[1]于涵,王丽娜,郭东华,等.牛坏死杆菌43K OMP的截短表达及多克隆抗体的制备[J].黑龙江农业科学,2022,(04):68-72.[doi:10.11942/j.issn1002-2767.2022.04.0068]
　YU Han,WANG Li-na,GUO Dong-hua,et al.Expression and Polyclonal Antibody Preparation of Truncated 43K OMP Protein from Fusobacterium necrophrum[J].HEILONGJIANG AGRICULTURAL SCIENCES,2022,(04):68-72.[doi:10.11942/j.issn1002-2767.2022.04.0068]
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牛坏死杆菌43K OMP的截短表达及多克隆抗体的制备

《黑龙江农业科学》[ISSN:1002-2767/CN:23-1204/S] 卷: 期数: 2022年04 页码: 68-72 栏目: 出版日期: 2022-04-10
Title:
Expression and Polyclonal Antibody Preparation of Truncated 43K OMP Protein from Fusobacterium necrophrum
作者:
于涵; 王丽娜; 郭东华; 贺显晶
黑龙江八一农垦大学 动物科技学院，黑龙江 大庆 163319
Author(s):
YU Han; WANG Li-na; GUO Dong-hua; HE Xian-jing
College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,China
关键词:
牛坏死杆菌; 43K OMP; 原核表达; 多克隆抗体
Keywords:
Fusobacterium necrophrum; 43K OMP;  prokaryotic expression;  polyclonal antibody
DOI:
10.11942/j.issn1002-2767.2022.04.0068
文献标志码:
A
摘要:
为制备牛坏死杆菌截短43K OMP多克隆抗体，以A25菌株基因组DNA为模板，特异性扩增43K OMP基因的4个相互重叠的截短片段，构建原核表达载体，利用大肠杆菌原核表达系统对截短基因进行表达并纯化蛋白，同时免疫家兔制备多克隆抗体。结果表明：43K OMP基因截短片段经IPTG诱导成功表达，蛋白大小分别为32 kDa、30 kDa、28 kDa和30 kDa，运用蛋白制备的多克隆抗体43K OMP-1、43K OMP-2、43K OMP-3和43K OMP-4的效价分别为1∶25 600、1∶51 200、1∶25 600和1∶51 200，且制备的抗体能识别天然43K OMP。
Abstract:
In order to prepare the polyclonal antibody of Fusobacterium necrophrum,so as to promote the research of genetic engineering vaccine from Fusobacterium necrophrum,the study used A25 gene DNA as template to amplify four fragments of 43K OMP gene overlap each other,and constructed prokaryotic expression vector.The purified protein was immunized to rabbits by prokaryotic expression system of E.coli,and then the polyclonal antibody was prepared and identified.The results showed that the truncated 43K OMP proteins were successfully expressed,and the length of the truncated protein size was 32 kDa,30 kDa,28 kDa and 30 kDa respectively.The polyclonal antibodies 43K OMP-1,43K OMP-2,43K OMP-3 and 43K OMP-4 prepared by the protein were 1∶25 600,1∶51 200,1∶25 600,1∶51 200.The prepared antibody can recognize natural 43K OMP,which lays a foundation for the research of genetic engineering vaccine of Fusobacterium necrophrum.

参考文献/References:

［1］PILLAI D K,AMACHAWADI R G,BACA G,et al.Leukotoxic activity of Fusobacterium necrophorum of cattle origin[J].Anaerobe,2019,56:51-56.

［2］MENON S,PILLAI D K,NARAYANAN S.Characterization of Fusobacterium necrophorum subsp.necrophorum outer membrane proteins[J].Anaerobe,2018,50:101-105. ［3］FRANCIS A M,JEON S J,CUNHA F,et al.Draft genome sequences of two Fusobacterium necrophorum strains isolated from the uterus of dairy cows with metritis[J].Microbiology Resource Announcements,2019,8(17):e00201-19.

［4］祁世荣.奶牛五种恶性乳房炎诊治总结[J].中国奶牛,1999(2):44-46.

［5］任士飞,徐建生,董国雄,等.细菌黏附研究进展[J].中国预防兽医学报,2004,26(3):238-240.

［6］KUMAR A,PETERSON G,NAGARAJA T G,et al.Outer membrane proteins of Fusobacterium necrophorum subsp.necrophorum and subsp.funduliforme[J].Journal of Basic Microbiology,2014,54(8):812-817.

［7］SUN D,ZHANG H,LV S,et al.Identification of a 43-kDa outer membrane protein of Fusobacterium necrophorum that exhibits similarity with poreforming proteins of other Fusobacterium species[J].Journal of Medical Microbiology,2013,95(1):27-33.

［8］KUMAR A,GART E,NAGARAJA T G,et al.Adhesion of Fusobacterium necrophorum to bovine endothelial cells is mediated by outer membrane proteins[J].Veterinary Microbiology,2012,162(2-4):813-818.

［9］蒋剑成,张思瑶,汪锋锋,等.牛坏死杆菌43 kDa OMP对小鼠免疫效果的评价[C]//中国畜牧兽医学会兽医病理学分会第二十四次学术研讨会，中国病理生理学会动物病理生理学专业委员会第二十三次学术研讨会，中国实验动物学会实验病理学专业委员会第三次学术研讨会，中国兽医病理学家第三次学术研讨会论文集,2018:41.

［10］吕思文,张红,孙东波,等.牛坏死杆菌43ku外膜蛋白截短片段的原核表达[J].中国畜牧兽医,2014,41(4):56-61.

［11］RIORDAN T.Human Infection with Fusobacterium necrophorum(Necrobacillosis),with a focus on Lemierre′s Syndrome[J].Clinical Microbiology Reviews,2007,20(4):622. ［12］JOLOBE O M P.Fusobacterium necrophorum hepatic abscess and colorectal cancer[J].The American Journal of Medicine,2020,133(7):e392.

［13］NAGARAJA T G,NARAYANAN S K,STEWART G C,et al.Fusobacterium necrophorum infections in animals:Pathogenesis and pathogenic mechanisms[J].Anaerobe,2005,11(4):239-246.

［14］UMANA A,SANDERS B E,YOO C C,et al.Utilizing whole Fusobacterium genomes to identify,correct,and characterize potential virulence protein families[J].Journal of Bacteriology,2019,201(23)：1-20.

［15］LUDLAM H A,MILNER N J,BRAZIER J S,et al.lktA-encoded leukotoxin is not a universal virulence factor in invasive Fusobacterium necrophorum infections in animals and man[J].Journal of Medical Microbiology,2009,58(4):529-530.

［16］TADEPALLI S,STEWART G C,NAGARAJA T G,et al.Human Fusobacterium necrophorum strains have a leukotoxin gene and exhibit leukotoxic activity[J].Journal of Medical Microbiology,2008,57(2):225.

［17］KWAKU A K,YOSHITAKA G,NAOAKI M,et al.The erythrocyte receptor for Fusobacterium necrophorum hemolysin:Phosphatidylcholine as a possible candidate[J].Fems Microbiology Letters,1998(1):65.

［18］吕思文,张虹,郭东华.牛羊坏死杆菌保护性抗原的筛选与鉴定[C]//中国畜牧兽医学会，中国病理生理学会.2013年中国畜牧兽医学会兽医病理学分会暨中国病理生理学会动物病理生理学专业委员会学术研讨会论文集，2013：296-301.

［19］MACHADO V S,BICALHO M L D S,ROSSI R,et al.Subcutaneous immunization with inactivated bacterial components and purified protein of Escherichia coli,Fusobacterium necrophorum and Trueperella pyogenes prevents puerperal metritis in holstein dairy cows[J].PLoS One,2014,9(3):91734-91734.

［20］郭东华,王君伟,孙玉国,等.牛腐蹄病坏死梭杆菌菌株白细胞毒素基因的原核表达及其免疫活性分析[J].中国预防兽医学报,2007,29(6):435-438.

［21］NAKAGAKI H,SEKINE S,TERAO Y,et al.Fusobacterium nucleatum envelope protein FomA is immunogenic and binds to the salivary statherin-derived peptide[J].Infection & Immunity,2010,78(3):1185-1192.

［22］GLAZER S,SOL A,ABED J,et al.Fap2 of Fusobacterium nucleatum is a galactose inhibitable adhesin,involved in coaggregation,cell adhesion and preterm birth[J].Infection & Immunity,2015,83(3):1104-1113.

［23］朱必凤,杨旭夫,彭凌,等.副猪嗜血杆菌外膜蛋白的免疫原性[J].中国兽医学报,2013,33(6):838-842，848.

［24］KUMAR A,PETERSON G,NAGARAJA T G,et al.Outer membrane proteins of Fusobacterium necrophorum subsp.necrophorum and subsp.funduliforme[J].Journal of Basic Microbiology,2014,54(8):812-817.

［25］徐晶,陈立志,李淑艳,等.坏死梭杆菌44.5 ku外膜蛋白的免疫原性分析[J].中国兽医科学,2016(4):512-515.

［26］HE X,WANG L,LI H,et al.Screening of BHK-21 cellular proteins that interact with outer membrane protein 43K OMP of Fusobacterium necrophorum[J].Anaerobe,2020，63：102184.

［27］HE X J,JIANG K,XIAO J W,et al.Interaction of 43K OMP of Fusobacterium necrophorum with fibronectin mediates adhesion to bovine epithelial cells［J］.Veterinary Microbiology,2022,266：109335.

备注/Memo

收稿日期：2022-01-21

基金项目：黑龙江省大学生创新创业项目（202110223068）；黑龙江八一农垦大学三纵科研支持计划青年创新人才项目（ZRCQC202103）。

第一作者：于涵（2000-），女，本科生，专业方向为奶牛腐蹄病综合防控技术研发。E-mail：1178416907@qq.com。

通信作者：贺显晶（1985-），女，博士，副教授，从事动物厌氧菌疾病的综合防治研究。E-mail:xianjinghe@126.com。

更新日期/Last Update: 2022-07-14