APEC强毒力岛核心基因irp2、fyuA敲除对其致病性影响的研究

The relationship between the core gene irp2、fyuA deletion of the high pathogenicity island and the pathogenicity of APEC

采用Red同源重组技术分别构建出APEC irp2单基因敲除株△AE17和irp2、fyuA双基因敲除株△△AE17。运用活菌计数法分别观察AE17、△AE17、△△AE17黏附鸡胚成纤维细胞DF-1的能力。并且应用Real-time PCR检测AE17、△AE17和△△AE17中的luxs,pfs,tsh,ibeA,stx2f,iss,ompA,fimC 8个毒力基因转录水平。结果成功构建出基因敲除株△AE17和△△AE17,它们黏附DF-1细胞的能力分别下降为AE17的66.04%和53.54%。同时,Real-time PCR结果显示,△AE17的luxs,iss,ompA和fimC等4个毒力基因的转录水平均极显著下降(P<0.01),△△AE17的luxs,pfs,tsh,iss,ompA和fimC等6个毒力基因的转录水平极显著的下降(P<0.01)。结果表明强毒力岛中核心基因的敲除能够减弱APEC黏附DF-1的能力,并且使其毒力基因的转录水平有所降低。irp2、fyuA双基因敲除较irp2单基因敲除对APEC的致病性影响更显著。

Abstract：The the Red homologous recombination method was used to construct the irp2 gene dele- tion strain △AE17 and the irp2, fyuA genes deletion strain △△AE17. Viable bacteria counting method was used to evaluate the effects of AE17, △AE17 and △△AE17 on the capability of APEC to adhere and invade DF-lcell. Their effects on the mRNA levels of the luxs,pfs,tsh,ibeA, stx2f,iss,ompA and fimC virulence genes were analyzed using real-time PCR. The results showed that succeed constructed APEC strains △AE17 and △△AE17 were. The adherences of △AE17 and △△AE17 were decreased by 66.04% and 53.54%. Real-time PCR showed that the transcrip- tion of luxs,iss,ompA, fimC genes of △AE17 was decreased and the transcription of luxs, pfs, tsh,iss,ompA,fimC genes of △△AE17 was decreased. The invasion to DF 1 cells of the bacteria were decreased and transcription of the virulence genes were decreased by the core gene deletion of high pathogenicity island in the APEC. The influence of irp2, fyuA genes deletion is more impor- tant to the pathogenicity in APEC when compared with the irp2 gene deletion.