非洲猪瘟病毒无标签p30-ELISA抗体检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2022.05.019

摘要/Abstract

摘要： 非洲猪瘟(African swine fever, ASF)是由非洲猪瘟病毒(African swine fever virus, ASFV)引起猪的一种急性、热性、出血性、高度接触性传染病，临床症状以败血症、皮炎和关节炎为特征，高发病率和高死亡率。为建立临床检测ASFV抗体的间接ELISA检测方法，本研究扩增了ASFV-CP204L基因，通过pET-30a原核表达系统表达p30蛋白，使用Ni-NTA纯化表达产物，通过肠激酶切除外源性蛋白，得到无His-组氨酸标签的p30蛋白，以此为诊断抗原，建立间接ELISA方法。结果显示：表达的无标签p30重组蛋白大小约为30 ku，与ASF阳性猪血清具有较好的反应原性；确定ELISA抗原包被浓度为1 μg·mL^-1，根据ROC曲线下面积确定S/P值>0.398判定为阳性，批内、批间变异系数均<10%；与PCV2、CSFV、PRV-gE、PRRSV阳性血清无交叉反应与INGENASA商品化试剂盒总符合率为97.78%。用该方法分别检测标准阳性血清、动物感染试验血清和收集的区域性临床血清644份，该方法最低可检测到1∶512倍稀释的标准阳性血清样品；检测感染动物血清，其中80%(4/5)的试验动物在第10天抗体为阳性。644份临床猪血清样品中抗体阳性率为7.61%，其中，母猪、后备母猪、仔猪、保育猪和育肥猪抗体阳性率分别为3.03%、0%、4.94%、7.55%和28.7%。本试验建立的ASFV-p30间接ELISA方法具有良好的特异性、灵敏度和重复性，可应用于ASFV的抗体检测，为ASF的诊断和流行病学调查提供了技术手段。

关键词: 非洲猪瘟, CP204L基因, p30蛋白, 原核表达, ELISA抗体检测

Abstract: African Swine fever (ASF), caused by infection with the ASF virus (ASFV), is one of the most serious viral diseases affecting pigs. It is featured by acute, febrile, hemorrhagic, high morbidity and mortality, etc. The aim of this paper is to develop an efficient and clinically convenient indirect ELISA antibody detection method for ASFV. Here, the ASFV-CP204L gene was amplified from positive samples, and the p30 protein was expressed through the pET-30a prokaryotic expression system. The expressed product was further purified with Ni-NTA, and the His protein was removed by enterokinase. Thus, an indirect ELISA method was established based on the purified recombinant tag-free p30 protein. An approximate 30 ku fusion p30 tag-free protein was observed. The results showed that the protein expressed in this study has good reactogenicity with ASF-positive pig serum. It was finally determined that the ELISA antigen coating concentration was 1 μg·mL^-1. The positive value (S/P) was more than 0.398 by the area under the ROC curve. The coefficients of inter-and intra-batches were less than 10%, indicating the good repeatability of the method. This method can detect at least 512-fold diluted standard positive serum samples. In the detection of experimentally infected sera, 80% of the samples were seroconversion on the 10th day. In this study, a total of 644 pig serum samples were collected. The results showed that the positive rates were 7.61%. Among them, the positive rates of antibodies in sows, gilts, piglets, nursery pigs and fattening pigs were 3.03%, 0%, 4.94%, 7.55% and 28.7%, respectively. The indirect ELISA method based on the tag-free ASFV-p30 protein has good specificity, sensitivity and reproducibility, and can be initially applied to the detection of antibodies of ASFV. It provides a technical tool for epidemiologically serological investigation of ASF.

Key words: African swine fever, CP204L gene, p30 protein, prokaryotic expression, ELISA antibody detection

中图分类号:

于学祥, 陈晓雨, 李栋凡, 孙琪, 库旭钢, 范盛先, 杨汉春, 何启盖. 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.

YU Xuexiang, CHEN Xiaoyu, LI Dongfan, SUN Qi, KU Xugang, FAN Shengxian, YANG Hanchun, HE Qigai. Establishment and Application of an Indirect ELISA Antibody Detection Method Based on African Swine Fever Virus Tag-free p30 Protein[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(5): 1517-1526.

参考文献 32

[1]	DE LA TORRE A, BOSCH J, IGLESIAS I, et al. Assessing the risk of african swine fever introduction into the European union by wild boar[J]. Transbound Emerg Dis, 2015, 62(3):272-279.
[2]	ZAKARYAN H, REVILLA Y. African swine fever virus:current state and future perspectives in vaccine and antiviral research[J]. Vet Microbiol, 2016, 185:15-19.
[3]	GAUDREAULT N N, MADDEN D W, WILSON W C, et al. African swine fever virus:an emerging DNA arbovirus[J]. Front Vet Sci, 2020, 7:215.
[4]	MAI N T A, VU X D, NGUYEN T T H, et al. Molecular profile of African swine fever virus (ASFV) circulating in Vietnam during 2019-2020 outbreaks[J]. Arch Virol, 2021, 166(3):885-890.
[5]	GE S Q, LI J M, FAN X X, et al. Molecular characterization of African swine fever virus, China, 2018[J]. Emerg Infect Dis, 2018, 24(11):2131-2133.
[6]	王清华, 任炜杰, 包静月, 等. 我国首例非洲猪瘟的确诊[J]. 中国动物检疫, 2018, 35(9):1-4.WANG Q H, REN W J, BAO J Y, et al. The first outbreak of African swine fever was confirmed in China[J]. China Animal Health Inspection, 2018, 35(9):1-4. (in Chinese)
[7]	EDELSTEN R M, CHINOMBO D O. An outbreak of African swine fever in the southern region of Malawi[J]. Rev Sci Tech, 1995, 14(3):655-666.
[8]	EBWANGA E J, GHOGOMU S M, PAESHUYSE J. African swine fever in Cameroon:a review[J]. Pathogens, 2021, 10(4):421.
[9]	BISIMWA P N, ISHARA L K, WASSO D S, et al. Detection and genetic characterization of African swine fever virus (ASFV) in clinically infected pigs in two districts in South Kivu province, Democratic Republic Congo[J]. Heliyon, 2021, 7(3):e06419.
[10]	LIU H, SHI K C, SUN W C, et al. Development a multiplex RT-PCR assay for simultaneous detection of African swine fever virus, classical swine fever virus and atypical porcine pestivirus[J]. J Virol Methods, 2020, 287:114006.
[11]	LI L, REN Z W, WANG Q H, et al. Infection of African swine fever in wild boar, China, 2018[J]. Transbound Emerg Dis, 2019, 66(3):1395-1398.
[12]	GE S Q, LIU Y T, LI L, et al. An extra insertion of tandem repeat sequence in African swine fever virus, China, 2019[J]. Virus Genes, 2019, 55(11):843-847.
[13]	ZHAO D M, LIU R Q, ZHANG X F, et al. Replication and virulence in pigs of the first African swine fever virus isolated in China[J]. Emerg Microbes Infect, 2019, 8(1):438-447.
[14]	SUN E C, ZHANG Z J, WANG Z L, et al. Emergence and prevalence of naturally occurring lower virulent African swine fever viruses in domestic pigs in China in 2020[J]. Sci China Life Sci, 2021, 64(5):752-765.
[15]	张艳艳, 张静远, 杨金金, 等. 1株非洲猪瘟病毒自然变异毒株的鉴定[J]. 中国兽医学报, 2021, 41(2):199-207.ZHANG Y Y, ZHANG J Y, YANG J J, et al. Identification of a natural variant of African swine fever virus in China[J]. Chinese Journal of Veterinary Science, 2021, 41(2):199-207. (in Chinese)
[16]	BELTRÁN-ALCRUDO D, KRAMER S A, KRAMER C, et al. African swine fever:detection and diagnosis[R]. Rome:FAO, 2017.
[17]	SÁNCHEZ-VIZCAÍNO J M, MUR L, GÓMEZ-VILLAMANDOS J C, et al. An update on the epidemiology and pathology of African swine fever[J]. J Comp Pathol, 2015, 152(1):9-21.
[18]	杨文兵, 邹亚文, 蒋一凡, 等. 非洲猪瘟血清学诊断靶点的研究进展[J]. 畜牧兽医学报, 2021, 52(5):1208-1217.YANG W B, ZOU Y W, JIANG Y F, et al. Advances research on African swine fever serological diagnostic targets[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5):1208-1217. (in Chinese)
[19]	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.WU J, WANG X X, WU Y T, et al. Prokaryotic expression of p30 gene of African swine fever virus and establishment of Indirect ELISA antibody detection method[J]. China Animal Husbandry & Veterinary Medicine, 2018, 45(12):3555-3562. (in Chinese)
[20]	靳雯雯, 杨俊兴, 花群俊, 等. 非洲猪瘟病毒抗体检测间接ELISA方法的建立[J]. 中国兽医学报, 2014, 34(7):1043-1046.JIN W W, YANG J X, HUA Q J, et al. Development of an indirect enzyme-linked immunosorbent assay for detection antibody against African swine fever virus[J]. Chinese Journal of Veterinary Science, 2014, 34(7):1043-1046. (in Chinese)
[21]	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.GENG X, SUN M X, WANG S J, et al. Prokaryotic expression of African swine fever p72 protein and development of indirect ELISA method[J]. Chinese Journal of Preventive Veterinary Medicine, 2021, 43(9):946-951. (in Chinese)
[22]	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.SHI L, TIAN Z C, YANG J F, et al. Comparison of the antigenicity of African swine fever virus p35 protein as diagnostic antigen[J]. Chinese Journal of Biotechnology, 2021, 37(1):187-195. (in Chinese)
[23]	邬旭龙, 彭彬, 姜睿姣, 等. 以原核表达的非洲猪瘟病毒pK205R蛋白为包被抗原的间接ELISA抗体检测方法的建立[J]. 中国预防兽医学报, 2016, 38(10):800-803.WU X L, PENG B, JIANG R J, et al. Development of an indirect ELISA for detection of antibody against African swine fever virus (ASFV) with prokaryotic expressed ASFV pK205R protein as the coating antigen[J]. Chinese Journal of Preventive Veterinary Medicine, 2016, 38(10):800-803. (in Chinese)
[24]	周晓慧, 肖景景, 张鑫宇, 等. 非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用[J]. 畜牧兽医学报, 2020, 51(10):2472-2480.ZHOU X H, XIAO J J, ZHANG X Y, et al. Preparation and preliminary application of highly immunogenic recombinant CD2v antigen of African swine fever virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10):2472-2480. (in Chinese)
[25]	BLOME S, FRANZKE K, BEER M. African swine fever-a review of current knowledge[J]. Virus Res, 2020, 287:198099.
[26]	DAN L, LIU Y G, QI X L, et al. African swine fever virus MGF-110-9L-deficient Mutant has attenuated virulence in pigs[J]. Virol Sin, 2021, 36(2):187-195.
[27]	ASAMBE A, SACKEY A K B, TEKDEK L B. Prevalence of African swine fever virus and classical swine fever virus antibodies in pigs in Benue State, Nigeria[J]. Trop Anim Health Prod, 2018, 50(3):689-692.
[28]	GÓMEZ-PUERTAS P, RODRÍGUEZ F, OVIEDO J M, et al. The African Swine Fever Virus Proteins p54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody-mediated protective immune response[J]. Virology, 1998, 243(2):461-471.
[29]	JIA N, OU Y W, PEJSAK Z, et al. Roles of African swine fever virus structural proteins in viral infection[J]. J Vet Res, 2017, 61(2):135-143.
[30]	LITHGOW P, TAKAMATSU H, WERLING D, et al. Correlation of cell surface marker expression with African swine fever virus infection[J]. Vet Microbiol, 2014, 168(2-4):413-419.
[31]	BARDERAS M G, RODRíGUEZ F, GÓMEZ-PUERTAS P, et al. Antigenic and immunogenic properties of a chimera of two immunodominant African swine fever virus proteins[J]. Arch Virol, 2001, 146(9):1681-1691.
[32]	荣明轩, 徐保娟, 南文龙, 等. 非洲猪瘟病毒抗体间接ELISA检测方法的建立与应用[J]. 中国动物检疫2021, 28(5):109-118.RONG M X, XU B J, NAN W L, et al. Establishment and application of the iELISA for detection of antibodies against ASFV[J]. China Animal Health Inspection, 2021, 28(5):109-118. (in Chinese)