猪链球菌2型05Z33强毒株转录调控因子Rgg基因敲除突变体的构建及毒力分析

Construction and Virulence Evaluation of Gene Knock-Out Mutant of a Transcriptional Regulator Rgg of Virulent Strain 05ZYH33 of Streptococcus suis Type 2

目的:构建猪链球菌2型强毒株05ZYH33转录调控因子Rgg的基因敲除突变体,观察其生物学性状,并在动物感染实验中比较敲除株与野生株的毒力差异,为进一步研究猪链球菌转录调控因子在致病中的作用提供实验基础。方法:分别以猪链球菌2型05ZYH33基因组和pSET1质粒为模板,扩增基因SSU05_1997两侧各约500 bp的片段为上下游同源臂,氯霉素(Cm)抗性基因为中间片段,采用重叠PCR方法连接3个片段;连接产物先克隆到T载体上,再经过酶切克隆到温度敏感自杀载体pSET4S上;将构建的基因敲除载体pSET4S-1997电转化入05ZYH33感受态细胞,通过改变培养温度筛选出基因敲除突变体05Z33△rgg;对敲除株和野生株的生物学性状及小鼠和猪的致病性进行了初步比较。结果:PCR分析和测序结果均显示基因SSU05_1997完全被Cm抗性基因所替代,基因敲除突变体构建成功;05ZYH33△rgg对小鼠和猪的致病性与野生株相比无明显差异。结论:转录调控因子Rgg可能和猪链球菌2型的毒力无关。

Objective： To construct gene knock-out mutant of an transcriptional regulator gene SSU05_1997 of Streptococcus suis type 2（SS2） virulent strain 05Z33,characterize its biological feature and compare the virulence with the wild-type virulent strain in an animal model in order to provide an experimental basis for studying the role of SS2＇s transcriptional regulators in pathogenesis.Methods： Genomic DNA of S.suis type 2 05Z33 and pSET1 plasmid were used as template to amplify flanking fragments about 500 bp of gene SSU05_1997 and the chromosomal（Cm） resistance cassette respectively.Cm cassette with flanking homology regions were connected by an overlap extension PCR method.The connected product was first cloned to T vector,then to temperature sensitive suicide vector pSET4S after digestion.The plasmid pSET4S-1997 was transformed into wild strain 05Z33,and the gene knock-out mutant strain 05Z33△rgg was screened by changing culture temperature.Biological characteristics and virulence of mutant strain 05Z33 △rgg in mice and pigs were compared with wild-type strain 05ZYH33.Results： PCR analysis and sequencing confirmed a completely replacement of coding gene SSU05_1997 by Cm resistance cassette and a successful construction of gene knockout mutant.There were no significant differences in virulence in mice and pigs between mutant strain 05ZYH33△rgg and wild-type strain 05Z33.Conclusion： Transcriptional regulator Rgg may not relevant with virulence in S.suis type 2.