基因芯片技术对赖型钩端螺旋体溶血素基因体内诱导差异表达的研究

Research on the Expression of Hemolysin Genes of Leptospira in vivo by Genechip

目的探讨经宿主体内环境诱导前后赖型钩端螺旋体溶血素致病基因表达的差异。方法以赖型钩端螺旋体56601株的染色体DNA为模板,PCR扩增出溶血素基因片段为探针,并通过VersArray Chipwriter系统构建基因芯片。提取感染宿主前后钩端螺旋体RNA,逆转录为cDNA,用随机引物PCR扩增,以HEX和CY5双色荧光标记并与基因芯片进行杂交,使用Genepix 4000B扫描仪扫描芯片,通过检测芯片上的荧光信号比值来研究钩端螺旋体溶血素基因在宿主体内的表达情况。结果成功构建赖型钩端螺旋体56601株溶血素基因芯片,芯片检测发现在宿主感染后钩体溶血素基因LA1029(Ratio=0.65)、LA1027(Ratio=0.53)具有明显上调表达(Ratio<0.67);LA3540(Ratio=1.88)、LA3937(Ratio=5.58)、LA1029(Ratio=3.00)在宿主肝脏中的表达比在血液中有明显增高(Ratio>1.5),而LA4004的表达在宿主肝脏中要明显低于血液中(Ratio=0.67);LA3937(Ratio=2.28)和LA1029(Ratio=2.20)在宿主肾脏中的表达要明显高于血液中(Ratio>1.5)。结论钩体溶血素基因的体内外表达和不同器官间的表达存在差异,这些差异表达的溶血素基因及产物在钩体病的致病中可能起重要的作用。

Objective To explore the expression of hemolysin genes of Leptospira in infected host.Methods Amplified the gene segment of hemolysin genes from the genome of Leptospira by PCR for gene probe.Manufacture genechip by the VersArray Chipwriter systerm.The total RNAs of Leptospira before and after infection host were extracted,reversely transcribed to cDNA,after the random PCR,the products were marked with HEX and CY5 respectively,and hybridized to genechip to demonstrate the expression of hemolysin genes of Leptospira.Results The hemolysin genes LA1029（Ratio=0.65）,LA1027（Ratio=0.53） were up-regulated after infection of host;LA3540（Ratio=1.88）,LA3937（Ratio=5.58）,LA1029（Ratio=3.00） were up-regulated and LA4004（Ratio=0.67） was down-regulated in live than in blood;LA3937（Ratio=2.28）,LA1029（Ratio=2.20） were up-regulated in kidney than in blood.Conclusion The expression level of hemolysin genes exist observable differences with inducement in vivo and in different organs.These suggested that these genes are probably involved in the pathogenesis and and disease progression.