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猪链球菌2型中国强毒株05ZYH33 ofs 基因敲除突变株构建及其生物学特性研究

ISSN号：1002-2694
期刊名称：中国人兽共患病学报
时间：2014.3.15
页码：223-230
分类：R378.1[医药卫生—病原生物学;医药卫生—基础医学]
作者机构：[1]温州医学院检验医学院、生命科学学院,温州325035, [2]南京军区军事医学研究所,南京210002
相关基金：国家自然科学基金（No.81172794;No.81071317;No.31170124;No.81171527）;江苏省自然科学基金资助项目（No.BK2011098;No.BK2011097）;南京军区医学科技创新课题（ZX39）资助
相关项目：新现人兽共患病原菌猪链球菌组氨酸三聚体家族蛋白对细菌致病性的作用及促进细菌免疫逃避的机制研究

关键词：猪链球菌2型, ofs, 基因敲除, 生物特性, 毒力, Streptococcus suis 2 , ofs, gene knock-out, biological characteristics, virulence assay

中文摘要：

目的探究猪链球菌血清浑浊因子ofs基因在强致病性2型猪链球菌致病过程中的作用。方法利用同源重组基因敲除方法构建中间为壮观霉素抗性基因，两侧为ofs编码基因上、下游同源序列的基因敲除质粒，将构建好的质粒电转化入猪链球菌感受态，同源重组筛选ofs基因敲除突变株，通过组合PCR、Southern杂交和DNA测序对疑似突变株进行验证。生物学功能实验研究比较ofs突变株和野生株05ZYH33在革兰氏染色、菌落溶血活性、生长速率和毒力等方面的差异。结果组合PCR、Southern杂交和DNA测序结果均证实ofs基因敲除突变株05ZYH33△ofs构建成功，体外实验结果显示ofs基因缺失后菌株在革兰氏染色和菌落溶血活性及形态、生长速率等方面未发生改变，但小鼠毒力实验数据结果表明敲除突变株△ofs加的毒力降低。结论猪链球菌2型ofs基因与细菌的毒力相关，本实验构建的突变株05ZYH33 △ofs为进一步研究猪链球苗2型的致病机理奠定某础。

英文摘要：

To study the functionality of the opacity factor of S. suis （ofs） in pathogenicity of Chinese virulent strain 05ZYH33, we constructed a gene knock-out mutant of ofs gene based on the principle of homologous recombination. Recombi- nant gene knock-out vector was constructed consisting of Spc＇cassette with the flanking homology regions of gene ofs. The re- suiting mutant strains were further confirmed by PCR analysis, Southern hybridization and DNA sequencing. To better assess the role of ofs gene in the virulence of 05ZYH33, gram strain analysis, hemolytic activity, growth characteristics, and experi- mental infection of mice was adopted. The results suggested that the mutant of 05ZYH33 ofs gene was successfully construc- ted. Analysis of biological characteristics showed that there was no obvious distinction in gram strain analysis, hemolytic activi- ty and growth rates between the mutant and the wild type strain 05ZYH33. Virulence assays with murine model confirmed that the mutant was a low virulent strain. These observations indicate that ofs is critical for the full virulence of 05ZYH33. The construction of the mutant strain 05ZYH33△ofs laid the foundation for the further study on the mechanisms of S. suis 2 during infection.

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