副猪嗜血杆菌CDT三顺反子的表达、组装及生物学活性  

Tricistronic expression,assembly and biological activity of CDT of Haemophilus parasuis

作　　者：李军星[1] 余斌[1] 徐丽华[1] 苏菲[1] 叶十一 袁秀芳[1] LI Junxing;YU Bin;XU Lihua;SU Fei;YE Shiyi;YUAN Xiufang(Instituteof Animal Husbandry and Veterinary Science,Zhejiang Academy of Agricultural Sciences,Hangzhou 310021,China)

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《中国兽医学报》2024年第2期283-289,共7页Chinese Journal of Veterinary Science

基　　金：浙江省自然科学基金资助项目(LY21C180002)。

摘　　要：副猪嗜血杆菌是猪的常见病原菌,以异源三聚体形式分泌到胞外环境的细胞致死性膨胀毒素(CDT)是该菌重要的毒力因子,制备完整的重组CDT对于其功能研究至关重要。本研究构建了可用于同时表达CDT 3个亚基的三顺反子重组质粒,优化了完整毒素三聚体的体外组装及纯化方法,并验证了重组三聚体毒素的生物学活性。结果显示,CDT 3个亚基以CdtA/CdtB/CdtC-his方式组合时,按0.1 g/L等体积混合,在PBS中以尿素浓度梯度透析时组装效果最好。构建的重组三顺反子质粒在大肠杆菌中可以同时表达出比例相当的3个CDT亚基,并且均以包涵体形式存在,经包涵体离心粗纯化后,即可用于重组三聚体CDT的组装。组装的三聚体CDT经亲和层析及分子筛层析纯化,得到高纯度的完整毒素。完整毒素可以引起Marc145细胞体积明显膨胀,且分裂周期停滞于G2/M期的细胞比例显著增加。结果表明,本研究建立了一种简单有效的副猪嗜血杆菌重组CDT完整毒素制备方法,所制备的CDT具有良好的生物学活性。Abstrsact: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 purification of the recombinant CDT holotoxin were optimized,and the bioactivity of the reconstituted holotoxin 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 inclusion 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 Marcl45 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 effective method for preparation of recombinant CDT holotoxin of H.parasuis,and the holotoxin showed good bioactivity in cellular toxicity assays.

关 键 词：副猪嗜血杆菌 细胞致死性膨胀毒素 三顺反子质粒 完整毒素 

分 类 号：S852.61[农业科学—基础兽医学]