猪流行性腹泻病毒单克隆抗体的制备及特性分析

王隆柏; 王晨燕; 吴学敏; 车勇良; 陈如敬; 周伦江

doi:10.19303/j.issn.1008-0384.2017.08.002

猪流行性腹泻病毒单克隆抗体的制备及特性分析

doi: 10.19303/j.issn.1008-0384.2017.08.002

福建省农业科学院畜牧兽医研究所/福建省畜禽疫病防治工程技术研究中心, 福建福州 350013

基金项目:

福建省自然科学基金项目 2015J01112

福建省科技创新平台建设项目 2014N2003

福建省科技计划项目——省属公益类科研院所基本科研专项 2014R1023-7

详细信息

作者简介:
王隆柏(1977-), 男, 副研究员, 主要从事分子生物学与免疫学研究(E-mail:wanglongbai@163.com)

通讯作者:
周伦江(1973-), 男, 研究员, 主要从事猪病防控技术研究(E-mail:lunjiang@163.com)

中图分类号: S852.65
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出版历程
- 收稿日期: 2016-10-13
- 修回日期: 2017-03-25
- 刊出日期: 2017-08-28

Preparation and Characterization of Monoclonal Antibodies Against Porcine Epidemic Diarrhea Virus

Institute of Animal Husbandry and Veterinary Medicine, FuJian Academy of Agriculture Sciences/Agricultural Industrialization Research Institute, FuJian Academy of Agriculture Sciences, Fuzhou, Fujian 350013, China

摘要

摘要: 为制备猪流行性腹泻病毒（PDEV）的单克隆抗体，并鉴定单克隆抗体特性。本试验采用差速和蔗糖梯度离心纯化PEDV抗原，免疫Balb/c小鼠，将免疫鼠的脾细胞与SP2/0细胞进行融合，经ELISA方法筛选和细胞克隆，得到能分泌鼠抗PEDV单克隆抗体杂交瘤细胞株，制备出相应的单克隆抗体，并分析其特性。试验结果：获得2株能稳定分泌抗PEDV的单克隆抗体，命名为E1和H6株，其中E1单隆抗体为IgG2a亚型，H6单隆抗体为IgM亚型，2株单克隆抗体均具有IFA、ELISA和Western bloting特性。E1杂交瘤细胞株的细胞上清液和腹水的ELISA抗体效价分别2⁶和10⁵，H6杂交瘤细胞株的细胞上清液和腹水的ELISA抗体效价分别2⁴和10⁴。2株单克隆抗体与Vero细胞、猪传染性胃肠炎病毒（TGEV）、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）均无交叉反应。Western bloting测定结果表明，E1株单克隆抗体能识别PEDV的M蛋白，H6单克隆抗体能识别PEDV的N蛋白。PEDV单克隆抗体的成功研制，为PEDV免疫诊断、表位识别及蛋白研究奠定了良好基础。
- 猪流行性腹泻病毒 /
- 单克隆抗体 /
- 制备 /
- 特性
Abstract: This study aimed to obtain the monoclonal antibodies against porcine epidemic diarrhea virus(PEDV) for antibody characterization as well asdisease control.The antigen of PEDV was purifiedby means of differential centrifugation and sucrose gradientcentrifugation. Spleen cells of the Balb/c mice immunized with viral antigen were fused with SP2/0 myeloma cells.Hybridoma cell lines secreting the monoclonal antibodies were isolated by using the indirect ELISA and sub-cloning approach to provide the source for the target antibody.Subsequently, specificities of the antibody were determined by means of IFA, ELISA and the Western blotting. The results obtained the following. (a) Two isolated hybridoma cell lines that could steadily secretethe specific McAbs against PEDV were designated as E1 and H6. (b) The isotyping analysis showed that E1 belonged to IgG2a; and H6, IgM sub-type. The McAbs from them could both bind to the antigen of PED in ELISA, IFA and the western blot analyses. (c) ELISA titers of the supernatants of E1 and H6 were 2⁶ and 2⁴ respectively; while those of the ascites fluids of E1 and H6, 10⁵ and 10⁴ respectively. (d) The McAbs from E1 and H6 showed no cross-reactivity with Vero cells, CSFV, TGEV or PRRSV.(e) The Western blotting confirmed that E1 McAbs recognized M protein; and, H6 McAbs, N protein. It appeared that two monoclonal antibodies with a high specificity against PEDV were successfully prepared, and further studies on PEDV immunodiagnosis, epitope discernment and the proteins became possible.
- porcine epidemic diarrhea virus /
- monoclonal antibody /
- preparation /
- characterization

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图 1 病变Vero细胞

Figure 1. Cytopathic effect on Vero cells

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图 2 正常Vero细胞

Figure 2. Normal Vero cells

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图 3 PEDV单克隆抗体IFA染色结果

注：A为E1单克隆抗体，B为H6单克隆抗体，C为阴性对照。

Figure 3. IFA of two McAbs on Vero cellsinfected by PEDV

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参考文献(16)

[1]	CHANG S H, BAE J L, KANG T J, et al.Identification of the epitope region capable of inducing neutralizing antibodies against the porcine epidemic diarrhea virus[J]. Cells, 2002, 14:295-299. http://www.ncbi.nlm.nih.gov/pubmed/12442904
[2]	殷震, 刘景华.动物病毒学[M].北京:科学出版社, 1997:681-688.
[3]	PENSAERT M B, DE BOUCK P. A new coronavirus-like particle associated with diarrhea in swine[J].Arch Virol, 1978, 58(3):243-247. doi: 10.1007/BF01317606
[4]	CHASEY D, CARTWRIGHT S F. Virus-like particles associated with porcine epidemic diarrhea[J]. Res vet Sci, 1978, 25(2):255-256. http://europepmc.org/abstract/MED/103154
[5]	宣华, 邢德坤, 王殿派, 等.应用猪胎肠单层细胞培养猪流行性腹泻的研究[J].兽医大学学报, 1984, 4(3):202-208. http://www.cnki.com.cn/Article/CJFDTOTAL-ZSYX198403001.htm
[6]	TAKAHASHI K, OKADA K, OHSHIMA K. An Outbreak of Swine Diarrhea of a New-type Associated with Coronavirus-like Particles in Japan[J]. Vet Sci, 1983, 45(6):829-832. http://europepmc.org/abstract/MED/6323804
[7]	CHEN J F, SUN D B, WANG C B, et al. Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China[J]. Virus Genes, 2008, 36(2):355-364. doi: 10.1007/s11262-007-0196-7
[8]	PARK S J, MOON H J, YANG J S, et al. Sequence analysis of the partial spike glycoprotein gene of porcine epidemic diarrhea viruses isolated in Korea[J]. Virus Genes, 2007, 35(2):321-332. doi: 10.1007/s11262-007-0096-x
[9]	PURANAVEJA S, POOLPERM P, LERTWATCHARASA-RAKUL P, et al. Chinese-like strain of porcine epidemic diarrhea virus, Thailand[J]. Emerg Infect Dis, 2009, 15(7):1112-1115. doi: 10.3201/eid1507.081256
[10]	陈仕龙, 林天龙, 陈少莺, 等.猪繁殖与呼吸综合征病毒单克隆抗体的制备与特性分析[J], 西北农林科技大学学报:自然科学版, 2008, 36(8):46-50. http://www.cnki.com.cn/Article/CJFDTOTAL-XBNY200808009.htm
[11]	KNIPE D M, HOWLEY P M, GDMN D E, et al.Chapter 36:Corona vimses in Fields Virology[M].4thed, Philadelphia:Lippincott Williams aJld Wilkins, 2001:1187-1197.
[12]	FA N J H, L I Y J. Cloning and Sequence Analysis of the Mgene of Porcine Epidemic Diarrhea Virus LJB/03[J].Virus Genes, 2005, 30:69-73. doi: 10.1007/s11262-004-4583-z
[13]	LEE H K, YEO S G. Cloning and sequence analysis of the nucleocapsid gene of Porcine epidemic diarrhea virus Chinju99[J]. Virus Genes, 2003, 26(2):207-212. doi: 10.1023/A:1023447732567
[14]	丁壮, 李佑明.抗猪流行性腹泻病毒单克隆抗体杂交瘤细胞株的建立及初步应用[J].兽医大学学报, 1989, 9(1):60-67. http://www.cnki.com.cn/Article/CJFDTOTAL-ZSYX198901011.htm
[15]	裴敦国, 葛俊伟, 马广鹏, 等.抗猪流行性腹泻病毒N蛋白单克隆抗体制备及部分特性鉴定[J].东北农业大学学报, 2008, 39(7)84-89. http://www.cnki.com.cn/Article/CJFDTOTAL-DBDN200807021.htm
[16]	张志榜, 陈建飞, 时洪艳, 等.猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报, 2011, 33(7):568-570. http://www.cnki.com.cn/Article/CJFDTOTAL-ZGXQ201107018.htm