副猪嗜血杆菌细胞致死膨胀毒素的细胞毒性研究

Cytotoxicity of the Cytolethal Distending Toxin of Haemophilus parasuis

本研究旨在原核表达副猪嗜血杆菌细胞致死膨胀毒素(Cytolethal distending toxin,CDT),并作用于猪髋动脉内皮细胞(Pig iliac endothelial cells,PIEC),以研究其细胞毒性。根据本实验室完成的副猪嗜血杆菌SH 0165株(血清5型)全基因组序列,针对cdtA、cdtB和cdtC基因序列设计引物,扩增的基因片段大小分别约为681、834和531bp。将靶基因克隆到原核表达载体pET28a中,再转化到E.coli BL21(DE3),IPTG诱导表达3h,SDS-PAGE和Western blot检测证实表达产物以包涵体形式存在,大小分别约36、34和28ku。通过体外重构毒素与PIEC细胞作用3h,继续培养72h观察细胞形态学变化。结果表明,CdtABC全毒素致PIEC细胞膨胀、空泡形成、细胞凋亡等,而其他试验组变化不显著。结果提示,CDT毒素可能在细菌感染与致病中发挥着重要作用。

The study was aimed at prokaryotic expressing cytolethal distending toxin of Haemophilus parasuis,and acted on Pig iliac endothelial cells（PIEC） to investigate toxin effects.According to the complete genome sequence of Haemophilus parasuis SH0165（serovar 5）,which finished by our laboratory,we designed a set of specific primer,the cdtA,cdtB and cdtC gene was amplified by PCR,respectively.The 681,834 and 531 bp amplified DNA fragment was cloned into pET-28a and the expression were induced in E.coli BL21（DE3） by IPTG for 3 hours.Results of Sodium docecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blot assay showed that the cdtA,cdtB and cdtC gene was expressed in the form of inclusion body,and the recombinant protein was about 36,34 and 28 kDa.Recombinant toxin acted on PIEC for 3 hours in vitro,and observed the morphological change after another culture of 72 hours.These results showed that CdtABC holotoxin induced PIEC distension,vacuolization and apoptosis,while the other test groups changed unobviously.The study indicated that CDT may play an important role in the infection and pathopoiesis of bacterial.