微量凝集试验用大肠杆菌O50、O60、O117、O131定型血清的研制

doi:10.16431/j.cnki.1671-7236.2020.08.029

摘要/Abstract

摘要： 为进一步完善中国大肠杆菌菌体微量凝集试验定型血清库，改良微量凝集试验用大肠杆菌O抗原定型血清的制备工艺，本试验选取大肠杆菌O50、O60、O117、O131血清型进行定型血清制备方法的探究。首先用大肠杆菌不同血清型的参考菌株制备灭活抗原，而后多次免疫家兔以采血获得粗血清，用微量凝集试验的方法确定不同粗血清的交叉凝集素及凝集效价，再通过交叉凝集素吸收法结合血清稀释法消除非特异性凝集，最终研制出特异性良好的微量凝集试验用大肠杆菌O抗原定型血清。本研究确定了大肠杆菌O50、O60、O117、O131定型粗血清的主要交叉凝集素，最终成功研制出特异性良好的微量凝集试验用大肠杆菌O50、O60、O117、O131定型血清共4种，凝集效价在1：256至1：2 048之间，其中微量凝集试验用大肠杆菌O50、O60定型血清无交叉凝集素，为单因子定型血清，微量凝集试验用大肠杆菌O117、O131定型血清存在1~2种非特异性的交叉凝集素O14和O107。本研究作为大肠杆菌O抗原定型血清制备工艺摸索过程中的重要部分，为完善中国大肠杆菌菌体微量凝集试验定型血清库，进一步改良微量凝集试验用大肠杆菌O抗原定型血清的制备方法提供了重要理论依据。

关键词: 大肠杆菌; O抗原; 微量凝集试验; 血清制备

Abstract: In order to improve and perfect the E.coli specific anti-O-antigen sera pool for micro-agglutination test in China,and further improve the preparation process of mono-specific anti-O-antigen serum,E.coli O50,O60,O117 and O131 serotypes were selected to investigate the method of preparing sera.First,inactivated antigens were prepared by reference strains of E.coli.Crude sera were extracted from rabbits after repeated immunizations by inactivated antigens.The method of micro-agglutination was used in the preparation process to determine the cross-lectins and agglutination value of different sera.The non-specific agglutination was eliminated by absorbing non-specific lectins and diluting the serum.Finally,E.coli specific anti-O-antigen sera for micro-agglutination test with good specificity were developed.The main cross-lectins of four kinds of E.coli specific anti-O-antigen sera were identified.Four kinds of sera with good specificity which could be used for E.coli O antigen micro-agglutination test were prepared,which agglutination titers were between 1:256 and 1:2 048.E.coli O50 and E.coli O60 serotypes sera were mono-specific anti-O-antigen sera.E.coli O117 and E.coli O131 serotypes sera contained 1-2 nonspecific cross-lectins,O14 and O107.This study was an important part of exploring the preparation process of mono-specific anti-O-antigen serum.The important theoretical bases were provided for improving the serum library of E.coli bacterial micro-agglutination test in China,and further improved the preparation method of E.coli O antigen serum for micro-agglutination test.

Key words: Escherichia coli; O antigen; micro-agglutination test; serum preparation

中图分类号:

S852.5⁺3

辛凌翔, 王秀丽, 张一帜, 李建, 张媛. 微量凝集试验用大肠杆菌O50、O60、O117、O131定型血清的研制[J]. 中国畜牧兽医, 2020, 47(8): 2596-2602.

XIN Lingxiang, WANG Xiuli, ZHANG Yizhi, LI Jian, ZHANG Yuan. Development of E.coli O50,O60,O117,O131 Serotyped Sera for Micro-agglutination Test[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(8): 2596-2602.

参考文献

[1] 高玉斌,赵格,邹明.不同地区动物源致病性大肠杆菌的分子特性与表型分析[J].中国动物检疫,2018,35(9):62-66. GAO Y B,ZHAO G,ZOU M.Analysis on molecular characteristics and phenotypes of animal-origin pathogenic E.coli in different areas[J].China Animal Health Inspection,2018,35(9):62-66.(in Chinese)
[2] DESROCHES M,ROYER G,ROVHE D,et al.The odyssey of the ancestral Escherich strain through culture collections:An example of allopatric diversification[J].mSphere,2018,3(1):e00553-17.
[3] STENUZT R,WEINTRAUB A,WIDMALM G.The structures of Escherichia coli O-polysaccharide antigens[J].FEMS Microbiology Reviews,2006,30:382-403.
[4] 王磊杰.哈尔滨地区猪源致病性大肠杆菌耐药性分析及基因分型研究[D].哈尔滨:东北农业大学,2013. WANG L J.Resistance analysis and genotype research of pathogenic Escherichia coli derived from swines in Harbin[D].Harbin:Northeast Agricultural University,2013.(in Chinese)
[5] 央珍,聂奎.重庆市部分地区鸭源大肠杆菌的O抗原血清型及耐药性[J].西南大学学报(自然科学版),2011,33(8):52-56. YANG Z,NIE K.Serotype identification and antibiotics susceptibility test of duck Escherichia coli from some duck farms in Chongqing[J].Journal of Southwest University(Natural Science Edition),2011,33(8):52-56.(in Chinese)
[6] 张玉霞,徐怀英.家禽大肠杆菌分型方法研究进展[J].家畜生态学报,2011,32(5):98-100. ZHANG Y X,XU H Y.Research progress on typing of E.coli from poultry[J].Journal of Domestic Animal Ecology,2011,32(5):98-100.(in Chinese)
[7] 诸明欣,张焕容,汤承,等.四川阿坝州牦牛源溶血性大肠杆菌血清型鉴定及毒力相关基因检测[J].中国预防兽医学报,2018,40(1):9-13. ZHU M X,ZHANG H R,TANG C,et al.Serotype identification and virulence-related genes detection of hemolytic E.coli isolated from yak in Sichuan Aba state[J].Chinese Journal of Preventive Veterinary Medicine,2018,40(1):9-13.(in Chinese)
[8] 蔺旭光,马德慧,康桂英,等.犊牛腹泻大肠埃希菌O-抗原血清型鉴定[J].中国生物制品学杂志,2018,31(1):55-58. LIN X G,MA D H,KANG G Y,et al.Identification of serogroup of E.coli O-antigen from calves with diarrhea[J].Chinese Journal of Biologicals,2018,31(1):55-58.(in Chinese)
[9] 宋晓莉,成大荣,刘晓明,等.临床腹泻肉羊源大肠杆菌流行病学调查及耐药性分析[J].黑龙江畜牧兽医,2019,23:71-75. SONG X L,CHENG D R,LIU X M,et al.Epidemiological investigation and drug resistance analysis of E.coli from sheep with clinical diarrhea[J].Heilongjiang Animal Science and Veterinary Medicine,2019,23:71-75.(in Chinese)
[10] 张媛,刘博,王秀丽,等.大肠杆菌菌体O抗原单因子定型血清的制备[J].中国兽药杂志,2016,50(11):9-16. ZHANG Y,LIU B,WANG X L,et al.Preparation of mono-specific anti-O-antigen serum of E.coli[J].Chinese Journal of Veterinary Drug,2016,50(11):9-16.(in Chinese)
[11] 张媛,王秀丽,刘博,等.大肠杆菌O抗原定型血清标定用抗原的制备[J].中国兽药杂志,2016,50(12):12-18. ZHANG Y,WANG X L,LIU B,et al.The preparation of E.coli's antigen which calibrate the titer of the mono-factor stereotype serum[J].Chinese Journal of Veterinary Drug,2016,50(12):12-18.(in Chinese)
[12] 辛凌翔,魏财文,张媛,等.5种大肠杆菌O抗原定型血清的制备[J].中国兽医杂志,2019,55(5):21-25. XIN L X,WEI C W,ZHANG Y,et al.Preparation of 5 kinds of E.coli specific anti-O-antigen sera[J].Chinese Journal of Veterinary Drug,2019,55(5):21-25.(in Chinese)
[13] 房海.大肠埃希氏菌[M].石家庄:河北科学技术出版社,1997. FANG H.Escherichia coli[M].Shijiazhuang:Hebei Science Technology Publishing House,1997.(in Chinese)
[14] 黄涛,山珊,黄艳梅,等.大肠埃希氏菌分型方法的研究进展[J].微生物学通报,2020,47(3):892-902. HUANG T,SHAN S,HUANG Y M,et al.Advances in typing methods for Escherichia coli[J].Microbiology China,2020,47(3):892-902.(in Chinese)
[15] DEBROY C,FRATAMICO P M,YAN X H,et al.Correction:Comparison of O-antigen gene clusters of all O serogroups of Escherichia coli and proposal for adopting a new nomenclature for O-typing[J].PLoS One,2016,11(4):e0154551.
[16] 申进玲,吴瑜凡,崔思宇,等.成簇的规律间隔短回文重复序列在致病菌中分型研究及应用[J].生物技术,2017,27(2):186-191. SHEN J L,WU Y F,CUI S Y,et al.Study of clustered regularly interspaced short palindromic repeat sequence subtyping and its application in pathogens[J].Biotechnology,2017,27(2):186-191.(in Chinese)
[17] LGUCHI A,LYODA S,KIKUCHI T,et al.A complete view of the genetic diversity of the Escherichia coli O-antigen biosynthesis gene cluster[J].DNA Research,2015,22(1):101-107.
[18] 刘璨颖,张济培,王丙云.大肠杆菌O-抗原血清型鉴定研究进展[J].中国人兽共患病学报,2016,32(10):928-933. LIU C Y,ZHANG J P,WANG B Y.Research progress on identification of Escherichia coli O-antigen serogroups[J].Chinese Journal of Zoonoses,2016,32(10):928-933.(in Chinese)
[19] MOSAVIE M,BLANDY O,JAUNEIKAITE E,et al.Sampling and diversity of Escherichia coli from the enteric microbiota in patients with Escherichia coli bacteraemia[J].BMC Research Notes,2019,12:335.
[20] 冯敏燕,林树乾,房玉波,等.山东省鸡源致病性大肠杆菌O-抗原混合血清型分析[J].中国兽医学报,2017,37(9):1676-1679. FENG M Y,LIN S Q,FANG Y B,et al.Analysis of mixed O-antigen serotypes of E.coli isolated from chickens in Shandong province[J].Chinese Journal of Veterinary Science,2017,37(9):1676-1679.(in Chinese)
[21] 许亚卓,杨春柳,刘文鑫,等.O_139,142,26混合血清型大肠杆菌的O-抗原基因簇的破译[J].东北农业大学学报,2010,41(2):101-105. XU Y Z,YANG C L,LIU W X,et al.Sequence and analysis of O-antigen gene cluster in Escherichia coli O_139,142,26[J].Journal of Northeast Agricultural University,2010,41(2):101-105.(in Chinese)
[22] AGHAMOHAMMAD S,BADMASTI F,SHIRAZI A S,et al.Considerable rate of putative virulent phylo-groups in fecal carriage of extended-spectrum β-lactamase producing Escherichia coli[J].Infection,Genetics and Evolution,2019,73:184-189.