高致病性猪链球菌双组分系统Ihk/Irr双基因缺失突变株的构建

目的构建Ⅱ型猪链球菌05ZYH33菌株双组分系统Ihk/Irr的双基因缺失突变株.方法首先对双组分系统Ihk/Irr基因进行序列分析;选取Ihk/Irr基因的相关片段克隆至p ET30a表达载体,进行可溶表达并注射家兔制备抗体;分别将ihk基因上游irr基因下游各1 kb的片段扩增,将两个片段连接起来;克隆至温敏型p JRS233的穿梭质粒中;利用两步同源重组法获得突变体;分别提取突变株的基因组DNA,RNA及菌体总蛋白,利用PCR,RT-PCR,Western-blot方法验证突变体.结果成功制备了双组分系统Ihk/Irr可溶性表达的蛋白,成功构建了重组的温敏型ikr-p JRS233重组质粒,成功将重组质粒转入猪链球菌05ZYH33中并获得突变株,基因组PCR,RTPCR及Western-blot分别证实了双组分系统Ihk/Irr双基因被成功敲除.结论成功构建重组温敏型的穿梭质粒,导入到猪链球菌05ZYH33菌株中经两步同源重组获得突变株,在基因组DNA,RNA及蛋白水平上验证了双组分系统Ihk/Irr双基因缺失株的成功构建.

Construction of Two-component System Ihk/Irr Double Gene Deletion Mutant Strain in Highly Pathogenic Streptoccus Suis

Objective To construct two-component Ihk/Irr double gene deletion mutant of Streptococcus suis typeⅡ strain 05ZYH33. Method Firstly,the sequence analysis of Ihk/Irr gene in two component system was performed. The related fragments of Ihk/Irr gene were cloned into pET30a expression vector for soluble expression and injected into rabbits to prepare antibodies.The fragments of 1 kb from upstream of irr gene and 1 kb from downstream of ihk gene were amplified and connected. Then,the fragment was cloned into shuttle plasmid of thermosensitive pJRS233,and the mutant of Streptococcus suis was obtained by two step homologous recombination method at different temperatures.The genomic DNA,RNA and total protein of the mutant strain were extracted,and the mutants were verified by PCR,RT-PCR and Western-blot. Results The protein was successfully expressed in soluble Ihk/Irr two-component system,and the recombinant thermosensitive type ikr-pJRS233 was prepared. The recombinant plasmid was transformed into Streptococcus suis 05ZYH33,and the mutant strains were subcultured by two step homologous recombination method,respectively.Genomic PCR,RT-PCR and Western-blot all confirmed that the two-component system Ihk/Irr double gene was successfully knocked out. Conclusion A recombinant thermosensitive shuttle plasmid was successfully constructed and imported into Streptococcus suis 05ZYH33 strain. The mutant was obtained through two steps homologous recombination.The reconstructs of two component Ihk/Irr double gene deletion strain were verified on genomic DNA,RNA and protein level.