[1]吴植,卢会鹏,王安平,等.非洲猪瘟病毒无标签p35蛋白的制备及间接ELISA抗体检测方法的建立[J].江苏农业学报,2023,(05):1209-1216.[doi:doi:10.3969/j.issn.1000-4440.2023.05.013]
　WU Zhi,LU Hui-peng,WANG An-ping,et al.Preparation of tag-free p35 of African swine fever virus and development of an indirect enzyme linked immunosorbent assay (ELISA) antibody detection method[J].,2023,(05):1209-1216.[doi:doi:10.3969/j.issn.1000-4440.2023.05.013]
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非洲猪瘟病毒无标签p35蛋白的制备及间接ELISA抗体检测方法的建立()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2023年05期
页码:
1209-1216
栏目:
畜牧兽医·水产养殖
出版日期:
2023-08-31

文章信息/Info

Title:
Preparation of tag-free p35 of African swine fever virus and development of an indirect enzyme linked immunosorbent assay (ELISA) antibody detection method
作者:
吴植; 卢会鹏; 王安平; 谢军; 曹世诺; 徐艳; 朱善元
（江苏农牧科技职业学院/江苏省兽用生物制药高技术研究重点实验室/江苏现代畜牧与新兽药工程技术中心，江苏泰州225300）
Author(s):
WU Zhi; LU Hui-peng; WANG An-ping; XIE Jun; CAO Shi-nuo; XU Yan; ZHU Shan-yuan
（Jiangsu Agri-Animal Husbandry Vocational College/Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals/Engineering Technology Research Center for Modern Animal Science and Novel Veterinary Pharmaceutic Development, Taizhou 225300, China）
关键词:
非洲猪瘟; 类弹性蛋白; 无标签p35蛋白; 间接ELISA方法
Keywords:
African swine fever; elastin-like polypeptide; tag-free p35 protein; indirect ELISA
分类号:
S852.65+1
DOI:
doi:10.3969/j.issn.1000-4440.2023.05.013
文献标志码:
A
摘要:
为了进一步提高非洲猪瘟病毒（ASFV）间接酶联免疫吸附试验（ELISA）抗体检测方法的特异性，本研究构建了类弹性蛋白（ELP）标签与ASFV p35融合表达载体，利用相变循环分离纯化融合蛋白后，用烟草蚀纹病毒蛋白酶（TEVP）切除ELP标签，制备获得无标签p35蛋白，以此为包被抗原，通过一系列的条件摸索和优化，建立ASFV间接ELISA抗体检测方法。结果显示，ELP-p35融合蛋白的相对分子质量大小约为80 000；制备获得的无标签p35蛋白能够被非洲猪瘟阳性血清所识别；抗原包被最佳质量浓度为2.00 μg/ml，待检血清最佳稀释比例为1∶200，二抗最佳稀释比例为1∶10 000，阴性和阳性判定阈值OD450为0.171；与口蹄疫病毒（FMDV）、猪圆环病毒2型（PCV2）、猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）和伪狂犬病毒（PRV）等阳性血清无交叉反应，特异性好；批内、批间试验结果显示变异系数都小于5.000%，重复性好；可检测400倍稀释的血清，敏感性较高；与商品化检测试剂盒（ING）检测结果的符合率高达100%。结果表明，用本研究制备的ASFV无标签p35蛋白作为包被抗原建立的间接ELISA方法可用于ASFV抗体的检测，为该病的进一步精准检测提供了技术手段。
Abstract:
To further improve the specificity of indirect enzyme linked immunosorbent assay (ELISA) method for detection of African swine fever virus (ASFV) antibody, fusion expression vector containing elastin-like polypeptide (ELP) tag and ASFV p35 was constructed. After separating and purifying the fusion protein by inverse transition cycling, the tag-free p35 protein was obtained by cutting off the ELP tag by tobacco etch virus protease (TEVP). Using the purified recombinant protein as coating-antigen, the study aimed to establish an indirect ELISA method for detecting ASFV antibody through a series of condition exploration and purification. The results showed that, the relative molecular mass of ELP-p35 fusion protein was 80 000, and the obtained tag-free p35 protein could be recognized by positive serum of ASFV. It was found that the optimum antigen coating mass concentration was 2.00 μg/ml, the optimum dilution rate for sera to be tested was 1∶200, the optimum dilution rate for HRP-IgG was 1∶10 000, and the cut-off value of OD450 was 0.171. The method showed no cross-reaction with positive sera of foot-and-mouth disease virus (FMDV), porcine circovirus type 2 (PCV2), porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV) and pseudorabies virus (PRV), which showed high specificity. Coefficients of variation of the intra- and inter-assay were both <5.000% and showed good repeatability. Besides, the method could detect serum diluted 400 times, which showed high sensitivity. Compared with commercialized detection kits (ING), the coincidence rate of testing results of tag-free p35-ELISA method was 100%. The results indicated that the established indirect ELISA method by using tag-free p35 protein as coating antigen of ASFV can be applied in detection of ASFV-specific antibodies, which can provide technological tool for accurate detection of ASFV.

参考文献/References:

[1]ALONSO C, BORCA M, DIXON L, et al. ICTV virus taxonomy profile: asfarviridae[J]. Journal Of General Virology, 2018,99(5):613-614.
[2]DIXON L K, SUN H, ROBERTS H. African swine fever[J]. Antiviral Research,2019,165:34-41.
[3]GE S Q, LI J M, FAN X F, et al. Molecular characterization of African swine fever virus, China[J]. Emerging Infectious Diseases,2018,24(11):2131-2133.
[4]ZHOU X, LI N, LUO Y, et al. Emergence of African swine fever in China[J]. Transboundary and Emerging Diseases,2018,65(6):1482-1484.
[5]DE VILLIERS E P, GALLARDO C, ARIAS M, et al. Phylogenomic analysis of 11 complete African swine fever virus genome sequences[J]. Virology,2010,400(1):128-136.
[6]DIXON L K, CHAPMAN D A, NETHERTON C L, et al.African swine fever virus replication and genomics[J]. Virus Research,2013,173(1):3-14.
[7]ALEJO A, MATAMOROS T, GUERRA M, et al. A proteomic atlas of the African swine fever virus particle[J]. Journal of Virology,2018,92(23): e01293.
[8]EULLIO A, NUNES-CORREIA I, SALAS J, et al. African swine fever virus p37 structural protein is localized in nuclear foci containing the viral DNA at early post-infection times[J]. Virus Research,2007,130(1/2):18-27.
[9]DIXON L K, ABRAMS C C, BOWICK G, et al. African swine fever virus proteins involved in evading host defence systems[J]. Veterinary Immunology and Immunopathology,2004,100(3/4):117-134.
[10]WANG G G, XIE M J, WU W,et al. Structures and functional diversities of ASFV proteins[J]. Viruses,2021,13(11):2124.
[11]QUINTERO J C, WESLEY R D, WHYARD T C, et al. In vitro and in vivo association of African swine fever virus with swine erythrocytes[J].American Journal of Veterinary Research,1986,47(5):1125-1131.
[12]FRANZONI G, GRAHAM S P, GIUDICI S D, et al. Characterization of the interaction of African swine fever virus with monocytes and derived macrophage subsets[J].Veterinary Microbiology,2017,198:88-98.
[13]郭怡德，李冰，蔡汝健，等.非洲猪瘟病毒蛋白功能研究进展[J].动物医学进展,2020,41(10):96-101.
[14]储珺，赵普，张婕妮，等.非洲猪瘟病毒pp62蛋白单克隆抗体的制备和鉴定[J].中国兽医科学,2022,52(5):579-585.
[15]欧云文，刘俐君，贾宁，等.非结构蛋白在非洲猪瘟病毒感染中作用[J].病毒学报, 2021,37(4):910-921.
[16]李景生，徐军，刘陶莹，等.类弹性蛋白多肽及其应用研究进展[J].生物技术世界,2015(6):16-17.
[17]LIU W J, WU Q, XU B, et al. Single-step purification of recombinant proteins using elastin-like peptide-mediated inverse transition cycling and self-processing module from Neisseria meningitides FrpC[J]. Protein Expression And Purification,2014,98:18-24.
[18]LI G Y, XIAO Z Z, LU H P, et al. A simple method for recombinant protein purification using self-assembling peptide-tagged tobacco etch virus protease[J].Protein Expression and Purification,2016,128:86-92.
[19]肖景景，卢会鹏，李洋洋，等.非洲猪瘟病毒无标签重组K205R抗原制备与应用[J].扬州大学学报(农业与生命科学版),2019,40(3):90-94,100.
[20]ZHONG K, ZHU M, YUAN Q, et al. Development of an indirect ELISA to detect African swine fever virus pp62 protein-specific antibodies[J].Frontiers in Veterinary Science,2022,8:798559.
[21]吴竞，王西西，吴映彤，等.非洲猪瘟病毒p30基因的原核表达及间接ELISA抗体检测方法的建立[J].中国畜牧兽医, 2018, 45(12):3555-3562.
[22]庚辛，孙明霞，王淑杰,等.非洲猪瘟病毒p72蛋白间接ELISA抗体检测方法的建立[J].中国预防兽医学报, 2021, 43（9）:946-951.
[23]施磊，田占成，杨吉飞，等.非洲猪瘟病毒p35蛋白作为诊断抗原的抗原性比较[J].生物工程学报,2021,37(1):187-195.
[24]于学祥,陈晓雨,李栋凡,等.非洲猪瘟病毒无标签p30-ELISA抗体检测方法的建立及应用[J].畜牧兽医学报, 2022，53(5):1517-1526.

备注/Memo

备注/Memo:
收稿日期：2022-10-12 基金项目：泰州市科技支撑计划（农业）项目（TN202001）；江苏省重点研发计划（现代农业）重点项目（BE2020407）；科技部“科技助力经济2020”重点专项（SQ2020YFF0419717）作者简介：吴植（1980-），男，江苏盐城人，硕士，副教授，主要从事畜禽疫病防控技术研究。（Tel）13951163801；（E-mail）yzwuzhi@163.com 通讯作者：朱善元，（E-mail）jstzzsy@126.com
更新日期/Last Update: 2023-09-13