一株粗糙型牛种布鲁氏菌诱导株的构建及鉴定

doi:10.16431/j.cnki.1671-7236.2020.11.004

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (11): 3445-3453.doi: 10.16431/j.cnki.1671-7236.2020.11.004

一株粗糙型牛种布鲁氏菌诱导株的构建及鉴定

孙浩杰^1,2, 任小侠¹, 秦玉明¹, 朱良全¹, 蒋卉¹, 孙石静¹, 丁家波¹, 辛凌翔¹, 王楠¹, 李晓宁¹, 李巧玲¹, 毛开荣¹, 蔡亚南², 徐磊¹

1. 中国兽医药品监察所国家/OIE布鲁氏菌病参考实验室, 北京 100081;
2. 吉林农业大学动物科学技术学院, 长春 130118

收稿日期:2020-05-31 出版日期:2020-11-20 发布日期:2020-11-20
通讯作者: 蔡亚南, 徐磊 E-mail:caiyanan0925@163.com;609697832@qq.com
作者简介:孙浩杰(1993-),男,江苏徐州人,硕士,研究方向:布鲁氏菌病疫苗,E-mail:sunhaojie0227@163.com;任小侠(1988-),女,北京人,硕士,研究方向:人畜共患病生物制品,E-mail:npprxx@126.com
基金资助:
国家重点研发计划"牛羊重要疫病免疫防控新技术研究"（2017YFD0500900）和"现代奶牛高效安全养殖技术应用与示范"（2018YFD0501600）

Construction and Identification of an Induced Strain of Rough Brucella abortus

SUN Haojie^1,2, REN Xiaoxia¹, QIN Yuming¹, ZHU Liangquan¹, JIANG Hui¹, SUN Shijing¹, DING Jiabo¹, XIN Lingxiang¹, WANG Nan¹, LI Xiaoning¹, LI Qiaoling¹, MAO Kairong¹, CAI Yanan², XU Lei¹

1. National/OIE Reference Laboratory for Brucellosis, China Institute of Veterinary Drugs Control, Beijing 100081, China;
2. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China

Received:2020-05-31 Online:2020-11-20 Published:2020-11-20

摘要/Abstract

摘要： 本试验旨在研究布鲁氏菌病疫苗的新型研制方法，并通过op诱导剂成功诱导出一株粗糙型牛种布鲁氏菌弱毒株，命名为RB71。试验检测了RB71相关基因的缺失情况，并对其脂多糖完整性、生长特性、遗传稳定性以及在小鼠巨噬细胞（RAW264.7）中生存能力等与光滑型菌株进行了比较研究。结果显示，试验成功获得了诱导突变株，其缺失片段大小为15 070 bp；热凝集试验阳性，能被结晶紫染色，吖啶黄凝集试验阳性；对提取脂多糖进行银染，结果显示O链缺失，诱导株RB71脂多糖不完整；连续传代30次，PCR检测未发现基因回复突变；在体外相同培养条件下，诱导株RB71生长速度显著低于亲本株A19；入侵RAW264.7细胞72 h时，其胞内存活率与亲本株相比极显著下降（P<0.01）。综上所述，本试验开发出一种能高效诱导光滑型布鲁氏菌变异为粗糙型布鲁氏菌的试剂及诱导方法，成功获得一株具有良好遗传稳定性的粗糙型减毒布鲁氏菌RB71诱导株，该诱导株在RAW264.7细胞内的存活能力显著变弱，这为新型弱毒布鲁氏菌粗糙型疫苗的研制奠定技术基础。

关键词: 诱导; 粗糙型; 布鲁氏菌; 疫苗

Abstract: The purpose of the experiment was to study a new development method of brucellosis vaccine,and to successfully induce a rough Brucella abortus attenuated strain named RB71 by op inducer.Deletions of RB71-related genes were detected,The LPS integrity,growth characteristics,genetic stability,and viability in murine macrophage RAW264.7 cells were compared with those in smooth strains.The results showed that the mutant was successfully induced in the test,and the size of the missing fragment was 15 070 bp.The heat agglutination test was positive,which could be stained by crystal violet,and the acridine yellow agglutination test was positive.The extraction of lipopolysaccharide for silver staining showed that the O chain was deleted,and the induced strain RB71 lipopolysaccharide was incomplete.No gene mutation was detected by PCR after 30 consecutive passages.Under the same culture conditions in vitro,the growth rate of the induced strain RB71 was significantly lower than that of the parent strain A19.When infected with RAW264.7 macrophages in mice for 72 h,the intracellular survival rate was significantly reduced than that of the parent strain (P<0.01).In summary,this experiment successfully obtained an induced strain of Brucella RB71 with good genetic stability through the op inducer mutagenesis technique the survival ability of the induced strain in RAW264.7 cells was significantly weakened,which laid the technical foundation for the development of a new attenuated Brucella rough vaccine.

Key words: induce; rough; Brucella; vaccines

中图分类号:

S852.61

孙浩杰, 任小侠, 秦玉明, 朱良全, 蒋卉, 孙石静, 丁家波, 辛凌翔, 王楠, 李晓宁, 李巧玲, 毛开荣, 蔡亚南, 徐磊. 一株粗糙型牛种布鲁氏菌诱导株的构建及鉴定[J]. 中国畜牧兽医, 2020, 47(11): 3445-3453.

SUN Haojie, REN Xiaoxia, QIN Yuming, ZHU Liangquan, JIANG Hui, SUN Shijing, DING Jiabo, XIN Lingxiang, WANG Nan, LI Xiaoning, LI Qiaoling, MAO Kairong, CAI Yanan, XU Lei. Construction and Identification of an Induced Strain of Rough Brucella abortus[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3445-3453.

参考文献

[1] POETSCH A,MARCHESINI M I.Proteomics of Brucella[J].Proteomes,2020,8(2):8.
[2] 赵建东,解松林,项朝荣,等.山羊不同途径免疫S2株布鲁氏菌病活疫苗后的抗体消长规律[J].黑龙江畜牧兽医,2018,4:136-137. ZHAO J D,XIE S L,XIANG C R,et al.Regularity of antibody variability in goats immunized with S2 strain Brucella[J].Heilongjiang Animal Science and Veterinary Medicine,2018,4:136-137.(in Chinese)
[3] 葛瑞华,龚小林,潘乳玲.布鲁氏菌病及动物疫苗的研究进展[J].当代畜牧,2017,6:53-55. GE R H,GONG X L,PAN R L.Current application research advances of brucellosis and vaccines[J].Contemporary Animal Husbandry,2017,6:53-55.(in Chinese)
[4] 王珊珊,楚遵锋,胡莉萍,等.家畜布鲁氏菌病的综合防控及净化措施[J].山东畜牧兽医,2019,40(6):82-85. WANG S S,CHU Z F,HU L P,et al.Comprehensive prevention,control and purification measures for livestock brucellosis[J].Shandong Journal of Animal Science and Veterinary Medicine,2019,40(6):82-85.(in Chinese)
[5] 涂明亮,葛兴农,雷元元,等.布病疫苗不同免疫方法对比评价[J].农业开发与装备,2019,8:114-115. TU M L,GE X N,LEI Y Y,et al.Comparative evaluation of different immunization methods for brucellosis vaccine[J].Agricultural Development & Equipments,2019,8:114-115.(in Chinese)
[6] 熊流新,黄健,张涛,等.布鲁氏菌外膜蛋白Omp16的原核表达、纯化及多克隆抗体制备[J].现代医药卫生,2018,34(5):669-673. XIONG L X,HUANG J,ZHANG T,et al.Prokaryotic expression,purification and polyclonal antibody preparation of outer membrane proteins Omp16 from Brucella[J].Journal of Modern Medicine & Health,2018,34(5):669-673.(in Chinese)
[7] 张淑琴.布氏杆菌病疫苗生产工艺的改进及效果评价研究[D].乌鲁木齐:新疆农业大学,2011. ZHANG S Q.Improvement of production process for the brucellosis vaccine and effect evaluation[J].Urumqi:Xinjiang Agricultural University,2011.(in Chinese)
[8] 农业部兽用生物制品规程委员会主编.中华人民共和国兽用生物制品规程[M].北京:化学工业出版社,2000. COMMITTEE OF VETERINARY BIOLOGICS OUTLINES OF MINISTRY OF AGRICULTURE.Regulations of the People's Republic of China on Veterinary Biological Products[M].Beijing:Chemical Industry Press,2000.(in Chinese)
[9] 张阁.利用比较基因组学筛选粗糙型布鲁氏菌RM57株致弱相关基因[D].北京:中国兽医药品监察所,2017. ZHANG G.Screening of weaken related genes of Brucella RM57 by comparative genomics[D].Beijing:China Institute of Veterinary Drug Control,2017.(in Chinese)
[10] 徐先栋,谢珍玉,王世锋,等.脂多糖快速银染检测方法的改良[J].生物技术通报,2009,3:95-97. XU X D,XIE Z Y,WANG S F,et al.A modified silver-staining method for lipopolysaccharides detection[J].Biotechnology Bulletin,2009,3:95-97.(in Chinese)
[11] 李小明,田波,杨儒爱,等.布鲁氏菌病的危害及流行情况概述[J].兽医导刊,2019,5:29-30. LI X M,TIAN B,YANG R A,et al.Overview of the hazards and prevalence of brucellosis[J].Veterinary Orientation,2019,5:29-30.(in Chinese)
[12] 丁家波,冯忠武.动物布鲁氏菌病疫苗应用现状及研究进展[J].生命科学,2013,25(1):91-99. DING J B,FENG Z W.Current application of brucellosis vaccines and its research advances[J].Chinese Bulletin of Life Sciences,2013,25(1):91-99.(in Chinese)
[13] 景志刚,严家瑞,范伟兴.布鲁氏菌病疫苗研究进展[J].中国人兽共患病学报,2016,32(2):188-199. JING Z G,YAN J R,FAN W X.Research progress of brucellosis vaccines[J].Chinese Journal of Zoonoses,2016,32(2):188-199.(in Chinese)
[14] 曹小安,邱昌庆,秦天达.布鲁氏菌毒力基因的研究状况[J].中国人兽共患病学报,2009,25(7):689-693. CAO X A,QIU C Q,QIN T D.Research status of Brucella virulence genes[J].Chinese Journal of Zoonoses,2009,25(7):689-693.(in Chinese)
[15] 王小蕾,吴清民.布鲁菌脂多糖的研究进展[J].中国预防兽医学报,2016,38(1):82-86. WANG X L,WU Q M.Research progress of Brucella lipopolysaccharide[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(1):82-86.(in Chinese)
[16] 邓丽芳,黄亚宁,罗雯.布鲁菌病疫苗研制进展[J].陕西农业科学,2012,58(2):130-134. DENG L F,HUANG Y N,LUO W.Development of brucellosis vaccine[J].Shaanxi Journal of Agricultural Sciences,2012,58(2):130-134.(in Chinese)
[17] 丁家波.基因缺失粗糙型布鲁氏菌rS2-ΔWboA株的构建及生物学特性研究[C].中国畜牧兽医学会兽医公共卫生学分会第三次学术研讨会论文集[A].2012. DING J B.Construction and biological characteristics of the gene deletion rough Brucella rS2-ΔWboA strain[C].Proceedings of the Third Symposium of Veterinary Public Health Branch of China Animal Husbandry and Veterinary Association[A].2012.(in Chinese)
[18] HOU H,LIU X,PENG Q.The advances in brucellosis vaccines[J].Vaccine,2019,37(30):3981-3988.
[19] 刘志国,王妙,崔步云,等.布鲁氏菌胞内存活及疫苗研究进展[J].中国人兽共患病学报,2019,35(5):68-77. LIU Z G,WANG M,CUI B Y,et al.Brucella intracellular survival and vaccine research progress[J].Chinese Journal of Zoonoses,2019,35(5):68-77.(in Chinese)
[20] 王温,吕学增,岩枫,等.布鲁氏菌M-111菌种对绵羊免疫效力的研究[J].中国人兽共患病学报,1990,6(3):63. WANG W,LYU X Z,YAN F,et al.Study on the immunological efficacy of Brucella M-111 strain on sheep[J].Chinese Journal of Zoonoses,1990,6(3):63.(in Chinese)
[21] 丁家波,王芳,杨宏军,等.一株中等毒力牛种布鲁氏菌的鉴定和毒力测定[J].中国农业科学,2014,47(13):2652-2658. DING J B,WANG F,YANG H J,et al.Identification and virulence assessment for a strain of Brucella abortus[J]. Scientia Agricultura Sinica,2014,47(13):2652-2658.(in Chinese)

一株粗糙型牛种布鲁氏菌诱导株的构建及鉴定

Construction and Identification of an Induced Strain of Rough Brucella abortus

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	韩冰, 汤思淇, 朱大帷, 李海玲, 冯亚莉, 苏超凡, 徐艺娜, 冷昊钰, 王永涛, 张月美, 太晓春, 张莹. 新型疫苗佐剂研究进展[J]. 中国畜牧兽医, 2023, 50(6): 2460-2467.
[2]	余新刚, 袁凯健, 穆宣儒, 王红彩, 戚南山, 张浩吉. 贾第虫包囊壁形成及囊壁蛋白研究进展[J]. 中国畜牧兽医, 2023, 50(5): 2061-2068.
[3]	陈天宝, 王娅妮, 刘宝玲, 王刚, 王晓虎, 柳旭辉, 蔡汝健, 贺东生. 中药抗猪繁殖与呼吸综合征病毒的作用机制研究进展[J]. 中国畜牧兽医, 2023, 50(5): 2114-2129.
[4]	陈吉敏, 李辉煌, 赖连杰, 杨银华, 杨悦, 林瑞意. 鸭坦布苏病毒检测方法及疫苗研究进展[J]. 中国畜牧兽医, 2023, 50(4): 1329-1339.
[5]	刘新月, 栗利芳, 王振豹, 张文亮, 张欣, 赵洪涛, 李鹏宇, 郑昊天, 肖进, 齐鹏. 猪圆环病毒2型合成肽疫苗佐剂筛选与免疫保护性研究[J]. 中国畜牧兽医, 2023, 50(3): 1068-1080.
[6]	刘馨, 潘吉脉, 胡安东, 曾成容, 杨颖, 程振涛, 陈江凤, 姜海波, 文明. 黄芪多糖对杂交鲟用嗜水气单胞菌灭活疫苗的影响[J]. 中国畜牧兽医, 2023, 50(3): 1177-1184.
[7]	李琦, 田野, 黄涛, 刘存霞. 纳米疫苗在禽流感防控中的研究进展[J]. 中国畜牧兽医, 2023, 50(2): 684-692.
[8]	魏春燕, 郭嘉, 朱德馨, 张伟, 朱嘉乐, 邓兴梅, 贾思锋, 刘良波, 张辉. 布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响[J]. 中国畜牧兽医, 2023, 50(1): 26-36.
[9]	徐景峨, 李婷, 陈汝佳, 余波, 张亚楠, 杨莉, 赵滨, 蒲龄. 鸭疫里氏杆菌二价灭活疫苗制备及免疫效力研究[J]. 中国畜牧兽医, 2023, 50(1): 290-299.
[10]	刘宏莹, 王婧瑜, 崔珍珍, 马青霞, 汪婷婷, 李健明, 时坤, 宫庆龙, 杜锐, 冷雪. 中药有效成分对动物疫苗的免疫佐剂效果及相关机理研究进展[J]. 中国畜牧兽医, 2023, 50(1): 390-397.
[11]	高也凡, 宋哈楠, 王育南, 吴月, 牛锐利, 宗宪春, 关伟军. 北京油鸡角膜缘干细胞分离培养及分化潜能研究[J]. 中国畜牧兽医, 2022, 49(9): 3372-3381.
[12]	韩瑞, 杨行, 张文波, 陈佳玲, 周晓丽. 猪链球菌自溶素的原核表达及其抗体间接ELISA检测方法的建立[J]. 中国畜牧兽医, 2022, 49(9): 3508-3519.
[13]	赵逢淼, 郭婷, 周雅坪, 赵红梅, 王宇琛, 田广原, 孙雅婕, 卞宇晨, 于佳梁, 郝永清. 牛病毒性腹泻病毒对新西兰白兔致病性分析及E2蛋白免疫效果的评价[J]. 中国畜牧兽医, 2022, 49(9): 3549-3558.
[14]	贾新月, 马静, 张艳艳, 马勋, 王正荣, 薄新文. 动物寄生虫口服疫苗研究进展[J]. 中国畜牧兽医, 2022, 49(9): 3569-3580.
[15]	邓兴梅, 曹树珠, 郭嘉, 朱德馨, 赵天艺, 柴迎锦, 张伟, 史超, 贾思锋, 张辉. 长链非编码RNA Gm35082-202对布鲁氏菌引起的细胞焦亡的影响[J]. 中国畜牧兽医, 2022, 49(9): 3599-3609.