施马伦贝格病毒N蛋白原核表达及间接ELISA抗体检测方法的建立

doi:10.16431/j.cnki.1671-7236.2025.08.036

Abstract

Abstract: 【Objective】 The objective of this study was to establish an indirect ELISA (iELISA) method with good specificity and sensitivity for detecting antibodies against Schmallenberg virus (SBV). 【Method】 The full-length sequence of the SBV N protein-coding gene was cloned into pET-28a(+) plasmid to construct recombinant plasmid pET-28a-SBV.The target protein was expressed using the Escherichia coli expression system and purified using a His-tagged nickel column.After identification by SDS-PAGE and Western blotting,the purified SBV N protein was used as the detection antigen for coating to establish an iELISA method.The coating conditions,sealing fluid,sealing time,and incubation time of serum were optimized.The negative and positive critical value,specificity,sensitivity,and repeatability of the method were evaluated. 【Result】 SDS-PAGE results showed that the molecular weight of SBV N protein was 30 ku.Western blotting results demonstrated that the purified SBV N protein specifically reacted with SBV positive serum.The optimal conditions for the established SBV iELISA method were as follows:SBV N antigen coating concentration of 4 μg/mL,coating at 4 ℃ for 12 h,sealing with 1% gelatin at 37 ℃ for 1 h,serum dilution of 1∶200,incubation at 37 ℃ for 1 h,and enzyme-labeled secondary antibody dilution of 1∶6 000 with incubation at 37 ℃ for 1 h.The negative and positive critical value was 0.371.The iELISA method specifically reacted with SBV positive serum and showed no cross-reactivity with positive sera against BPIV3,BRV,BCoV,IBRV,BVDV and AKAV.The method exhibited high sensitivity,with a clear reaction observed even at a serum dilution of 1∶1 280.The coefficient of variation of inter-batch and intra-batch repeated tests was less than 10%.Compared with the commercial kit,the total concordance rate was 94.57%. 【Conclusion】 This study established an SBV iELISA method using SBV N protein as the detection antigen.The method demonstrated good specificity and sensitivity and could be applied for serological detection of SBV,providing an additional option for rapid diagnosis of SBV.

Key words: Schmallenberg virus(SBV); N protein; indirect ELISA(iELISA); prokaryotic expression

CLC Number:

S852.65^＋9.5

LI Yang, SUN Ruixue, CHEN Tianjie, LIU Sitong, CAO Jiahui, ZHAO Jianjun. Prokaryotic Expression of Schmallenberg Virus N Protein and Establishment of Indirect ELISA Antibody Detection Method[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3877-3887.

References

[1] DE REGGE N.Akabane,Aino and Schmallenberg virus—Where do we stand and what do we know about the role of domestic ruminant hosts and Culicoides vectors in virus transmission and overwintering?[J].Current Opinion in Virology,2017，27：15-30.
[2] WINDHABER S,XIN Q,LOZACH P Y，et al.Orthobunyaviruses:From virus binding to penetration into mammalian host cells[J].Viruses,2021,13(5):872.
[3] ELLIOTT M.Orthobunyaviruses:Recent genetic and structural insights[J].Nature Reviews Microbiology，2014,12(10):673-685.
[4] ENDALE A,FABURAY B,WILSON W,et al.Schmallenberg disease—A newly emerged Culicoides-borne viral disease of ruminants[J].Viruses，2019,11(11)：1065.
[5] FREITAS N,ENGUEHARD M,DENOLLY S,et al.The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity[J].PLoS Pathogens,2020,16(9):e1008850.
[6] SHÍ X,KOHL A,LÉONARD V,et al.Requirement of the N-terminal region of Orthobunyavirus nonstructural protein NSm for virus assembly and morphogenesis[J].Journal of Virology,2006，80(16):8089-8099.
[7] MOHAMED M,MCLEES A,ELLIOTT R M.Viruses in the anopheles A,anopheles B,and tete serogroups in the Orthobunyavirus genus (family Bunyaviridae) do not encode an NSs protein[J].Journal of Virology，2009，83(15)：7612-7618.
[8] KHALIL J,KATO H,FUJITA T.The role of non-structural protein NSs in the pathogenesis of severe fever with thrombocytopenia syndrome[J].Viruses,2021，13(5)：876.
[9] MONTEIRO G,VUREN P,SCHREUR P,et al.Mutation of adjacent cysteine residues in the NSs protein of Rift Valley fever virus results in loss of virulence in mice[J].Virus Research,2018，249：31-44.
[10] 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,purification,and preparation of polyclonal antibodies for the N protein of Schmallenberg virus[J].Acta Veterinaria et Zootechnica Sinica,2013,44(10):1629-1636.(in Chinese)
[11] XU L,CAO C,YANG Z,et al.Identification of a conservative site in the African swine fever virus p54 protein and its preliminary application in a serological assay[J].Journal of Veterinary Science,2022,23(4):e55.
[12] 王程,夏应菊,王海东,等.非洲猪瘟病毒P54蛋白原核表达及间接ELISA检测方法建立[J].中国畜牧兽医,2024,51(10):4440-4449. WANG C,XIA Y J,WANG H D,et al.Prokaryotic expression of African swine fever virus P54 protein and establishment of an indirect ELISA detection method[J].China Animal Husbandry & Veterinary Medicine,2024,51(10):4440-4449.(in Chinese)
[13] VARELA M,SCHNETTLER E,CAPORALE M,et al.Schmallenberg virus pathogenesis,tropism and interaction with the innate immune system of the host[J].PLoS Pathogens,2013,9(1):e1003133.
[14] DE REGGE N,DEBLAUWE I,DE DEKEN R,et al.Detection of Schmallenberg virus in different Culicoides spp.by Real-time RT-PCR[J].Transboundary and Emerging Diseases,2012,59(6):471-475.
[15] FISCHER M,SCHIRRMEIER H,WERNIKE K,et al.Development of a pan-Simbu Real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems[J].Virology Journal,2013,10:327.
[16] BOUWSTRA R J,KOOI E A,DE KLUIJVER E P,et al.Schmallenberg virus outbreak in the Netherlands:Routine diagnostics and test results[J].Veterinary Microbiology,2013,165(1-2):102-108.
[17] 徐浩钧,刘颖,梅晨,等.基于外膜蛋白的鼻气管鸟杆菌抗体间接ELISA检测方法的建立[J].中国畜牧兽医,2024,51(10):4420-4428. XU H J,LIU Y,MEI C,et al.Establishment of an indirect ELISA detection method for antibodies against Ornithobacterium rhinotracheale based on outer membrane protein[J].China Animal Husbandry & Veterinary Medicine,2024,51(10):4420-4428.(in Chinese)
[18] NÄSLUND K,BLOMQVIST G,VERNERSSON C,et al.Development and evaluation of an indirect enzyme-linked immunosorbent assay for serological detection of Schmallenberg virus antibodies in ruminants using whole virus antigen[J].Acta Veterinaria Scandinavica,2014,56:1-8.
[19] VAN DER HEIJDEN H,BOUWSTRA R J,MARS M H,et al.Development and validation of an indirect enzyme-linked immunosorbent assay for the detection of antibodies against Schmallenberg virus in blood samples from ruminants[J].Research in Veterinary Science,2013,95(2):731-735.
[20] 王贞钧.蓝舌病病毒VP7蛋白、施马伦贝格病毒N蛋白的原核表达及iELISA检测方法的建立[D].长春:吉林农业大学,2015. WANG Z J.Prokaryotic expression of Bluetongue virus VP7 protein，Schmallenberg virus N protein anddevelopment of indirect ELISA[D].Changchun:Jilin Agricultural University,2015.(in Chinese)
[21] ZHAI S L,LV D H,WEN X H,et al.Preliminary serological evidence for Schmallenberg virus infection in China[J].Tropical Animal Health and Production,2018,50:449-453.
[22] LAZUTKA J,ZVIRBLIENE A,DALGEDIENE I,et al.Generation of recombinant Schmallenberg virus nucleocapsid protein in yeast and development of virus-specific monoclonal antibodies[J].Journal of Immunology Research，2014，2014(1):160316.
[23] 宋姗姗，张永宁，吴绍强，等.施马伦贝格病毒核衣壳蛋白在昆虫杆状病毒表达系统中的表达与纯化[J].中国兽医科学，2016，46(4)：442-448. SONG S S,ZHANG Y N,WU S Q,et al.Expression and purification of Schmallenberg virus nucleocapsid protein using the Bac-to-Bac baculovirus expression system[J].Chinese Veterinary Science，2016，46(4):442-448.(in Chinese)

Prokaryotic Expression of Schmallenberg Virus N Protein and Establishment of Indirect ELISA Antibody Detection Method

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments

[1]	WANG Xianjiong, JIN Yu, XIA Ying, ZHANG Bingyin, DONG Siyan, JIN Chunmei, YU Longzheng. Preparation and Identification of Monoclonal Antibodies Against Theileria orientalis P33 Protein [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3800-3809.
[2]	ZHANG Mingliang, WANG Pei, MA Lei, YAO Chang, PANG Rong, WENG Shaoting, MA Shengming, LIAN Kaiqi. Bioinformatics Analysis of Flagellar Protein FliC of Salmonella Typhimurium and Its Effect on Mouse Macrophages [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3820-3829.
[3]	FENG Xiujuan, REN Jichao, CUI Zeyun, ZHAO Xueqing, LI Jiaxin, ZHANG Yang, ZHANG Wei, BAYIN Chahan·Gailike, GUO Qingyong, LI Yongchang. Bioinformatics Analysis and Preparation of Polyclonal Antibodies Against Babesia bovis SBP2 Protein [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3847-3856.
[4]	CHEN Xuke, PU Na, ZHANG Yuxia, ZHAO Wenqing, ZHAO Jiaxin, HUANG Juncheng, WEI Linying, ZHANG Yanyan, SUN Yan, BO Xinwen, WANG Zhengrong. Bioinformatics Analysis of Methionine Aminopeptidase Ⅱ of Echinococcus granulosus and Its Differential Expression in Different Stages of the Worm [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3286-3296.
[5]	ZHANG Pian, CHEN Jing, ZHANG Xiaoxiao, MAI Xiaopeng, TANG Ke, XIANG Hua, WANG Gang, LUO Shengjun, MA Huihai, YUAN Ziguo, WANG Xiaohu. Expression of GRA1 Protein of Toxoplasma gondii and Establishment of an Indirect ELISA Detection Method [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3308-3320.
[6]	SI Wei, TAN Xiyu, XU Lingyun, LUO Tingrong, LI Xiaoning, GU Jinyan. Prokaryotic Expression of HA1 Protein of Swine Influenza Virus Subtype H3N2 and Preparation of Polyclonal Antibodies [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(4): 1739-1749.
[7]	ZHAO Wenqing, BO Xinwen, PU Na, ZHANG Yuxia, CHEN Xuke, ZHANG Yanyan, SUN Yan, QI Meng, WANG Zhengrong. Bioinformatics Analysis and Prokaryotic Expression of EgBAL Protein of Echinococcus granulosus [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(2): 801-810.
[8]	HAN Fengye, LIU Ying, PAN Chenfan, ZHANG Qianyi, CHEN Xiaoyun, ZHU Zhen, YIN Chunsheng, WEN Yongjun, WANG Fengxue, DU Jige. Expression and Preparation of Monoclonal Antibodies of Clostridium perfringens Alpha-Toxin Mutant [J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(1): 442-450.
[9]	GUO Chenyun, SONG Jinxing, WANG Mengxiang, SUN Junru, YAN Ruoqian, XIE Caihua, WU Yanan, ZHANG Gaiping. Prokaryotic Expression,Purification and Polyclonal Antibodies Preparation of African Swine Fever Virus B602L Protein [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(9): 3980-3989.
[10]	CHENG Jing, CHEN Peilin, GUO Yu, CUI Jinqiang, JIANG Bo, ZHOU Linyi, LIU Wenxiao, LI Huanrong, LI Yongqing. Prokaryotic Expression and Monoclonal Antibody Preparation of Bovine Promyelocytic Leukemia Protein [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(9): 4014-4024.
[11]	CUI Yanyan, WANG Guan, HAO Junfang, LI Zhiqiang, YU Fuchang, SHI Ke, XI Li, QI Meng. Prokaryotic Expression of Anaplasma phagocytophilum MSP4 Protein and Preparation of Its Polyclonal Antibodies [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(9): 4036-4042.
[12]	LI Rong, FU Guowen, ZENG Qin, WU Jiao, YUAN Jiarui, LIU Yabo, SILANG Yuzhen, FAN Yueyuan. Analysis of the Differences of Chemokine-binding Protein Gene Between Ovine Contagious Pustular Dermatitis Virus Oral and Visceral Infection Strains and Their Prokaryotic Expression [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(8): 3506-3518.
[13]	YUAN Liang, HAN Xiaoxi, GONG Yingchao, FAN Xianan, HAO Beili, HAO Shuangshuang, LI Changwen, CHANG Yicong, LI Rui, LIU Fangping. Bioinformatics Analysis and Prokaryotic Expression of SipD Protein of Salmonella Enteritidis in Chickens [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 2998-3007.
[14]	FU Mingzhi, LI Xinxin, LIU Xuewei, LI Huanrong. Prokaryotic Expression of Porcine Secretory Leukocyte Protease Inhibitor and Preparation of Its Monoclonal Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(6): 2556-2565.
[15]	XIANG Yong, LI Linlin, ZHANG Junqin, DONG Jiawen, HUANG Yunzhen, ZHAI Qi, XU Zhihong, LIAO Ming, SUN Minhua. Prokaryotic Expression of VP27 Protein of Goose Astrovirus Genotype 1 and Preparation and Identification of Its Polyclonal Antibody [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(6): 2576-2584.