[1]杨 溢?,杨玮枫?,张 东,等.抗沙门菌PEG菌毛单克隆抗体的制备及初步临床应用[J].中国预防兽医学报,2018,(05):438-442.[doi:0.3969/j.issn.1008-0589.201708016]
 YANG Yi?,YANG Wei-feng?,ZHANG Dong,et al.Preparation and preliminary application of monoclonal antibodies against PEG fimbriae of Salmonella[J].Chinese journal of preventive veterinary medicine,2018,(05):438-442.[doi:0.3969/j.issn.1008-0589.201708016]
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抗沙门菌PEG菌毛单克隆抗体的制备及初步临床应用()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2018年05
页码:
438-442
栏目:
免疫学
出版日期:
2018-05-15

文章信息/Info

Title:
Preparation and preliminary application of monoclonal antibodies against PEG fimbriae of Salmonella
文章编号:
1008-0589(2018)5-0438-05
作者:
 

杨 溢12?杨玮枫3?张 东12孟 霞12潘夏雨4袁天梅4朱春红5*朱国强12*

 (1.扬州大学 兽医学院,江苏 扬州 225009;2.江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏 扬州 225009;
3. 上海出入境检验检疫局 虹口办事处,上海 200000;4. 广西园丰牧业集团股份有限公司,广西 钦州 535400;
5. 江苏省家禽科学研究所,江苏 扬州 225125)
Author(s):
 

YANG Yi12? YANG Wei-feng3? ZHANG Dong12 MENG Xia12 PAN Xia-yu4 YUAN Tian-mei4 ZHU Chun-hong5* ZHU Guo-qiang12*

 (1. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China; 2. Important Disease Animal and Zoonosis Prevention and Control Collaborative Innovation Center of Jiangsu Province, Yangzhou 225009, China; 3. Shanghai Entery-Exit Inspection and Quarantine Bureau Hongkou Office, Shanghai 200000, China; 4. Guanxi Yuanfeng Animal Husbandary Group Co., Ltd, Qinzhou 535400, China;
5. Jiangsu Institute of Poultry Sciences, Yangzhou 225125, China)
关键词:
 沙门菌PegA菌毛原核表达MAb玻板凝集
Keywords:
 Salmonella  pegA fimbriae  prokaryotic expression  MAb  slide agglutination test
分类号:
S852.61
DOI:
0.3969/j.issn.1008-0589.201708016
文献标志码:
A
摘要:
为制备抗沙门菌PEG菌毛单克隆抗体(MAb),本研究从鸡白痢沙门菌(CVCC526)基因组中扩增菌毛蛋白基因pegA,将其克隆至原核表达载体pET-22b(+)中构建重组质粒pET-pegA,转化至大肠杆菌BL21(DE3)感受态细胞中,经IPTG诱导表达并纯化重组蛋白rPegA。应用该蛋白免疫BALB/c小鼠,取其脾细胞与SP2/0细胞融合,经间接ELISA筛选后获得1株稳定分泌MAb的杂交瘤细胞株,命名为3D12,抗体亚类鉴定其属于IgG1亚类,腹水效价为1∶64 000。通过western blot和玻板凝集试验检测结果显示,该MAb与肠炎沙门菌、鸡白痢沙门菌和鸡伤寒沙门菌呈阳性反应,而与鼠伤寒沙门菌、大肠杆菌CE2、CE7、CE53、DH5α 均不发生反应。以PEG菌毛MAb建立玻板凝集方法,检测本实验室临床分离保存的72份禽源沙门菌,同时以商品化沙门菌诊断血清作为平行对照,结果两种方法符合率达97.2 %。本研究研制的MAb可用于沙门菌PEG菌毛基础研究和快速检测。
Abstract:
To prepare the monoclonal antibodies (MAb) against PEG fimbriae of Salmonella, the fimbriae gene pegA of Salmonella was amplified from the S.pullorum (CVCC526) genome, and cloned into pET-22b(+) to construct recombinant plasmid pET-pegA, which was transformed into E.coli BL21 to express the recombinant PegA (rPegA). Then BALB/c mice were immunized with the purified rPegA, and the spleen cells from the immunized mice were fused with SP2/0 cells. As the result, one hybridoma cell line stably secreted MAb was obtained by indirect ELISA screening, named 3D12. The subclass of the MAb was identified as IgG1, which specifically reacted with S.enteritidis, S.pullorum and S.gallinarum verified by western blot analysis and plate agglutination test, but it was unable to react with S.typhimurium and E.coli. Additionally, the method of slide agglutination test was established based on the PEG fimbriae MAb, and 72 clinical samples were tested along with commercial diagnostic sera for Salmonella as a parallel control, the coincidence rate between these two detection methods was 97.2%. The MAb prepared in this study could be used for the basic research and the rapid detection for Salmonella PEG fimbriae.

参考文献/References:

[1]王杰,陈颖钰,彭清洁,等. 沙门菌检测方法研究进展[J]. 动物医学进展,2016,37(4):94-98.
[2]张江英. 抗沙门氏菌鞭毛素单克隆抗体的研制及相关应用[D]. 扬州:扬州大学,2014.
[3]Thomson N R, Clayton D J, Windhorst D, et al. Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 provides insights into evolutionary and host adaptation pathways [J]. Genome Res, 2008, 18(10): 1624-1637.
[4]Yue M, Rankin S C, Blanchet R T, et al. Diversification of the Salmonella fimbriae: a model of macro- and microevolution [J]. PLoS One, 2012, 7(6): e38596.
[5]段小丽,董立伟,张江英,等. 肠炎沙门氏菌SEF14菌毛单克隆抗体的制备及初步应用[J]. 上海畜牧兽医通讯,2013,(1):2-6.
[6]肖增鸿,黄昭亮,林月霞,等. 腹水型单克隆抗体纯化方法的研究[J]. 中国医药生物技术,2013,8(6):425-428.
[7]龚建森,王鑫,韩先干,等. 不同培养条件对禽源沙门菌菌毛基因表达的影响[J]. 畜牧兽医学报,2014,45(1):116-122.
[8]郭志燕,周明旭,朱国强. 细菌Ⅰ型菌毛结构及其致病性研究进展[J]. 中国预防兽医学报,2014,36(7):577-581.
[9]朱春红,孟霞,厚华艳,等. 沙门氏菌菌毛研究进展[J]. 中国动物传染病学报,2013,(2):68-74.
[10]Barrow P A, Thomson N R, Deacon V L, et al. Analysis of the role of 13 major fimbrial subunits in colonisation of the chicken intestines by Salmonella enterica serovar Enteritidis reveals a role for a novel locus [J]. Bmc Microbiology, 2008, 8: 228.
[11]Silva C A, Blondel C J, Quezada C P, et al. Infection of mice by Salmonella enterica serovar Enteritidis involves additional genes that are absent in the genome of serovar Typhimurium [J]. Infect Immun, 2012, 80(2): 839-849.
[12]杨玮枫,朱春红,李芙蓉,等. 沙门氏菌PEG菌毛研究进展[J]. 中国预防兽医学报,2017,39(4):329-332.
 (本文编辑:李     娜)

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更新日期/Last Update: 2018-05-16