Truncated Expression of Porcine deltacoronavirus M Protein and Establishment of Indirect ELISA
SONG Dai-Li1, YANG Fu-Sheng1, CHEN Yu-Qiao1, SUN Fan-Yuan1, CHEN Rui1, CAO San-Jie1,2,3, HUANG Xiao-Bo1,2,3,*
1 Research Center of Swine Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang 611130, China; 2 Sichuan Science-observation Experimental station of Veterinary Drugs and Veterinary Diagnostic Technology, Ministry of Agriculture and Rural affairs, Wenjiang 611130, China; 3 National Teaching and Experiment Center of Animal, Sichuan Agricultural University, Wenjiang 611130, China
Abstract：Porcine deltacoronavirus (PDCoV) is a new swine (Sus scrofa) enteropathogenic coronavirus, which can cause severe dehydration, vomiting and watery diarrhea in piglets, with worldwide distribution. In order to develop an indirect ELISA method for rapid detection of PDCoV antibody, based on the bioinformatics analysis of PDCoV M gene sequence, the M gene located at 76~651 bp was amplified and inserted into the expression vector pET-32a to construct the recombinant plasmid pET-32a-M. The pET-32a-M was transformed into Escherichia coil Transetta (DE3) and the recombinant M protein was expressed, and a target truncated protein with a size of 35 kD was obtained. Western blot test showed that M protein had good reactivity with porcine PDCoV positive serum. An indirect ELISA method was constructed based on purified recombinant M truncated protein, and the critical value was determined to be 0.271, and had no cross reaction with Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndrome virus (PRRSV), Porcine circovirus type 2 (PCV-2), Pseudorabies virus (PRV), Foot-and-mouth disease virus (FMDV), Porcine epidemic diarrhea virus (PEDV), Swine transmissible gastroenteritis virus (TGEV). The coefficient of variation of intra- and inter-batch repeatability tests were less than 10%, and the total coincidence rate with the indirect ELISA based on PDCoV recombinant S1-CTD protein as coating antigen was 96%. The above results showed that the established indirect ELISA method had good specificity, sensitivity, and repeatability. The positive rate of PDCoV antibody was 49.11% in 507 pig serum samples collected in some areas of Sichuan province from 2012 to 2018, which preliminarily proved that the disease had been seriously infected in Sichuan. This study established an indirect ELISA method based on recombinant M truncated protein, which could provide technical support for PDCoV epidemic diagnosis and serum epidemiological investigation, and is of certain significance for PDCoV prevention and control.
宋代丽, 杨富升, 陈钰乔, 孙凡媛, 陈汭, 曹三杰, 黄小波. 猪δ冠状病毒M蛋白的截短表达及间接ELISA方法的建立[J]. 农业生物技术学报, 2022, 30(2): 402-412.
SONG Dai-Li, YANG Fu-Sheng, CHEN Yu-Qiao, SUN Fan-Yuan, CHEN Rui, CAO San-Jie, HUANG Xiao-Bo. Truncated Expression of Porcine deltacoronavirus M Protein and Establishment of Indirect ELISA. 农业生物技术学报, 2022, 30(2): 402-412.
[1] 陈建飞, 王潇博, 焦贺勋, 等. 2016. 国内首株猪德尔塔冠状病毒(Porcine deltacoronavirus)的分离鉴定[J]. 中国预防兽医学报, 38(03): 171-174. (Chen J F, Wang X B, Jiao H X.2016. Isolation and identification of the first Porcine deltacoronavirus strain in China[J]. Chinese Journal of Preventive Veterinary Medicine, 38(03): 171-174.) [2] 付嘉钰, 陈汭, 黄小波, 等. 2020. 1株猪δ冠状病毒N蛋白单克隆抗体制备与鉴定[J]. 微生物学通报, 47(06): 1847-1856. (Fu J Y, Chen R, Huang X B, et al.2020. Preparation and identification of a monoclonal antibody against N protein of Porcine deltacoronavirus[J]. Microbiology China, 47(06): 1847-1856.) [3] 侯林杉, 贾敬亮, 顾文源, 等. 2019. 基于猪丁型冠状病毒重组S1蛋白间接ELISA抗体检测方法的建立与应用[J]. 畜牧兽医学报, 50(8): 1642-1648. (Hou L S, Jia J L, Gu W Y, et al.2019. Establishment and application of an indirect ELISA based on recombinant S1 protein for the detection of antibodies against Porcine deltacoronavirus[J]. Chinese Journal of Animal and Veterinary Sciences, 50(8): 1642-1648.) [4] 逄凤娇, 俞正玉, 何孔旺, 等. 2017. 猪丁型冠状病毒重组N蛋白间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报, 39(6): 461-465. (Pang F J, Yu Z Y, He K W, et al.2017. Development of an indirect ELISA for detection antibody to Porcine deltacoronavirus using recombinant N protein[J]. Chinese Journal of Preventive Veterinary Medicine, 39(6): 461-465.) [5] 逄凤娇, 张柏猛, 何孔旺, 等. 2016. 猪δ冠状病毒M基因RT-PCR检测方法的建立及应用[J]. 畜牧与兽医, 48(9): 50-53. (Pang F J, Zhang B M, He K W, et al.2016. Development and application of a RT-PCR method based on M-gene for detection of Porcine deltacoronavirus[J]. Animal Husbandry and Veterinary Medicine, 48(9): 50-53.) [6] 瞿欢, 李成, 陈汭, 等. 2021. 猪δ冠状病毒S1-CTD的截短表达及间接ELISA抗体方法的建立[J]. 生物技术通报, 37(05): 273-280. (Qu H, Li C, Chen R, et al.2021. Truncated expression of the S1-CTD fragment of Porcine deltacoronavirus and establishment of an indirect ELISA for detecting its antibody[J]. Biotechnology Bulletin, 37(05): 273-280.) [7] 宋亚兵, 徐帅飞, 苏丹萍, 等. 2018. 2012年~2016年广东省猪丁型冠状病毒的流行病学调查及分析[J]. 中国预防兽医学报, 40(10): 886-890. (Song Y B, Xu S F, Su D P, et al. Epidemiological survey and analysis of Porcine deltacoronavirus in Guangdong province from2012 to 2016[J]. Chinese Journal of Preventive Veterinary Medicine, 40(10): 886-890.) [8] 王经纬. 2017. 基于猪丁型冠状病毒S1蛋白抗原的间接ELISA方法的建立与初步应用及NS6蛋白多克隆抗体的制备[D]. 硕士学位论文, 浙江大学, 导师: 黄耀伟, pp. 27-28. (Wang J W.2017. Development of an indiect ELISA for detection of antibodies against Procine deltacoronavirus S1 protein and preparation of polyclonal antibodies against PDCoV NS6 protein[D]. Thesis for M.S., Zhejiang University, Suppervisor: Huang Y W, pp. 27-28.) [9] 王经纬, 雷喜梅, 覃盼, 等. 2017. 猪丁型冠状病毒荧光定量RT-PCR与S1蛋白间接ELISA检测方法的建立及应用[J]. 生物工程学报, 33(8): 1265-1275. (Wang J W, Lei X M, Qin P, et al.2017. Development and application of real-time RT-PCR and S1 protein-based indirect ELISA for Porcine deltacoronavirus[J]. Chinese Journal of Biotechnology, 33(8): 1265-1275.) [10] 张森, 杨伟, 汤德元, 等. 2020. 猪δ冠状病毒基因组学及诊断技术研究进展[J]. 中国兽医学报, 40(7): 1429-1432. (Zhang S, Yang W, Tang D Y, et al.2020. Advances in genomics and diagnostic techniques of Porcine δ coronavirus[J]. Chinese Journal of Veterinary Science, 40(7): 1429-1432.) [11] 郑兰兰, 朱静静, 王盼, 等. 2019. 猪δ冠状病毒TaqMan荧光定量RT-PCR检测方法的建立与初步应用[J]. 畜牧兽医学报, 50(6): 1261-1267. (Zheng L L, Zhu J J, Wang P, et al.2019. Development and application of a TaqMan fluorescent RT-PCR for specific detection of Porcine deltacoronavirus[J]. Chinese Journal of Animal and Veterinary Sciences, 50(6): 1261-1267.) [12] Boley P A, Alhamo M A, Lossie G, et al.2020. Porcine deltacoronavirus infection and transmission in poultry, United States[J]. Emerging Infectious Diseases, 26(2): 255-265. [13] Chen Q, Gauger P, Stafne M, et al.2015a. Pathogenicity and pathogenesis of a United States Porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets[J]. Virology, 482: 51-59. [14] Chen F Z, Zhu Y X, Wu M Z, et al.2015b. Full length genome characterization of Chinese Porcine deltacoronavirus strain CH/SXD1/2015[J]. Genome Announcements, 3(5): e01284. [15] Duan C, Ge X N, Wang J C, et al.2021. Ergosterol peroxide exhibits antiviral and immunomodulatory abilities against Porcine deltacoronavirus (PDCoV) via suppression of NF-κB and p38/MAPK signaling pathways in vitro[J]. International Immunopharmacology, 93: 107317. [16] Gimenez-Lirola L G., Zhang J, Carrillo-Avila J A, et al.2017. Reactivity of Porcine epidemic diarrhea virus structural proteins to antibodies against Porcine enteric coronaviruses: Diagnostic implications[J]. Clinical Microbiology, 55(5): 1426-1436. [17] Hu H, Jung K, Vlasova A N, et al.2015. Isolation and characterization of Porcine deltacoronavirus from pigs with diarrhea in the United States[J]. Journal of Clinical Microbiology, 53(5): 1537-1548. [18] Jung K, Hu H, Saif L J.2017. Calves are susceptible to infection with the newly emerged Porcine deltacoronavirus, but not with the Swine enteric alphacoronavirus, Porcine epidemic diarrhea virus[J]. Archives of Virology, 162(8): 2357-2362. [19] Kim O, Chae C.2000. In situ hybridization for the detection and localization of Porcine epidemic diarrhea virus in the intestinal tissues from naturally infected piglets[J]. Veterinary Pathology, 37(1): 62-67. [20] Lee S, Lee C.2014. Complete genome characterization of Korean Porcine deltacoronavirus strain KOR/KNU14-04/2014[J]. Genome Announcements, 2(6): e1114-e1191. [21] Li G W, Chen Q, Harmon K M, et al.2014. Full-length genome sequence of Porcine deltacoronavirus strain USA/1A/2014/8734[J]. Genome Announcements, 2(2): e00278. [22] Li W, Hulswit R J G, Kenney S P, et al.2018. Bosch Berend-Jan. Broad receptor engagement of an emerging global coronavirus may potentiate its diverse cross-species transmissibility[J]. Proceedings of the National Academy of Sciences of the USA, 115(22): E5135-E5143. [23] Liang Q Q, Zhang H L, Li B X, et al.2019. Susceptibility of chickens to Porcine deltacoronavirus infection[J]. Viruses, 11(6): 573. [24] Ma Y M, Zhang Y, Liang X Y, et al.2015. Origin, evolution, and virulence of Porcine deltacoronavirus in the United States[J]. mBio, 6(2): e00064. [25] Ma Y M, Zhang Y, Liang X Y, et a1.2016. Two-way antigenic cross-reactivity between Porcine epidemic diarrhea virus and Porcine deltacoronavirus[J]. Veterinary Microbiology, 186: 90-96. [26] Marthaler D, Raymond L, Jiang Y, et al.2014. Rapid detection, complete genome sequencing, and phylogenetic analysis of Porcine deltacoronavirus[J]. Emerging Infectious Diseases, 20(8): 1347-1350. [27] Qiu S X, Ying X, Su S J, et al.2019. Bioinformatics analysis of the extracellular region of NA protein in novel H7N9 Avian influenza virus and preparation of polyclonal antibodies[J]. Biotechnology Bulletin, 35(12): 85-93. [28] Saeng-Chuto K, Stott C J, Wegner M, et al.2017. Retrospective investigation and evolutionary analysis of a novel Porcine deltacoronavirus strain detected in Thailand from 2008 to 2015[J]. Archives of Virology, 162(7): 2103-2108. [29] Song D, Zhou X, Peng Q, et al.2015. Newly emerged Porcine deltacoronavirus associated with diarrhea in swine in China: Identification, prevalence and full-length genome sequence analysis[J]. Transboundary and Emerging Diseases, 62(6): 575-580. [30] Su M J, Li C Q, Guo D H, et al.2016. A recombinant nucleocapsid protein-based indirect enzyme-linked immunosorbent assay to detect antibodies against Porcine deltacoronavirus[J]. Journal of Veterinary Medical Science, 78(4): 601-606. [31] Suzuki T, Shibahara T, Imai N, et al.2018. Genetic characterization and pathogenicity of Japanese Porcine deltacoronavirus[J]. Infection, Genetics and Evolution, 61(5): 176-182. [32] Thachil A, Gerber P F, Xiao C, et al.2015. Development and application of an ELISA for the detection of Porcine deltacoronavirus IgG antibodies[J]. PLOS ONE, 10(4): e124363. [33] Wang, Y W, Yue H, Fang W H, et al.2015. Complete genome sequence of Porcine deltacoronavirus strain CH/Sichuan/S27/2012 from mainland China[J]. Genome Announcements, 3(5): e00945. [34] Woo P C, Lau S K, Lam C S, et al.2012. Discovery of seven novel mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus[J]. Journal of Virology, 86(7): 3995-4008. [35] Woo P C, Lau S K, Tsang C C, et al.2017. Coronavirus HKU15 in respiratory tract of pigs and first discovery of coronavirus quasispecies in 5'-untranslated region[J]. Emerging Microbes & Infections, 6(6): e53. [36] Zhang H, Liang Q, Li B, et al.2019. Prevalence, phylogenetic and evolutionary analysis of Porcine deltacoronavirus in Henan province, China[J]. Preventive Veterinary Medicine, 166(5): 8-15. [37] Zhao Y J, Qu H, Hu J F, et al.2019. Characterization and pathogenicity of the Porcine deltacoronavirus isolated in southwest China[J]. Viruses, 11(11): 1074.
