Tricistronic expression,assembly and biological activity of CDT of Haemophilus parasuis
Haemophilus parasuis(H.parasuis)is a common agent of swine.Cytolethal distending toxin(CDT)which forms a heterotrimer before secreting to the extracellular environment is an important virulence factor of the agent.It is vital to prepare the recombinant holotoxin for the functional study of CDT.A tricistronic expression vector which could be used to express all three subunits of CDT was constructed in this study.Meanwhile,parameters for assembly and purifica-tion of the recombinant CDT holotoxin were optimized,and the bioactivity of the reconstituted ho-lotoxin was verified in this study.The result showed that the assembly and purification effects was the best when the combination of CdtA/CdtB/CdtC-his was adopted,and when the three subunits was mixed at 0.1 g/L with equal volume and dialyzed in PBS with urea concentration gradient.Three CDT subunits were expressed simultaneously when the recombinant tricistronic plasmid was induced in E.coli,and the three recombinant subunits were all in the insoluble fraction as in-clusion bodies which could be used for reconstitution of CDT holotoxin after brief purification by centrifugation.Pure holotoxin was obtained from the reconstituted heterotrimer solution by affinity chromatography and molecular sieve chromatography.Obvious cell distention was observed when Marc145 cells were incubated with CDT holotoxin,and the ratio of cells which were blocked in the G2/M phase of the cell cycle increased remarkably.In this study,we established a simple and effec-tive method for preparation of recombinant CDT holotoxin of H.parasuis,and the holotoxin showed good bioactivity in cellular toxicity assays.
NETL
NSTL
万方数据
