牛种布鲁氏菌ClpS基因突变株的构建及其对毒力的影响

doi:10.16431/j.cnki.1671-7236.2020.11.041

中国畜牧兽医 ›› 2020, Vol. 47 ›› Issue (11): 3767-3773.doi: 10.16431/j.cnki.1671-7236.2020.11.041

牛种布鲁氏菌ClpS基因突变株的构建及其对毒力的影响

刘郁夫^1,2,4, 董浩³, 孙石静¹, 彭小薇¹, 陈瑞爱^2,4, 丁家波¹, 蒋卉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 102600;
2. 华农(肇庆)生物产业技术研究院有限公司, 肇庆 526238;
3. 中国动物疫病预防控制中心, 北京 102600;
4. 华南农业大学兽医学院, 广州 510642

收稿日期:2020-04-21 出版日期:2020-11-20 发布日期:2020-11-20
通讯作者: 蒋卉 E-mail:15011216921@163.com
作者简介:刘郁夫(1990-),男,广西贺州人,博士,研究方向:动物传染病学,E-mail:363995606@qq.com
基金资助:
国家重点研发计划（2016YFD0500902）

Construction of Brucella ClpS Gene Mutant Strain and Its Effect on Virulence

LIU Yufu^1,2,4, DONG Hao³, SUN Shijing¹, PENG Xiaowei¹, CHEN Ruiai^2,4, DING Jiabo¹, JIANG Hui¹

1. National/OIE Reference Laboratory for Brucellosis, China Institute of Veterinary Drug Control, Beijing 102600, China;
2. Zhaoqing Institute of Biotechnology Co., Ltd., Zhaoqing 526238, China;
3. China Animal Disease Control Center, Beijing 102600, China;
4. College of Veterinary Medicne, South China Agricultural University, Guangzhou 510642, China

Received:2020-04-21 Online:2020-11-20 Published:2020-11-20

摘要/Abstract

摘要： 试验旨在探究ClpS基因在布鲁氏菌中的作用，分析比较ClpS基因突变对布鲁氏菌毒力的影响。利用同源重组技术，构建布鲁氏菌ClpS基因突变株，通过检测细菌生长曲线、细菌LPS合成能力及其在巨噬细胞内的存活能力和小鼠模型中的毒力，比较亲本株2308和突变株ΔClpS两者之间的差异。结果显示，在相同的培养条件下，亲本株2308和突变株ΔClpS的细菌浓度无明显差异，且两者提取的LPS银染结果基本一致，表明ClpS基因突变不影响布鲁氏菌生长速度，不影响细菌LPS合成；在细胞感染模型中，突变株ΔClpS在感染后72 h的胞内存活能力极显著低于亲本株2308（P<0.01）；小鼠感染试验显示，在感染后1周，亲本株2308感染组和突变株ΔClpS感染组小鼠脾脏重量及细菌含量无显著差异，但在感染后4周，突变株ΔClpS感染组的小鼠脾脏细菌含量为10^3.93 CFU/g脾脏，显著低于亲本株2308（10^6.68 CFU/g脾脏，P<0.01），且突变株ΔClpS感染组的小鼠脾脏肿胀程度极显著低于亲本株2308（P<0.01）。综上所述，布鲁氏菌ClpS基因突变不影响细菌生长速度及细菌LPS合成能力，但ClpS基因突变可降低布鲁氏菌在小鼠脾脏内的定殖能力。

关键词: 布鲁氏菌; ClpS基因; 基因突变; 毒力

Abstract: The present study aimed to determine the role of ClpS gene,and to analyse the impact of ClpS mutation on the virulence of Brucella.A ClpS gene mutant strain,named ΔClpS was constructed by homologous recombination technology.The bacterial growth kinetics,the LPS synthesis ability and the survival ability of bacterial within macrophages as well as the virulence in mouse model were measured.In addition,the difference between parent strain 2308 and the mutant strain ΔClpS were compared.The results showed that under the same culture conditions,no difference in bacterial concentration was observed between 2308 and ΔClpS strains.The silver staining examination showed that the expression level of LPS extracted from two strains were similar,indicating ClpS gene mutation did not alter the growth rate and LPS synthesis ability of Brucella. In the cell infection assay,the survival ability of ΔClpS strain in cells was extremely significantly lower than that of 2308 strain at 72 h after infection (P<0.01).The results of mouse infection experiment showed that in the first week after infection,no significant difference in spleen weight and bacterial concentration between 2308 and ΔClpS strains infected mice was observed.However,at 4 weeks after infection,the bacterial concentration in spleen of ΔClpS infected mice was 10^3.93 CFU/g spleen,which was significantly lower than that of 2308 strain (10^6.68 CFU/g spleen,P<0.01).The spleen weight of ΔClpS infected mice was also remarkably lower than that of 2308 strain (P<0.01).In summary,the results suggested that the ClpS gene of Brucella did not play a role in Brucella growth rate and ability of LPS synthesis,whereas ClpS gene mutation decreased the ability of Brucella colonization in mouse spleen.

Key words: Brucella; ClpS gene; gene mutation; virulence

中图分类号:

S855.1

刘郁夫, 董浩, 孙石静, 彭小薇, 陈瑞爱, 丁家波, 蒋卉. 牛种布鲁氏菌ClpS基因突变株的构建及其对毒力的影响[J]. 中国畜牧兽医, 2020, 47(11): 3767-3773.

LIU Yufu, DONG Hao, SUN Shijing, PENG Xiaowei, CHEN Ruiai, DING Jiabo, JIANG Hui. Construction of Brucella ClpS Gene Mutant Strain and Its Effect on Virulence[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(11): 3767-3773.

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牛种布鲁氏菌ClpS基因突变株的构建及其对毒力的影响

Construction of Brucella ClpS Gene Mutant Strain and Its Effect on Virulence

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