用大肠杆菌制备O157多糖-菌毛蛋白结合物的研究

目的以大肠杆菌O157型多糖与菌毛蛋白PilE为模型,在大肠杆菌中重构脑膜炎奈瑟球菌的O-糖基化修饰系统,为建立一步生物交联法制备多糖-蛋白结合疫苗技术提供基础。方法用Red重组技术敲除大肠杆菌W3110的O抗原连接酶基因waaL,构建CLM24菌株。在该菌株中共表达脑膜炎奈瑟球菌菌毛蛋白基因pilE、寡糖转移酶基因pglL,构建O-糖基化修饰系统。分别转化大肠杆菌鼠李糖转移酶基因wbbL与大肠杆菌O157型多糖表达载体于构建了O-糖基化修饰系统的大肠杆菌,再利用Western印迹检测菌毛蛋白PilE的修饰情况。结果利用抗His-tag单抗进行Western印迹检测,16℃IPTG诱导条件下,转化大肠杆菌鼠李糖转移酶基因wbbL于构建了O-糖基化修饰系统的大肠杆菌,重组菌CLM24/pMMB66EH-pilE-his/pETtac28-wbbL-pglL与其他对照菌相比,不仅在相对分子质量为19×103处有特异性条带,而且在相对分子质量（40～60）×103处也出现了特异的梯状条带;转化大肠杆菌O157的O多糖基因簇克隆载体于构建了O-糖基化修饰系统的大肠杆菌,重组菌CLM24/pMMB66EHpilE-his/pETtac28-pglL/pACYC184-O157用抗O157多抗和抗His-tag单抗进行Western印迹检测,在相对分子质量（40～60）×103处均产生特异的梯状条带。结论在大肠杆菌中重建的O-糖基化修饰系统可以利用自身的O16型O多糖或异源的O157型O多糖修饰菌毛蛋白PilE,获得多糖-蛋白结合物。本研究为建立一步生物交联法制备多糖-蛋白结合疫苗提供了技术基础。

Production of O157 polysaccharides-pilin conjugates in Escherichia coli

Objective To reconstruct the Neisseria meningitides O-linked protein glycosylation system in Escherichia coli and to investigate whether PglL is able to covalently link O-polysaccharides of O157 to the acceptor protein PilE in order to facilitate the one step bioconjugation of novel conjugate vaccines.Methods The Red recombination system was used to delete the O-antigen ligase gene（waaL） of W3110 to gain CLM24.The N.meningitides O-linked protein glycosylation system was reconstructing by co-expressing the pilin gene pilE and the oligosaccharide transferase gene pglL of N.meningitides in CLM24.The rhamnose transferase gene wbbL or the O-polysaccharides of E.coli O157 was transferred to this system before the PilE modified by the O-polysaccharides was detected by Western blotting.Results The CLM24 / pMMB66EH-pilEhis / pETtac28-wbbL-pglL had a ladder-like band between（40-60） × 103besides a specific band at 19 × 10^3by Western blotting with anti-His tag monoclonal antibody.Similarly,the CLM24 / pMMB66EH-pilE-his / pETtac28-pglL / pACYC184O157 also had a ladder-like band between（40-60） × 103by Western blotting with anti-His tag monoclonal antibody or anti-E.coli O157 polyclonal antibody.Conclusion The N.meningitides O-linked protein glycosylation system is successfully transferred to E.coli.This system can modify the PilE by means of the homorganic O16 type O-polysaccharides or heterogenic O157 type O-polysaccharides.