类鼻疽伯克霍尔德菌bopA基因敲除株的构建及生物学特征
Construction and characterization of Burkholderia pseudomallei bopA gene knockout mutant
  
DOI:  10.13344/j.microbiol.china.180380
中文关键词:类鼻疽伯克霍尔德菌,bopA基因,基因敲除,生物学特征
英文关键词:Burkholderia pseudomallei, bopA gene, Gene knockout, Biological characteristics
基金项目:国家自然科学基金(81471914,81601832)
作者单位E-mail
卢晓雪 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038  
胡治强 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038  
梅国徽 2 陆军勤务学校门诊部 重庆 401331  
张雨 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038  
胡语涵 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038  
岳娟娟 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038  
向阳 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038 xyang_@hotmail.com 
毛旭虎 1 陆军军医大学(第三军医大学)药学与检验医学系临床微生物与免疫学教研室 重庆 400038 maoxh2012@hotmail.com 
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中文摘要:
      【背景】类鼻疽杆菌是一种能够引起人类疾病甚至死亡的胞内寄生菌,Ⅲ型分泌系统在该菌入侵上皮细胞、逃避宿主免疫以及毒力因子的分泌过程中发挥重要作用,其中bopA基因为TTSS-3基因编码的重要效应蛋白,在类鼻疽杆菌的免疫逃逸中发挥重要作用。【目的】构建类鼻疽杆菌bopA基因敲除菌株,并对其生物学特征进行初步研究。【方法】构建pK18mobSacB-ΔbopA自杀质粒,通过大肠杆菌S17-1λpair以接合的方式转入类鼻疽杆菌,利用同源重组敲除了bopA基因,并用蔗糖平板筛选出菌株,最后在细胞和动物水平检测敲除菌株的表型变化。【结果】构建了bopA敲除的类鼻疽菌株,并通过细胞和动物实验证实敲除bopA基因后,细菌的细胞侵袭和胞内存活以及体内定殖能力都显著降低。【结论】利用同源重组成功构建类鼻疽bopA基因敲除株,为深入研究该基因的作用靶点奠定了实验基础。
英文摘要:
      [Background] Burkholderia pseudomallei is an intracellular parasitic bacterium that can cause human disease and even death. The type III secretory system plays an important role in the bacterial invasion of epithelial cells, escaping host immunity and the secretion of virulence factors. The bopA gene is an important effector protein encoded by the TTSS-3 gene, and it plays an important role in the immune escape of the Burkholderia pseudomallei. [Objective] To construct Burkholderia pseudomallei bopA gene knockout mutant strain, and evaluate the biological characteristics of the mutant strain. [Methods] We constructed suicide plasmid pK18mobSacB-ΔbopA, and then transformed the plasmid into B. pseudomallei from Escherichia coli S17-1λpair by conjugation. bopA gene was knocked out by homologous recombination, and the mutant strain was selected by sucrose agar screening. The phenotypic variation of the mutant strain was finally evaluated at the cell and animal levels. [Results] We constructed B. pseudomallei bopA mutant strain successfully, and found the invasion rate, intracellular survival ability as well as the ability of colonization in vivo of mutant strain was significantly reduced. [Conclusion] We constructed B. pseudomallei bopA mutant strain by homologous recombination, to provide a basis for further understanding of this gene.
卢晓雪,胡治强,梅国徽,张雨,胡语涵,岳娟娟,向阳,毛旭虎.类鼻疽伯克霍尔德菌bopA基因敲除株的构建及生物学特征[J].微生物学通报,2019,46(2):233~242
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