| 一株粗糙型牛种布鲁氏菌诱导株的构建及鉴定 |
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| 引用本文： | 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. |
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| 作者姓名： | 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 |
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| 作者单位： | 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 |
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| 基金项目： | 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） |
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| 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。
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| 关 键 词： | 诱导 粗糙型 布鲁氏菌 疫苗 |
| 收稿时间： | 2020-05-31 |
Construction and Identification of an Induced Strain of Rough Brucella abortus |
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| 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 & Veterinary Medicine,2020,47(11):3445-3453. |
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| Authors: | 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 |
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| Institution: | 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 |
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| 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. |
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| Keywords: | induce rough Brucella vaccines |
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