2型猪链球菌中国强毒株05ZYH33 ofs N-片段基因敲除株的构建

Deletion of the N-terminal Fragment of ofs Gene in Streptococcus Suis Serotype 2 Chinese Highly Virulent Strain 05ZYH33

通过生物信息学分析2型猪链球菌(Streptococcus suisserotype 2,S.suis2)中国强毒株05ZYH33的基因组,预测并发现猪链球菌血清浑浊因子(Opacity factor ofS.suis,OFS)的编码基因ofs.为了构建ofsN-末端片段ofs37-683的基因敲除株,首先构建中间为壮观霉素抗性基因,两侧为ofs37-683上、下游同源序列的基因敲除质粒,并对该质粒进行PCR和酶切鉴定.根据同源重组的原理,通过电转化方法筛选得到ofs37-683基因敲除株.PCR、测序和RT-PCR分析结果均显示ofs37-683已完全被壮观霉素抗性基因替代,证实ofs37-683基因敲除株构建成功.该敲除株的获得为进一步研究ofs在猪链球菌2型致病机制中的作用奠定基础.

The gene of Opacity factor of S. suis （ors） was predicted in the genome of Streptococcus suis serotype 2 （ S. suis 2） Chinese highly virulent strain 05ZYH33 and its protein structure was characterized with Bioinformaties tools. To delete the N-terminal fragment of ofs in 05ZYH33, recombinant gene knock-out plasmid with a Spcr cassette flanked with homology arms was constructed. The plasmid was confirmed by PCR and restriction enzyme digestion. According to the principle of homologous recombination, the isogenic ofs^37-683deficient mutant was screened through electroporation transformation. PCR amplication, sequencing of its product and RT - PCR analysis confirmed that the target gene was replaced by Spcr cassette. This mutated 05ZYH33 provided a potent tool for assessing the role of ofs in the S. suis 2 infection.