猪轮状病毒VP6蛋白的真核表达及单克隆抗体制备和应用

doi:10.16431/j.cnki.1671-7236.2025.06.027

Abstract

Abstract: 【Objective】 The aim of this experiment was to prepare monoclonal antibody against Porcine rotavirus (PoRV) VP6 protein,and provide reference for the development of PoRV detection method.【Method】 VP6 gene was amplified with Vaccinia virus promoter P28 as the promoter of VP6 and G3 type PoRV nucleic acid as the template.VP6 gene fragment was cloned into pSWE178 plasmid by In-Fusion cloning method,and the recombinant plasmid pSWE178-P28-VP6 was constructed.Recombinant Suipoxvirus rSWE178-P28-VP6 expressing VP6 protein was obtained by homologous recombination and plaque purification techniques.The expression of VP6 protein was identified by SDS-PAGE.BALB/c mice were immunized with G9 PoRV as the immunogener.Spleen cells of immunized mice were fused with SP2/0 myeloma cells.VP6 protein expressed by recombinant virus was used as the detection antigen,and hybridoma cells secreting anti-VP6 monoclonal antibody were screened by indirect ELISA.Indirect immunofluorescence assay (IFA) was used to detect the reactivity of monoclonal antibody with G3,G4,G5 and G9 PoRV.One of the monoclonal antibody strains was selected for immunohistochemical (IHC) detection of porcine intestinal tissue artificially infected with PoRV.【Result】 On the basis of constructing the recombinant plasmid pSWE178-P28-VP6 expressing the PoRV VP6 protein,a recombinant Swinepox virus expressing the PoRV VP6 protein was successfully generated.The molecular weight of the expressed protein was 45 ku and which was soluble protein.Monoclonal antibody was prepared by hybridoma technique,and 29 hybridoma cell lines secreting anti-VP6 monoclonal antibody were screened.IFA results showed that 26 monoclonal antibodies could react with G3,G4,G5 and G9 PoRV,but the fluorescence brightness was different.IHC results showed that monoclonal antibody could produce specific immune response with PoRV-infected clinical samples.【Conclusion】 In this study,PoRV VP6 protein was successfully expressed by eukaryotic expression system.The hybridoma technique was used to select 29 strains of anti-VP6 monoclonal antibodies,of which 26 strains could react with G3,G4,G5 and G9 PoRV.This results laid a foundation for the establishment of PoRV immunological detection method.

Key words: Porcine rotavirus (PoRV); VP6 protein; eukaryotic expression; monoclonal antibody

CLC Number:

S858.292

WEI Huangsiwu, ZHANG Xingyi, HUANG Xiaohua, LIU Changjin, WU Wenjie, SHEN Zhengqiao, LUO Feng, DENG Shunzhou. Eukaryotic Expression of Porcine Rotavirus VP6 Protein and Preparation and Application of Its Monoclonal Antibody[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(6): 2750-2761.

References

[1] 余俊杰,刘夏飞,李杉,等.完全基于质粒的轮状病毒反向遗传技术进展[J].病毒学报,2024,40(4):897-906.YU J J,LIU X F,LI S,et al.Advances in plasmid-based reverse genetics of Rotavirus[J].Chinese Journal of Virology,2024,40(4):897-906.(in Chinese)
[2] 赵少若,黄甜,郝丽影,等.犬轮状病毒单克隆抗体制备及胶体金试纸条检测方法的建立[J].中国畜牧兽医,2022,49(11):4429-4437.ZHAO S R,HUANG T,HAO L Y,et al.Preparation of monoclonal antibody against Canine rotavirus and development of colloidal gold strip detection method[J].China Animal Husbandry & Veterinary Medicine,2022,49(11):4429-4437.(in Chinese)
[3] 刘小兰,刘昌锦,余文洋,等.猪轮状病毒江西株AY01的分离鉴定[J].中国畜牧兽医,2022,49(8):3151-3162.LIU X L,LIU C J,YU W Y,et al.Isolation and identification of Porcine rotavirus Jiangxi strain AY01[J].China Animal Husbandry & Veterinary Medicine,2022,49(8):3151-3162..(in Chinese)
[4] 李平.绵羊源G6P[1]型轮状病毒的分离鉴定及其对乳鼠致病性研究[D].阿拉尔：塔里木大学,2024.LI P.Isolation and characterization of Rotavirus type of G6P[1] of sheep origin and its pathogenicity to suckling mice[D].Alaer：Tarim University,2024.(in Chinese)
[5] 李昌文,关云涛,刘怀然,等.兔轮状病毒的分离与鉴定[J].中国比较医学杂志,2003,13(6):3.LI C W,GUAN Y T,LIU H R,et al.Isolation and identification of Rabbit rotavirus[J].Chinese Journal of Comparative Medicine,2003,13(6):3.(in Chinese)
[6] 刁有祥,冯涛,崔治中,等.鸡轮状病毒的分离鉴定及特性研究[J].畜牧兽医学报,2004,35(4):5.DIAO Y X,FENG T,CUI Z Z,et al.Isolation,identification and characterization of Chicken rotavirus[J].Acta Veterinaria et Zootechnica Sinica,2004,35(4):5.(in Chinese)
[7] 常继涛,崔久辉,李建军,等.我国部分地区牛轮状病毒的病原学调查及一株G10P
[11]型牛轮状病毒的分离鉴定[J].中国预防兽医学报,2008，10:755-759.CHANG J T,CUI J H,LI J J,et al.Etiological investigation of Bovine rotavirus in the some regions of China and isolation of a G10P
[11] Rotavirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2008，10:755-759.(in Chinese)
[8] 肖莉,牛小杰,刘庆庆,等.猪轮状病毒VP4基因重组腺病毒的构建及抗体水平评价[J].中国畜牧兽医,2023,50(1):260-269.XIAO L,NIU X J,LIU Q Q,et al.Construction of recombinant Adenovirus with VP4 gene of Porcine rotavirus and evaluation of antibody level[J].China Animal Husbandry & Veterinary Medicine，2023,50(1):260-269.(in Chinese)
[9] TAO R,CHANG X,ZHOU J,et al.Molecular epidemiological investigation of group A Porcine rotavirus in East China[J].Frontiers in Veterinary Science,2023,10:1138419.
[10] VLASOVA A N,AMIMO J O,SAIF L J.Porcine rotaviruses:Epidemiology,immune responses and control strategies[J].Viruses,2017,9(3):48.
[11] YAN W,HUANG S,ZHANG L,et al.Virus-like particles vaccine based on co-expression of G5 Porcine rotavirus VP2-VP6-VP7 induces a powerful immune protective response in mice[J].Veterinary Microbiology,2024,298:110241.
[12] AYALA-BRETON C,ARIAS M,ESPINOSA R,et al.Analysis of the kinetics of transcription and replication of the Rotavirus genome by RNA interference[J].Journal of Virology,2009,83(17):8819-8831.
[13] 刘昌锦,邓舜洲,罗锋,等.猪痘病毒TK、ORF121、ORF143基因缺失毒株的构建及其生物学特性的测定[J].中国畜牧兽医,2020,47(3):828-836.LIU C J,DENG S Z,LUO F,et al.Construction of Swinepox virus TK,ORF121 and ORF143 gene deletion strains and determination of their biological characteristics[J].China Animal Husbandry & Veterinary Medicine,2020,47(3):828-836.(in Chinese)
[14] 胡换仪,汪建中,刘昌锦,等.绿色荧光蛋白的真核表达及其单克隆抗体制备[J].中国畜牧兽医,2022,49(1):328-337.HU H Y,WANG J Z,LIU C J,et al.Eukaryotic expression of green fluorescent protein and preparation of its monoclonal antibody[J]. China Animal Husbandry & Veterinary Medicine,2022,49(1):328-337.(in Chinese)
[15] 张洪英,高艳,刘立奎,等.轮状病毒研究进展[J].畜牧兽医科技信息,2007，6:4-6.ZHANG H Y,GAO Y,LIU L K,et al.Research progress of Rotavirus[J].Chinese Journal of Animal Husbandry and Veterinary Medicine，2007，6:4-6.(in Chinese)
[16] 张怡婷,王洁清,张冠群,等.牛轮状病毒VP6蛋白间接ELISA检测方法的建立[J].中国兽医科学,2010,40(10):1039-1043.ZHANG Y T,WANG J Q,ZHANG G Q,et al.Development of an indirect ELISA for detection of Bovine rotavirus VP6 protein[J].Chinese Veterinary Science, 2010,40(10):1039-1043.(in Chinese)
[17] 原霖,张瑜,刘洋,等.A群猪轮状病毒VP6结构蛋白在杆状病毒系统中的表达及鉴定[J].中国预防兽医学报,2018,40(6):476-480.YUAN L,ZHANG Y,LIU Y,et al.Expression and identification of recombinant structural protein VP6 of Porcine rotavirus group A in Baculovirus expression system[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(6):476-480.(in Chinese)
[18] 陈淑华,库旭钢,闫贵伟,等.猪A群轮状病毒VP6的表达及IgG抗体ELISA检测方法的建立与初步应用[J].动物医学进展,2014,35(7):1-6.CHEN S H,KU X G,YAN G W,et al.Expression of VP6 and development of an ELISA for detection of IgG antibody against Porcine rotavirus group A[J].Progress in Veterinary Medicine,2014,35(7):1-6.(in Chinese)
[19] 王奕斐,洪大林,米雪,等.猪轮状病毒VP4、VP6蛋白原核表达及多克隆抗体的制备[J].中国兽医学报,2022,42(11):2171-2180.WANG Y F,HONG D L,MI X,et al.Prokaryotic expression of Porcine rotavirus VP4 and VP6 proteins and preparation of polyclonal antibodies[J].Chinese Journal of Veterinary Science,2022,42(11):2171-2180.(in Chinese)
[20] DA SILVA JUNIOR H C,DA SILVA E MOUTA JUNIOR S,DE MENDONÇA M C,et al.Comparison of two eukaryotic systems for the expression of VP6 protein of Rotavirus specie A:Transient gene expression in HEK293-T cells and insect cell-Baculovirus system[J].Biotechnology Letters,2012,34(9):1623-1627.
[21] 柳佳佳,梁海英,曾智勇,等.猪轮状病毒TaqMan实时荧光定量RT-PCR检测方法的建立与应用[J].中国兽医科学,2024,54(2):162-168.LIU J J,LIANG H Y,ZENG Z Y,et al.Establishment and application of TaqMan Real-time fluorescence quantitative RT-PCR for detection of Porcine rotavirus[J].Chinese Veterinary Science,2024,54(2):162-168.(in Chinese)
[22] 李木子,刘佳丽,王瑞翀,等.牛轮状病毒VP6蛋白单克隆抗体的制备及双抗体夹心ELISA方法的建立[J].中国兽医科学,2018,48(11):1352-1357.LI M Z,LIU J L,WANG R C,et al.Preparation of monoclonal antibodies against VP6 protein of Bovine rotavirus and establishment of double antibody sandwich ELISA[J].Chinese Veterinary Science,2018,48(11):1352-1357.(in Chinese)
[23] 林玥延.猫泛白细胞减少症病毒VP2的原核表达及单克隆抗体制备[D].武汉：华中农业大学,2023.LIN Y Y.Prokaryotic expression of Feline panleukopenia virus VP2 gene and its mAbs preparation[D].Wuhan：Huazhong Agricultural University,2023.(in Chinese)
[24] 张艺艺.豫西南地区猪轮状病毒的分离鉴定及单克隆抗体的制备[D].南阳：南阳师范学院,2023.ZHANG Y Y.Isolation and identification of Porcine rotavirus and generation of monoclonal antibodies in Southwestern Henan[D].Nanyang：Nanyang Normal University,2023.(in Chinese)
[25] 李妍花,周兵强,康笃利,等.猪轮状病毒胶体金检测试纸条的研制及应用[J].中国畜牧兽医,2024,51(3):1223-1231.LI Y H,ZHOU B Q,KANG D L,et al.Development and application of colloidal gold test strip for Porcine rotavirus[J]. China Animal Husbandry & Veterinary Medicine, 2024,51(3):1223-1231.(in Chinese)
[26] 赵颖.不同G型轮状病毒VP7蛋白表达及抗体制备和应用[D].兰州：甘肃中医药大学,2024.ZHAO Y.The expression of VP7 proteins of different G-genotype Rotaviruses and the generation and application of VP7 antibodies[D].Lanzhou：Gansu University of Chinese Medicine,2023.(in Chinese)

Eukaryotic Expression of Porcine Rotavirus VP6 Protein and Preparation and Application of Its Monoclonal Antibody

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