检测猪流行性腹泻病毒S1蛋白抗体的间接ELISA方法

doi:10.11843/j.issn.0366-6964.2017.06.013

摘要/Abstract

摘要：

为了建立检测猪流行性腹泻病毒（porcine epidemic diarrhea virus，PEDV）抗体的间接ELISA方法，初步分析PED灭活疫苗免疫母猪所产仔猪的血清母源特异抗体动态，笔者应用重组PEDV部分纤突（spike，S）蛋白作为检测抗原，建立了基于PEDV S1蛋白的间接ELISA抗体检测方法，并用该方法检测了产前4周用PED灭活疫苗免疫母猪的初乳、分娩当天血清及其仔猪1、7、14、21、28和35日龄（d）时血清中的PEDV抗体。结果显示：建立的间接ELISA方法能够特异地检测PEDV抗体，与猪瘟病毒、猪圆环病毒2型、猪繁殖与呼吸综合征病毒、伪狂犬病病毒、猪传染性胃肠炎病毒及猪轮状病毒的抗血清无交叉反应；敏感性高于免疫过氧化物酶单层试验（immunoperoxidase monolayer assay，IPMA），与IPMA检测结果的符合率为97.14%；批内和批间重复性试验的变异系数小于10%。免疫母猪初乳、分娩当天血清中的PEDV抗体阳性率为100%，初乳比血清中的平均抗体水平低；1 d仔猪血清PEDV抗体阳性率为50%，平均抗体水平低，与初乳中的抗体水平接近，7 d以后所有仔猪血清全部转为PEDV抗体阴性。建立了检测PEDV抗体的间接ELISA方法；产前用PEDV灭活疫苗给怀孕母猪免疫1次时，其所产仔猪血清中的母源特异抗体阳性率和抗体水平低，且维持时间短暂。

关键词: 猪流行性腹泻病毒, 重组PEDV S1蛋白, 间接ELISA, 初生仔猪, 母源抗体

Abstract:

The present study aimed to develop an indirect ELISA for detecting antibody to porcine epidemic diarrhea virus (PEDV) spike (S) protein and analyze the dynamic of maternal special antibodies in the serum from neonatal piglets born by sows immunized with PEDV inactivated vaccine. An indirect ELISA was established using recombinant partial PEDV spike (S1) protein as detecting antigen. And anti-PEDV antibodies in the immunized sows' colostrum, sera from sows at the delivery day and from the neonatal piglets at 1, 7, 14, 21, 28 and 35 days were detected with the established ELISA. Results suggested that the ELISA method could be used to detect the special antibodies against PEDV without cross reactions with those antibodies to classical swine fever virus, porcine circovirus type 2, porcine reproductive and respiratory syndrome virus, pseudorabies virus, porcine transmissible gastroenteritis virus, and porcine rotavirus. The sensitivity of ELISA was higher than that of the immunoperoxidase monolayer assay (IPMA) and the coincidence rate was 97.14% when the same serum samples were detected using the ELISA and IPMA. The coefficients of variation (CV) of repeated in-batch and batch-to-batch tests of ELISA were less than 10%. Positive rate of anti-PEDV antibody in the colostrum and serum of all immunized sows was 100% with a higher average antibody level at the delivery day in the serum than that in the colostrum, and the specific antibody positive rate of one-day old neonatal piglets was 50% with a lower average antibody level in serum similar to that in the colostrum. And anti-PEDV antibodies in all of the piglet serum turned negative after day 7. The present study shows that PEDV antibodies could be detected using the established ELISA. The low positive rate and levels of maternal special antibodies are present in the serum of neonatal piglets born by the sows immunized once with PEDV inactivated vaccine before farrowing.

Key words: porcine epidemic diarrhea virus, recombinant PEDV S1 protein, indirect ELISA, neonatal piglets, maternal antibody

中图分类号:

S852.659.6

姚作俊, 郝达仁, 白云, 颜国华, 王海敏, 宋勤叶, 李潭清. 检测猪流行性腹泻病毒S1蛋白抗体的间接ELISA方法[J]. 畜牧兽医学报, 2017, 48(6): 1085-1091.

YAO Zuo-jun, HAO Da-ren, BAI Yun, YAN Guo-hua, WANG Hai-min, SONG Qin-ye, LI Tan-qing. Indirect ELISA for Detecting Antibody to Porcine Epidemic Diarrhea Virus S1 Protein[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1085-1091.

参考文献

[1] PENSAERT M B, DE BOUCK P. A new coronavirus-like particle associated with diarrhea in swine[J]. Arch Virol, 1978, 58(3):243-247.
[2] LI W T, LI H, LIU Y B, et al. New variants of porcine epidemic diarrhea virus, China, 2011[J]. Emerg Infect Dis, 2012, 18(8):1350-1353.
[3] PAN Y F, TIAN X Y, LI W, et al. Isolation and characterization of a variant porcine epidemic diarrhea virus in China[J]. Virol J, 2012, 9:195.
[4] WOOD E N. An apparently new syndrome of porcine epidemic diarrhoea[J]. Vet Rec, 1977, 100(12):243-244.
[5] KUBOTA S, SASAKI O, AMIMOTO K, et al. Detection of porcine epidemic diarrhea virus using polymerase chain reaction and comparison of the nucleocapsid protein genes among strains of the virus[J]. J Vet Med Sci, 1999, 61(7):827-830.
[6] 宣华, 邢德坤, 王殿瀛, 等. 应用猪胎肠单层细胞培养猪流行性腹泻病毒的研究[J]. 中国兽医学报, 1984(3):202-208.
XUAN H, XING D K, WANG D Y, et al. Study on a culture of porcine epidemic diarrhea virus using fetal porcine intestinal epithelial cell[J]. Chinese Journal of Veterinary Science, 1984(3):202-208. (in Chinese)
[7] SAIF L J, PENSAERT M B, SESTACK K, et al. Coronaviruses[M]//STRAW B E, ZIMMERMAN J J, KARRIKER L A, et al. Diseases of Swine. 10th ed. Ames:Wiley-Blackwell, 2012:501-524.
[8] CAVANAGH D. Nidovirales:a new order comprising Coronaviridae and Arteriviridae[J]. Arch Virol, 1997, 142(3):629-633.
[9] KOCHERHANS R, BRIDGEN A, ACKERMANN M, et al. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence[J]. Virus Genes, 2001, 23(2):137-144.
[10] SONG D, PARK B. Porcine epidemic diarrhoea virus:a comprehensive review of molecular epidemiology, diagnosis, and vaccines[J]. Virus Genes, 2012, 44(2):167-175.
[11] WU X D, SHANG B, YANG R F, et al. The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells[J]. Cell Res, 2004, 14(5):400-406.
[12] DE HAAN C A M, TE LINTELO E, LI Z, et al. Cooperative involvement of the S1 and S2 subunits of the murine coronavirus spike protein in receptor binding and extended host range[J]. J Virol, 2006, 80(22):10909-10918.
[13] SUN D B, FENG L, SHI H Y, et al. Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein[J]. Vet Microbiol, 2008, 131(1-2):73-81.
[14] 丁振江, 库旭钢, 闫贵伟, 等. 猪流行性腹泻IgA抗体间接ELISA检测方法的建立[J]. 畜牧与兽医, 2014, 46(11):1-5.
DING Z J, KU X G, YAN G W, et al. Development of an indirect ELISA method for detection of IgA antibody against porcine epidemic diarrhea virus[J]. Animal Husbandry & Veterinary Medicine, 2014, 46(11):1-5. (in Chinese)
[15] 华耀, 王玮, 李郁, 等. 猪流行性腹泻病毒S蛋白主要抗原表位区的原核表达及间接ELISA检测方法的建立[J]. 微生物学通报, 2016, 43(2):434-443.
HUA Y, WANG W, LI Y, et al. Prokaryotic expression of the structural protein S of porcine epidemic diarrhea virus and establishment of an indirect ELISA for an detection of antibody against porcine epidemic diarrhea[J]. Microbiology China, 2016, 43(2):434-443. (in Chinese)
[16] DE ARRIBA M L, CARVAJAL A, POZO J, et al. Mucosal and systemic isotype-specific antibody responses and protection in conventional pigs exposed to virulent or attenuated porcine epidemic diarrhoea virus[J]. Vet Immunol Immunopathol, 2002, 85(1-2):85-97.
[17] LADEKJAER-MIKKELSEN A S, NIELSEN J, STADEJEK T, et al. Reproduction of postweaning multisystemic wasting syndrome (PMWS) in immunostimulated and non-immunostimulated 3-week-old piglets experimentally infected with porcine circovirus type 2 (PCV2)[J]. Vet Microbiol, 2002, 89(2-3):97-114.
[18] 时晓丽, 谈晨, 赵攀登, 等. 猪流行性腹泻病毒抗原捕获ELISA抗体检测方法的建立与应用[J]. 中国兽医科学, 2014, 44(12):1262-1267.
SHI X L, TAN C, ZHAO P D, et al. Development and application of an antigen capture ELISA for detection of antibodies against porcine epidemic diarrhea virus[J]. Chinese Veterinary Science, 2014, 44(12):1262-1267. (in Chinese)
[19] 张博, 李守军, 杨保收. PEDV流行株重组截短N蛋白间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2016, 43(6):1446-1452.
ZHANG B, LI S J, YANG B S. Establishment of indirect ELISA method for detecting recombinant truncated N protein of porcine epidemic diarrhea virus[J]. China Animal Husbandry & Veterinary Medicine, 2016, 43(6):1446-1452. (in Chinese)
[20] 李任峰,田香勤,乔松林,等. 仔猪回肠绒毛M细胞的形态、分布及其与猪流行性腹泻病毒感染的关系[J]. 畜牧兽医学报, 2016, 47(3):581-586.
LI R F,TIAN X Q,QIAO S L,et al. Morphological and distribution characteristics of villous M cells in the ileum of newborn piglets and the association with PEDV infection[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(3):581-586. (in Chinese)
[21] SONG Q Y, STONE S, DREBES D, et al. Characterization of anti-porcine epidemic diarrhea virus neutralizing activity in mammary secretions[J]. Virus Res, 2016, 226:85-92.
[22] BAEK P S, CHOI H W, LEE S, et al. Efficacy of an inactivated genotype 2b porcine epidemic diarrhea virus vaccine in neonatal piglets[J]. Vet Immunol Immunopathol, 2016, 174:45-49.
[23] OUYANG K, SHYU D L, DHAKAL S, et al. Evaluation of humoral immune status in porcine epidemic diarrhea virus (PEDV) infected sows under field conditions[J]. Vet Res, 2015, 46:140.
[24] PAUDEL S, PARK J E, JANG H, et al. Evaluation of antibody response of killed and live vaccines against porcine epidemic diarrhea virus in a field study[J]. Vet Q, 2014, 34(4):194-200.