3三株抗K205R蛋白的杂交瘤细胞株。经鉴定,这3株单克隆抗体(单抗)的亚类均为Ig G1型;单抗的腹水效价分别为1∶51 200、1∶409 600和1∶102 400。将这3株杂交瘤细胞连续培养20代,在第20代仍有很高的分泌抗体的能力,表明其稳定性较好。Western-blot检测结果表明,这3株单抗与K205R重组蛋白有良好的反应性和特异性。上述结果表明,本研究制备的这3株杂交瘤细胞株能够稳定分泌抗体,分泌的抗体特异性高,为非洲猪瘟诊断技术的研究奠定了基础。
The synthesized K205 R gene of African swine fever virus(ASFV) was cloned into the p ET-28 a(+)expression vector,and then expressed with IPTG induction.The recombinant K205 R protein was used to immunize BALB/c mice for monoclonal antibody(Mc Ab) preparation,and three hybridoma cell lines(coded6 A4,7 F5 and 7 G3) against the K205 R protein were harvested after chemical fusion and screening.The Ig G1 subtype of the three monoclonal antibodies were identified,and the ascites titers of the McAbs were1∶51 200,1∶409 600 and 1∶102 400,respectively.After continuously cultivation of the three hybridoma cell lines for 20 generations,they were still able to secrete antibodies at a high level,indicating the stability of the cell lines.The three Mc Abs showed good reactivity and specificity with the recombinant K205 R protein in Western-blot.The above-mentioned results showed that the three hybridoma cell lines against recombinant K205 R protein were successfully established for producing Mc Abs,and the Mc Abs had high specificity to K205 R protein of ASFV,which could be useful tool for the development of serological diagnostic tests for African swine fever.
The synthesized K205 R gene of African swine fever virus(ASFV) was cloned into the p ET-28 a(+)expression vector,and then expressed with IPTG induction.The recombinant K205 R protein was used to immunize BALB/c mice for monoclonal antibody(Mc Ab) preparation,and three hybridoma cell lines(coded6 A4,7 F5 and 7 G3) against the K205 R protein were harvested after chemical fusion and screening.The Ig G1 subtype of the three monoclonal antibodies were identified,and the ascites titers of the McAbs were1∶51 200,1∶409 600 and 1∶102 400,respectively.After continuously cultivation of the three hybridoma cell lines for 20 generations,they were still able to secrete antibodies at a high level,indicating the stability of the cell lines.The three Mc Abs showed good reactivity and specificity with the recombinant K205 R protein in Western-blot.The above-mentioned results showed that the three hybridoma cell lines against recombinant K205 R protein were successfully established for producing Mc Abs,and the Mc Abs had high specificity to K205 R protein of ASFV,which could be useful tool for the development of serological diagnostic tests for African swine fever.
引文
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[12]张鑫宇,陈宇,刘文俊,等.非洲猪瘟病毒E183L、B602L、K205R和A104R基因表达及诊断抗原筛选[J].中国兽医杂志,2014,50(3):3-5,9.ZHANG Xin-yu,CHEN Yu,LIU Wen-jun,et al.Expressing the K205R,E183L,A104R and B602L of ASFV&screening the proteins as diagnostic antigens in serological assays[J].Chinese Journal of Veterinary Medicine,2014,50(3):3-5,9.
[13]LACASTA A,BALLESTER M,MONTEAGUDO P L,et al.Expression library immunization can confer protection against lethal challenge with African swine fever virus[J].Journal of Virology,2014,88(22):13322-13332.
[14]LOKHANDWALA S,WAGHELA S D,BRAY J,et al.Adenovirus-vectored novel African swine fever virus antigens elicit robust immune responses in swine[J].PLoS ONE,2017,12(5).
[15]O"DONNELL V,RISATTI G R,HOLINKA L G,et al.Simultaneous deletion of the 9GL and UK genes from the African swine fever virus Georgia 2007 isolate offers increased safety and protection against homologous challenge[J].Journal of Virology,2016:JVI.01760-16.
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[17]BNER H,ALEXANDRA,PETERSEN B,et al.Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30gene(CP204L)[J].Scientific Reports,2018,8(1):1449.
[18]KOLLNBERGER S D,GUTIERREZCASTA?EDA B,FOSTERCUE-VAS M,et al.Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody[J].Journal of General Virology,2002,83(6):1331-1342.
[19]CUBILLOS C,MEZSEBASTIAN S G,MORENO N,et al.African swine fever virus serodiagnosis:A general review with a focus on the analyses of African serum samples[J].Virus Research,2013,173(1):159-167.
[20]杨晓红.非洲猪瘟病毒P72基因单克隆抗体的制备及其重组杆状病毒的构建[D].武汉:华中农业大学,2012.YANG Xiao-hong.Preparation of monoclonal antibodies against P72 gene of African swine fever virus and construction of the recombinant baculovirus[D].Wuhan:Huazhong Agricultural University,2012.(in Chinese)
[21]储德文.非洲猪瘟病毒p30重组蛋白的表达及单克隆抗体制备[D].扬州:扬州大学,2013.CHU De-wen.Expression and monoclonal antibody preparation of the recombinant p30 protein of African swine fever virus[D].Yangzhou:Yangzhou University,2013.(in Chinese)
[22]冯尧.非洲猪瘟病毒p54重组蛋白的原核表达及其单克隆抗体制备[D].扬州:扬州大学,2014.FENG Yao.Expression and monoclonal antibody preparation of the recombinant p54 protein of African swine fever virμs[D].Yangzhou:Yangzhou University,2014.(in Chinese)
[23]REIS A L,PARKHOUSE R M,PENEDOS A R,et al.Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus[J].Journal of General Virology,2007,88(9):2426-2434.
[2]RONISH B,HAKHVERDYAN M,STAHL K,et al.Design and verification of a highly reliable linear-afterthe-exponential PCR(LATE-PCR)assay for the detection of African swine fever virus[J].Journal of Virological Methods,172:8-15.
[3]QUEMBO C J,JORI F,VOSLOO W,et al.Genetic characterization of African swine fever virus from soft ticks at the wildlife/domestic interface in Mozambique and identification of a novel genotype[J].Transboundary and Emerging Diseases,2018,65(2):420-431.
[4]戈胜强,吴晓东,李金明,等.巴西非洲猪瘟根除计划的经验与借鉴[J].中国兽医学报,2017,37(5):961-964.GE Sheng-qiang,WU Xiao-dong,LI Jin-ming,et al.Experience and reference of Brazil's African sine fever eradication program[J].Chinese Journal of Veterinary Science,2017,37(5):961-964.(in Chinese)
[5]MONTGOMERY R E.On a form of swine fever occurring in British East Africa(Kenya Colony)[J].Journal of Comparative Pathology&Therapeutics,1921,34:159-191.
[6]KOLBASOV D,TITOV I,TSYBANOV S,et al.African swine fever virus,Siberia,Russia,2017[J].Emerging Infectious Diseases,2018,24(4):796-798.
[7]GALINDO I,ALONSO C.African swine fever virus:A review[J].Viruses,2017,9(5):103.
[8]FAUQUET C M,MAYO M A,MANILOFF J,et al.Virus Taxonomy.VIIIth Report of the International Committee on Taxonomy of Virus[M].San Diego:Elsevier Acadmic Press,2005:135-143.
[9]王清华,任炜杰,包静月,等.我国首例非洲猪瘟的确诊[J].中国动物检疫,2018,35(9):1-4.WANG Qing-hua,REN Wei-jie,BAO Jing-yue,et al.The first outbreak of African swine fever was confirmed in China[J].China Animal Health Inspection,2018,35(9):1-4.(in Chinese)
[10]TULMAN E R,DELHON G A,KU B K,et al.African swine fever virus[J].Virology Monographs Die Virus for schung Innzeldarstellungen,2009,9(2):1.
[11]GUTIERREZ-CASTANEDA B,REIS A A,PARKHOUSE R,et al.Expression,cellular localization and antibody responses of the African swine fever virus genes B602L and K205R[J].Archives of Virology,2008,153(12):2303-2306.
[12]张鑫宇,陈宇,刘文俊,等.非洲猪瘟病毒E183L、B602L、K205R和A104R基因表达及诊断抗原筛选[J].中国兽医杂志,2014,50(3):3-5,9.ZHANG Xin-yu,CHEN Yu,LIU Wen-jun,et al.Expressing the K205R,E183L,A104R and B602L of ASFV&screening the proteins as diagnostic antigens in serological assays[J].Chinese Journal of Veterinary Medicine,2014,50(3):3-5,9.
[13]LACASTA A,BALLESTER M,MONTEAGUDO P L,et al.Expression library immunization can confer protection against lethal challenge with African swine fever virus[J].Journal of Virology,2014,88(22):13322-13332.
[14]LOKHANDWALA S,WAGHELA S D,BRAY J,et al.Adenovirus-vectored novel African swine fever virus antigens elicit robust immune responses in swine[J].PLoS ONE,2017,12(5).
[15]O"DONNELL V,RISATTI G R,HOLINKA L G,et al.Simultaneous deletion of the 9GL and UK genes from the African swine fever virus Georgia 2007 isolate offers increased safety and protection against homologous challenge[J].Journal of Virology,2016:JVI.01760-16.
[16]REIS A L,ABRAMS C C,GOATLEY L C,et al.Deletion of African swine fever virus interferon inhibitors from the genome of a virulent isolate reduces virulence in domestic pigs and induces a protective response[J].Vaccine,2016,34(39):4698-4705.
[17]BNER H,ALEXANDRA,PETERSEN B,et al.Efficient inhibition of African swine fever virus replication by CRISPR/Cas9 targeting of the viral p30gene(CP204L)[J].Scientific Reports,2018,8(1):1449.
[18]KOLLNBERGER S D,GUTIERREZCASTA?EDA B,FOSTERCUE-VAS M,et al.Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody[J].Journal of General Virology,2002,83(6):1331-1342.
[19]CUBILLOS C,MEZSEBASTIAN S G,MORENO N,et al.African swine fever virus serodiagnosis:A general review with a focus on the analyses of African serum samples[J].Virus Research,2013,173(1):159-167.
[20]杨晓红.非洲猪瘟病毒P72基因单克隆抗体的制备及其重组杆状病毒的构建[D].武汉:华中农业大学,2012.YANG Xiao-hong.Preparation of monoclonal antibodies against P72 gene of African swine fever virus and construction of the recombinant baculovirus[D].Wuhan:Huazhong Agricultural University,2012.(in Chinese)
[21]储德文.非洲猪瘟病毒p30重组蛋白的表达及单克隆抗体制备[D].扬州:扬州大学,2013.CHU De-wen.Expression and monoclonal antibody preparation of the recombinant p30 protein of African swine fever virus[D].Yangzhou:Yangzhou University,2013.(in Chinese)
[22]冯尧.非洲猪瘟病毒p54重组蛋白的原核表达及其单克隆抗体制备[D].扬州:扬州大学,2014.FENG Yao.Expression and monoclonal antibody preparation of the recombinant p54 protein of African swine fever virμs[D].Yangzhou:Yangzhou University,2014.(in Chinese)
[23]REIS A L,PARKHOUSE R M,PENEDOS A R,et al.Systematic analysis of longitudinal serological responses of pigs infected experimentally with African swine fever virus[J].Journal of General Virology,2007,88(9):2426-2434.
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