施马伦贝格病毒核衣壳蛋白的表达及单克隆抗体制备

doi:10.11843/j.issn.0366-6964.2016.06.026

Abstract

Abstract:

The objective of this study was to express and purify the nucleocapsid (N) protein of Schmallenberg virus (SBV) with the native conformation，and to prepare its monoclonal antibodies (McAbs).To this end，the SBV-N gene with an inserted coding sequence for an N-terminal hexahistidine (6×His) tag was cloned into the pFastBac^TM 1 vector.The resulting recombinant donor plasmid，pFastBac-His-SBV-N，was then transformed into DH10Bac E.coli competent cells.After selection with the blue/white screen，the recombinant bacmid，rBacmid-His-SBV-N，was transfected into Sf9 insect cells to generate recombinant baculoviruses that express the His-SBV-N fusion protein.After amplifying the recombinant baculoviral stocks on Sf9 cells，His-SBV-N fusion protein was purified using nickel nitrilotriacetic acid (Ni-NTA) agarose.The purified protein was then used to immunize BALB/c mice to prepare SBV-N-specific McAbs，and an indirect double-antibody binding ELISA was applied to detect whether the generated McAbs recognize different antigenic sites.The screened McAbs that recognize different antigenic sites were labeled with horseradish peroxidase (HRP) using the sodium periodate oxdization method.Finally，four McAbs (2A11，2E1，4H11 and 6E12) recognizing different antigenic sites were successfully obtained and labeled with HRP.The results of isotype identification demonstrated that 2A11 belongs to IgG1，and 2E1，4H11 and 6E12 belong to IgG2b.Indirect immunofluorescence assays revealed that all of the four McAbs reacted with the BHK-21 cell line stably expressing the SBV-N protein (BHK-21-EGFP-SBV-N).Western blot analyses further showed that the four HRP-conjugated McAbs reacted positively with the His-SBV-N fusion protein.Taken together，the successful preparation of His-SBV-N fusion protein and its McAbs provide valuable biological materials that can be used in the serological diagnosis of Schmallenberg disease.

CLC Number:

S852.659.5

ZHANG Yong-ning，SONG Shan-shan，WU Shao-qiang，LIN Xiang-mei. Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac^® Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(6): 1280-1286.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2016.06.026

https://www.xmsyxb.com/EN/Y2016/V47/I6/1280

References

［1］HOFFMANN B，SCHEUCH M，HÖPER D，et al.Novel orthobunyavirus in cattle，Europe，2011 ［J］.Emerg Infect Dis，2012，18(3)：469-472.
［2］HERDER V，WOHLSEIN P，PETERS M，et al.Salient lesions in domestic ruminants infected with the emerging so-called Schmallenberg virus in Germany［J］.Vet Pathol，2012，49(4)：588-591.
［3］RASMUSSEN L D，KRISTENSEN B，KIRKEBY C，et al.Culicoids as vectors of Schmallenberg virus ［J］.Emerg Infect Dis，2012，18(7)：1204-1206.
［4］LIEVAART-PETERSON K，LUTTIKHOLT S，PEPERKAMP K，et al.Schmallenberg disease in sheep or goats：Past，present and future ［J］.Vet Microbiol，2015，181(1-2)：147-153.
［5］WERNIKE K，CONRATHS F，ZANELLA G，et al.Schmallenberg virus-two years of experiences ［J］.Prev Vet Med，2014，116(4)：423-434.
［6］张永宁，吴绍强，林祥梅.施马伦贝格病研究进展［J］.畜牧兽医学报，2014，45(7)：1029-1037.
ZHANG Y N，WU S Q，LIN X M.The research progress of Schmallenberg disease ［J］.Acta Veterinaria et Zootechnica Sinica，2014，45(7)：1029-1037.（in Chinese）
［7］DOCEUL V，LARA E，SAILLEAU C，et al.Epidemiology，molecular virology and diagnostics of Schmallenberg virus，an emerging orthobunyavirus in Europe ［J］. Vet Res，2013，44：31.
［8］WERNIKE K，BROCCHI E，CORDIOLI P，et al.A novel panel of monoclonal antibodies against Schmallenberg virus nucleoprotein and glycoprotein Gc allows specific orthobunyavirus detection and reveals antigenic differences ［J］.Vet Res，2015，46：27.
［9］BRÉARD E，LARA E，COMTET L，et al.Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies ［J］.PLoS One，2013，8(1)：e53446.
［10］BILK S，SCHULZE C，FISCHER M，et al.Organ distribution of Schmallenberg virus RNA in malformed newborns ［J］.Vet Microbiol，2012，159(1-2)：236-238.
［11］张永宁，吴绍强，吕继洲，等.施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备［J］.畜牧兽医学报，2013，44(10)：1629-1636.
ZHANG Y N，WU S Q，LYU J Z，et al.Prokaryotic expression and purification of the nucleocapsid protein of Schmallenberg virus and preparation of its polyclonal antibodies ［J］.Acta Veterinaria et Zootechnica Sinica，2013，44(10)：1629-1636.（in Chinese）
［12］ZHANG Y，WU S，SONG S，et al.Preparation and characterization of a stable BHK-21 cell line constitutively expressing the Schmallenberg virus nucleocapsid protein ［J］.Mol Cell Probes，2015，29(4)：244-253.
［13］ZHANG Y，WU S，WANG J，et al.Expression and purification of the nucleocapsid protein of Schmallenberg virus，and preparation and characterization of a monoclonal antibody against this protein ［J］.Protein Expr Purif，2013，92(1)：1-8.
［14］FRIGUET B，DJAVADI-OHANIANCE L，PAGES J，et al.A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site.Application to hybridomas specific for the beta 2-subunit of Escherichia coli tryptophan synthase ［J］.J Immunol Methods，1983，60(3)：351-358.
［15］DOMINGUEZ M，HENDRIKX P，ZIENTARA S，et al.Preliminary estimate of Schmallenberg virus infection impact in sheep flocks-France ［J］.Vet Rec，2012，171(17)：426.
［16］MARTINELLE L，DAL POZZO F，GAUTHIER B，et al.Field veterinary survey on clinical and economic impact of Schmallenberg virus in Belgium ［J］.Transbound Emerg Dis，2014，61(3)：285-288.
［17］VELDHUIS A M，SANTMAN-BERENDS I M，GETHMANN J M，et al.Schmallenberg virus epidemic：impact on milk production，reproductive performance and mortality in dairy cattle in the Netherlands and Kleve district，Germany ［J］.Prev Vet Med，2014，116(4)：412-422.
［18］ROSSI S，VIAROUGE C，FAURE E，et al.Exposure of wildlife to the Schmallenberg virus in France (2011-2014)：higher，faster，stronger (than Bluetongue)［J］.Transbound Emerg Dis，2015.doi：10.1111/tbed.12371.
［19］WERNIKE K，HOFFMANN B，CONRATHS F J，et al.Schmallenberg virus recurrence，Germany，2014 ［J］.Emerg Infect Dis，2015，21(7)：1202-1204.
［20］MACHADO A M，MACHADO A R，MORELI M L，et al.Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli ［J］.Virol J，2011，8：218.
［21］CONTRERAS-GÓMEZ A，SÀNCHEZ-MIRÓN A，GARCÍA-CAMACHO F，et al.Protein production using the baculovirus-insect cell expression system ［J］.Biotechnol Prog，2014，30(1)：1-18.

[1]	MENG Lingzhai, CHEN Chunli, YU Mengmeng, WANG Zhanxin, WANG Suyan, LIU Peng, HE Tana, GUO Ru, CHEN Yuntong, LIU Changjun, QI Xiaole, WU Zhiqiang, GAO Yulong. Pathogenicity of Avian Metapneumovirus Subtype B on Yellow Feather Broilers and Evaluation on Immune Effect of Inactivated Vaccine [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5154-5161.
[2]	JIANG Shengqiang, HU Jing, CHEN Hongying. Expression Analysis of CircRNAs in A549 Cells Infected with H1N1 Influenza A Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4724-4734.
[3]	ZHANG Ao, TAN Bin, LIU Kexin, LIU Jiali, ZHANG Shuqin. Genomic Characterization Analysis of a H1N1 Subtype Swine Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4866-4871.
[4]	HE Chenxiang, GAO Shandian, TIAN Zhancheng, DU Junzheng, WANG Jinming, GUAN Guiquan, YIN Hong. Development of a Differential Diagnostic ELISA Based on G_NS Protein to Distinguish BEFV Infected and Vaccinated Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4320-4326.
[5]	CHANG Yiming, TANG Cheng, YUE Hua. Molecular Epidemiological Investigation of Bovine Respiratory Syncytial Virus in Yaks [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2030-2041.
[6]	FANG Yuan, HOU Qiaodi, XIANG Chaohui, ZHAO Hongyi, QI Xuefeng. Regulatory Effects of IFITM3 on Proliferation of Peste des Petits Ruminants Virus (PPRV) in Goat Endometrial Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2200-2207.
[7]	LI Shun, LI Guizhen, YUAN Yaoxian, LI Kangjian, MA Chunquan, LI Shoujun, HUANG Liangzong, MA Jun. Effect of the Length of PA-X Gene on Replication and Pathogenicity of H3N2 Canine Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 272-280.
[8]	CUI Mingxian, WANG Xingbo, HUANG Yanming, BIAN Xiyi, FENG Mengke, YAN Yan, DONG Weiren, ZHOU Jiyong. Genetic Characterization and Evolution of Three Strains of H3N2 Avian Influenza Viruses [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4116-4122.
[9]	YU Mengmeng, BAO Yuanling, WANG Suyan, XIN Ziqi, LIU Peng, FENG Xiaoyan, MENG Lingzhai, GUO Ru, ZHANG Yanping, LIU Changjun, QI Xiaole, LI Junping, WANG Xiaomei, GAO Yulong. Isolation, Identification and Pathogenicity of Subtype B Avian Metapneumovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3540-3549.
[10]	ZHOU Yong, LI Zhixin, LU Hongwei, SUN Yan, LI Tian, DU Fanshu, PU Juan. Surveillance and Outbreak Analysis of H5 and H7N9 Subtypes of Highly Pathogenic Avian Influenza in China [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3093-3106.
[11]	PAN Chunrong, DENG Ruixue, HU Linjie, ZHU Xueliang, SUN Yuefeng, WANG Guirong, MENG Xuelian. Effect of Histone Deacetylase Inhibitors on Peste Des Petits Ruminants Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2307-2316.
[12]	CHEN Zixuan, ZHANG Nan, HU Qun, QUAN Keji, QIN Tao, CHEN Sujuan, PENG Daxin, LIU Xiufan. Mutation Analysis of Antigen Sites 145 and 153 on Hemagglutinin of H9N2 Avian Influenza Virus in China [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1165-1172.
[13]	DUAN Zhiqiang, XIE Lingling, CHEN Jiaqi, TANG Hong, WANG Yanbi, ZHAO Caiqin, ZHAO Jiafu. Research Progress on the Interactions of Newcastle Disease Virus M Protein with Host Proteins [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 32-42.
[14]	YAN Yayao, GU Min, LIU Xiufan. Advance in the Influence of Amino Acid Variation in HA Protein on the Biological Properties of H7N9 Subtype Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2093-2106.
[15]	JIANG Mei, CHEN Wu, ZHAI Junqiong, BU Wandi, XIE Yilun, LIU Canbin, SHAN Fen, LUO Manlin. Cloning and Sequence Analysis of H and F Genes of Canine Distemper Virus Strain from Red Panda [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1670-1676.

Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac^® Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac® Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

Expression of the Nucleocapsid Protein of Schmallenberg Virus Using the Bac-to-Bac^® Baculovirus Expression System and Preparation of Monoclonal Antibodies against This Protein