伴放线放线杆菌细胞致死性扩张毒素的细胞毒性研究

摘要

目的:体外诱导表达伴放线放线杆菌(Aqqregatibacter actinomycetemcomitans,Aa)细胞致死性扩张毒素(cytolethal distending toxin,CDT),观察其对中国仓鼠卵巢细胞(Chinese hamster ovary cell,CHO cell)和人宫颈癌上皮细胞(HeLa cell)的毒性作用.方法:诱导重组蛋白Aa CdtA、CdtB、CdtC的体外表达,采用镍亲和层析柱法纯化目的蛋白,体外重构Aa CDT全毒素.通过体外酶活性实验检测重组蛋白CdtB的生物学活性,并通过细胞克隆形成实验(Colony-forming experiment)及3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐[3-(4,5-dimethylthiazol-2-y1)-2,5-diphe nyltetrazolium bromide,MTT]比色法进一步观察CDT对CHO细胞和HeLa细胞的毒性作用.结果:由重组蛋白Aa CdtA、CdtB、CdtC体外重构获得的Aa CDT全毒素不仅抑制CHO细胞增殖,导致CHO细胞克隆形成单位(Colony-Forming Unit,CFU)数目减少甚至消失.也可抑制He-La细胞增殖并引起包括细胞体积增大及细胞核增大增多的细胞形态学改变,并且此增殖抑制作用呈浓度依赖性.结论:体外成功构建了具有生物学活性的Aa CDT全毒素,且Aa CDT毒性发挥需要三个亚基CdtA、CdtB、CdtC同时存在并构成三聚体.%AIM : To investigate the cytotoxicity of Aqqregatibacter actinomyceterncomitans cytolethal distending toxin(Aa CDT) holotoxin on CHO cells and HeLa cells.METHODS: The pET15b-cdtA, pET15b-cdtB and pET15b-cdtC vectors were transformed into Escherichia coli BL21.Expression of the recombinant Aa CdtA, CdtB,CdtC protein was induced by IPTG, and Ni affinity chromatogaphy was used to purify the recombinant proteins.Aa CDT holotoxin was reconstituted in vitro.Dnasel activity was used to verify the biological activity of rCdtB, while colony-forming experiment and MTI viable-cell assay were used to observe the cytotoxicity of Aa CDT.RESULTS : The Aa CDT holotoxin which was reconstituted by Aa rCdtA.rCdtB and rCdtC proteins could inhibit the proliferation of CHO cells and prevent the formation of colony-forming unit( CFU).The morphology of HeLa cells was changed and the cell proliferation was also inhibited by exposure to Aa CDT holoroxin.Moreover, the anti-proliferative activiW of Aa CDT on HeLa cells was in a dose-dependent manner.CONCLUSION : The biologically active Aa CDT holotoxin had been reconstituted successfuUy in vitro, and all of CdtA, CdtB and CdtC are required to form heterotnmer for the biological activity of Aa CDT.