[1]刘 韬?,魏文燕?,刘家星,等.鱼源鲁氏耶尔森菌mobBC基因无痕缺失株的构建及其毒性研究[J].中国预防兽医学报,2019,(09):891-898.[doi:0.3969/j.issn.1008-0589.201812032]
 LIU Tao?,WEI Wen-yan?,LIU Jia-xing,et al.Construction and virulence characterization of traceless-deleted mobBC mutant of Yersinia ruckeri[J].Chinese journal of preventive veterinary medicine,2019,(09):891-898.[doi:0.3969/j.issn.1008-0589.201812032]
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鱼源鲁氏耶尔森菌mobBC基因无痕缺失株的构建及其毒性研究()
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《中国预防兽医学报》[ISSN:1008-0589/CN:23-1417/S]

卷:
期数:
2019年09
页码:
891-898
栏目:
病原生物学
出版日期:
2019-10-25

文章信息/Info

Title:
Construction and virulence characterization of traceless-deleted mobBC mutant of Yersinia ruckeri
文章编号:
1008-0589(2019)09-0891-08
作者:
 

刘 韬1?魏文燕2?刘家星2汪开毓1*

 (1. 四川农业大学 鱼病研究中心,四川 成都 611130;2. 成都市农林科学院,四川 成都 610000)
Author(s):
 

LIU Tao1? WEI Wen-yan2? LIU Jia-xing2 WANG Kai-yu1*

 (1. Fisheries Department of Sichuan Agricultural University, Chengdu 611130, China;
2. Institute of fisheries of chengdu agriculture and forestry academy, Chengdu 610000, China)
关键词:
鲁氏耶尔森菌mobBC基因无痕缺失体内感染毒性
Keywords:
Yersinia ruckeri  mobBC gene  unmarked deletion  acute infection  virulence
分类号:
S941.41
DOI:
0.3969/j.issn.1008-0589.201812032
文献标志码:
A
摘要:
为探究鲁氏耶尔森菌(Yersinia ruckeri)的四型分泌系统(T4SS)中mobBC基因的作用,本研究构建了强毒力Y.ruckeri SC09菌株mobBC基因的无痕缺失株并研究了其生理变化和毒性影响。将mobBC基因上、下游同源臂克隆入自杀载体pLP12并转化供体菌β2163,得到pLP12-mobBC/β2163,进一步通过接合转移,将pLP12-mobBC导入受体菌株SC09中。利用抗性筛选和vmi480反向筛选获得无痕缺失株Y.ruckeri ΔmobBC,并进行PCR测序鉴定。同时构建回补株Y.ruckeriΔmobBC+pMobBC。通过电子显微镜观察缺失株形态变化,同时绘制野生型菌株、缺失株和回补株的生长曲线;将缺失株和野生型菌株感染模式小鼠,初步研究mobBC毒性;将野生型菌株、缺失株和回补株感染虹鳟鱼,进一步研究其对水生动物的体内毒性;对虹鳟鱼主要感染组织进行细菌计数并分析菌株在感染组织的分布差异;观察感染缺失株和野生型菌株的虹鳟鱼组织病理学和免疫组织化学差异,进一步分析毒性影响。结果显示,本实验构建了无痕缺失株Y.ruckeriΔmobBC,并回补了mobBC基因。缺失株与野生株超微结构对比变化不显著,但缺失株的生长性能弱于野生株和回补株,且表现出较弱的体内毒性和较轻的组织感染力。这提示mobBC基因可能是Y.ruckeri重要的毒力基因,该基因的缺失能够有效降低Y.ruckeri的增殖力和对宿主的感染力。本文首次研究了水产病原菌Y.ruckeri SC09的mobBC基因的毒性作用,为该病原菌的毒力研究增加了新的基础。
Abstract:
To investigate the toxicity of the mobBC gene in the type IV secretion system (T4SS) system of Yersinia ruckeri, this study constructed a traceless-deletion strain of the highly virulent Y.ruckeri SC09 strain and studied its physiological changes and toxic effects. The upper and lower homologous arms of the mobBC gene were cloned into the suicide vector pLP12 and further transformed into the donor strain β2163 to obtain pLP12-mobBC/β2163. The donor strain was introduced into the recipient strain SC09 by conjugative transfer. A traceless-deletion strain Y.ruckeriΔmobBC was obtained by resistance screening and reverse screening with vmi480 and identified by PCR sequencing. On this basis, the vector pBAD33cm-mobBC was constructed and Y.ruckeriΔmobBC was complemented to obtain the complement strain Y.ruckeriΔmobBC+pMobBC. The morphological changes of the knockout strains were observed by electron microscopy, and the growth curves of wild-type strains, knockout strains and complemented strains were drawn at the same time, and the mobBC toxicity was preliminarily confirmed by using the knockout strains and wild-type strains in the mice model, rainbow trout were infected with the three kinds of strains to further clarify their toxicity to aquatic animals, the bacteria load of the knockout strains and wild-type strains in the main infected tissues of rainbow trout were counted and the distribution of strains in infected tissues were analyzed, the pathological and immunohistochemical differences of rainbow trout were further analyzed for toxic effects. The results showed that this experiment constructed a trace-free strain Y.ruckeriΔmobBC and complemented the mobBC gene. No significant changes were observed in the ultrastructure of knockout strains and wild strains. However, the mutant strains showed a weaker proliferation than that of wild and complemented strains and showed weaker in vivo toxicity and lighter tissue infectivity. This suggests that the mobBC gene might be an important virulence gene of Y.ruckeri, and its deletion could effectively reduce the proliferative capacity of Y.ruckeri and the infectivity to the host. This research investigated the toxic effects of the mobBC gene of the important aquatic pathogen Y.ruckeri SC09 for the first time, which added a new foundation for the study of the virulence of the pathogen.

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             (本文编辑:彭永刚;英文编辑:于申业)

更新日期/Last Update: 2019-10-25