流行性出血病病毒VP7蛋白原核表达及其多克隆抗体的制备

doi:10.16431/j.cnki.1671-7236.2021.04.028

摘要/Abstract

摘要： 研究旨在表达流行性出血病病毒（EHDV） VP7蛋白并制备其多克隆抗体，为EHDV检测方法的建立提供材料。试验通过大肠杆菌进行VP7重组蛋白的诱导表达，通过镍离子亲和层析与透析进行重组蛋白的纯化与复性并免疫家兔制备多克隆抗体，通过间接ELISA、Western blotting与间接免疫荧光试验（IFA）分析多克隆抗体的效价与反应原性。结果显示，在37℃与IPTG （0.1 mmol/L）的诱导下，VP7重组蛋白（分子质量46 ku）以包涵体形式高效表达，纯化与复性处理后的重组蛋白纯度达94.52%，可与不同血清型的EHDV阳性血清特异性结合。制备的兔抗VP7蛋白多克隆抗体效价达1∶24 000；以制备的多克隆抗体为一抗，通过IFA与Western blotting检测到EHDV感染细胞中VP7蛋白的表达。本研究在大肠杆菌中实现了EHDV VP7蛋白的高效表达，制备的兔抗VP7蛋白多克隆抗体具有良好的反应原性与特异性，为EHDV诊断抗原的制备与血清学检测方法的建立提供了试验材料。

关键词: 流行性出血病病毒(EHDV); VP7蛋白; 原核表达; 多克隆抗体

Abstract: To provide basis for diagnostic methods of Epidemic hemorrhagic disease virus (EHDV),VP7 protein of EHDV was expressed and polyclonal antibody against the protein was prepared in present study.Recombinant VP7 protein expressed in E.coli was purified and renatured through Ni²⁺-NTA affinity chromatography and dialysis,respectively.Specific polyclonal antibodies against the expressed VP7 protein were obtained by immunized rabbits.The titer and reactogenicity of the prepared antibodies were evaluated through indirect ELISA,Western blotting and indirect immunofluorescence assay (IFA),respectively.The results showed that VP7 recombinant protein with molecular weight at approximately 46 ku was efficiently expressed in E.coli as inclusion body when induced at 37 ℃ with the concentration of IPTG at 0.1 mmol/L.After purification and renaturation,the purity of recombinant VP7 protein was 94.52%,which could be recognized by sera of different EHDV serotypes.The titer of the prepared rabbit polyclonal antibody against EHDV VP7 was 1∶24 000 and the VP7 protein expressed in the EHDV infected cells was detected by Western blotting and IFA using the prepared antibody.In summary,VP7 protein of EHDV was successfully expressed in E.coli and rabbit polyclonal antibody against VP7 protein with robust specificity and reactivity was prepared,which provided basic materials for the development of EHDV diagnostic antigen and the establishment of serological detection method.

Key words: Epizootic haemorrhagic disease virus (EHDV); VP7 protein; prokaryotic expression; polyclonal antibody

中图分类号:

S852.65

李占鸿, 宋子昂, 肖雷, 朱建波, 杨振兴, 李卓然, 廖德芳, 李华春, 杨恒. 流行性出血病病毒VP7蛋白原核表达及其多克隆抗体的制备[J]. 中国畜牧兽医, 2021, 48(4): 1405-1413.

LI Zhanhong, SONG Ziang, XIAO Lei, ZHU Jianbo, YANG Zhenxing, LI Zhuoran, LIAO Defang, LI Huachun, YANG Heng. Prokaryotic Expression and Polyclonal Antibody Preparation of Epizootic Haemorrhagic Disease Virus VP7 Protein[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(4): 1405-1413.

参考文献

[1] SAVINI G,AFONSO A.Epizootic heamorragic disease[J].Research in Veterinary Science,2011,91(1):1-17.
[2] MACLACHLAN N J,ZIENTARA S,SAVINI G,et al.Epizootic haemorrhagic disease[J].Revue Scientifique et Technique,2015,34(2):341-351.
[3] SPEDICATO M,CARMINE I,TEODORI L,et al.Innocuity of a commercial live attenuated vaccine for Epizootic hemorrhagic disease virus serotype 2 in late-term pregnant cows[J].Vaccine,2016,34(12):1430-1435.
[4] MUTLU T E,KADIR Y,CARRIE B,et al.Epizootic hemorrhagic disease in cattle,Western Turkey[J].Emerging Infectious Diseases,2009,15(2):317-319.
[5] YADIN H,BRENNER J,BUMBROV V,et al.Epizootic haemorrhagic disease virus type 7 infection in cattle in Israel[J].Veterinary Record,2008,162(2):53-56.
[6] CORBEL M J,MACMILLAN A P.Office International des Epizooties OIE Manual of Standards for Diagnostic Tests and Vaccines Paris:Office International des Epizooties O.I.E 2008[M].Manual of Standards for Diagnostic Tests and Vaccines,1996.
[7] MAAN N S,MAAN S,POTGIETER A C,et al.Development of Real-time RT-PCR assays for detection and typing of Epizootic haemorrhagic disease virus[J].Transboundary and Emerging Diseases,2017,64(6):1120-1132.
[8] 寇美玲,杨振兴,李乐,等.云南边境地区流行性出血热病毒血清学监测及血清型鉴定[J].中国畜牧兽医,2019,49(10):3065-3074. KOU M L,YANG Z X,LI L,et al.Serological investigation and serotype identification of Epidemic hemorrhagic virus in Yunnan border area[J].China Animal Husbandry & Veterinary Medicine,2019,46(10):3065-3074.(in Chinese)
[9] 杨振兴,朱建波,肖雷,等.流行性出血病病毒抗原捕获ELISA方法的建立[J].中国兽医学报,2019,39(1):1-7. YANG Z X,ZHU J B,XIAO L,et al.Establishment of antigen-captured ELISA detecting Epizootic haemorrhagic disease virus[J].Chinese Journal of Veterinary Science,2019,39(1):1-7.(in Chinese)
[10] 杨振兴,朱建波,廖德芳,等.流行性出血病病毒一步法RT-PCR检测方法的建立及应用[J].中国兽医科学,2019,49(3):280-286. YANG Z X,ZHU J B,LIAO D F,et al.Development and application of one-step RT-PCR method for the detection of Epizootic haemorrhagic disease virus[J].Chinese Veterinary Science,2019,49(3):280-286.(in Chinese)
[11] 杨振兴,朱建波,李占鸿,等.蓝舌病病毒和流行性出血病病毒双重荧光定量RT-PCR检测方法的建立及应用[J].中国兽医科学,2019,49(9):1104-1111. YANG Z X,ZHU J B,LI Z H,et al.Establishment and application of a duplex Real-time RT-PCR assay for simultaneous detection of Bluetongue virus and Epidemic haemorrhagic disease virus[J].Chinese Veterinary Science 2019,49(9):1104-1111.(in Chinese)
[12] 杨振兴,李占鸿,宋子昂,等.流行性出血病病毒血清分型RT-PCR方法的建立及应用[J].华中农业大学学报,2020,39(4):85-92. YANG Z X,LI Z H,SONG Z A,et al.Development and utilization of RT-PCR assay for determining serotypes of Epidemic hemorrhagic disease virus[J].Journal of Huazhong Agricultural University,2020,39(4):85-92.(in Chinese)
[13] 朱建波,杨振兴,肖雷,等.流行性出血病多克隆抗体C-ELISA检测方法的建立[J].畜牧兽医学报,2018,49(7):1140-1150. ZHU J B,YANG Z X,XIAO L,et al.Establishment of rapid competitive ELISA for detecting antibodies against Epidemic hemorrhagic disease virus[J].Chinese Journal of Animal and Veterinary Sciences,2018,49(7):1140-1150.(in Chinese)
[14] FAUQUET C M,MAYO M A,MANILOFF J,et al.Virus Taxonomy.Eighth Report of the International Committee on Taxonomy of Viruses[M].London:Elsevier/Academic Press,2005.
[15] ANTHONY S J,MAAN N S.Genetic and phylogenetic analysis of the core proteins VP1,VP3,VP4,VP6 and VP7 of Epizootic haemorrhagic disease virus (EHDV)[J].Virus Research,2009,145(2):187-199.
[16] FORZAN M,PIZZURRO F,ZACCARIA G,et al.Competitive enzyme-linked immunosorbent assay using baculovirus-expressed VP7 for detection of Epizootic haemorrhagic disease virus (EHDV) antibodies[J].Journal of Virological Methods,2017,284(27):212-216.
[17] MECHAM J O,WILSON W C.Antigen capture competitive enzyme-linked immunosorbent assays using baculovirus-expressed antigens for diagnosis of Bluetongue virus and Epizootic hemorrhagic disease virus[J].Journal of Clinical Microbiology,2004,42(2):518-523.
[18] LUO L Z,SABARA M I.Production of a recombinant major inner capsid protein for serological detection of Epizootic hemorrhagic disease virus[J].Clinical and Diagnostic Laboratory Immunology,2005,12(8):904-909.
[19] 杨文静,李玉鹏,梁冬梅,等.猪NLRP6蛋白的原核表达、纯化及其多克隆抗体的制备[J].中国畜牧兽医,2019,46(9):2707-2714. YANG W J,LI Y P,LIANG D M,et al.Prokaryotic expression,purification and polyclonal antibody preparation of porcine NLRP6 protein[J].China Animal Husbandry & Veterinary Medicine,2019,46(9):2707-2714.(in Chinese)
[20] 杨俊兴,花群义,陈焕春,等.鹿流行性出血病病毒VP7基因的克隆及原核表达[J].动物医学进展,2009,30(8):5-8. YANG J X,HUA Q Y,CHEN H C,et al.Cloning and prokaryotic expression of VP7 gene of Epidemic hemorrhagic disease virus of deer in E.coli[J].Progress in Veterinary Medicine,2009,30(8):5-8.(in Chinese)
[21] REDDY A,TIWARI A,KATARIS R,et al.Expression of major core protein gene VP7 of Bluetongue virus serotype 23[J].Journal of Immuno-logy and Immunopathology,2006,8(2):29-35.
[22] WU X,LIU Q,HE J,et al.Preparation and characterization of a monoclonal antibody against the core protein VP7 of the 25th serotype of Bluetongue virus[J].Monoclonal Antibodies in Immunodiagnosis and Immunotherapy,2015,34(2):116-121.
[23] IAFOLLA M A,MAZUMDER M,SARDANA V,et al.Dark proteins:Effect of inclusion body formation on quantification of protein expression[J].Proteins,2008,72(4):1233-1242.
[24] WINGFIELD P T,PALMER I,LIANG S M.Folding and purification of insoluble (inclusion body) proteins from Escherichia coli[J].Current Protocols in Protein Science,2014,78:6.5.1-6.5.30.