病原菌多糖基因簇在大肠杆菌中的克隆  

Pathogens Polysaccharide Gene Cluster Cloned in Escherichia coli

作　　者：王雯[1,2] 孙鹏[2] 徐敏锐[3] 徐威[1] 吴军[2] 

机构地区：[1]沈阳药科大学,辽宁沈阳110016 [2]军事医学科学院生物工程研究所,北京100071 [3]安徽大学,安徽合肥230601

出　　处：《生物技术通讯》2014年第6期753-759,共7页Letters in Biotechnology

基　　金：重大创新药物创制专项(2012ZX09301003-001-005)

摘　　要：目的：构建稳定的外源病原菌多糖基因簇克隆载体，为在糖基工程大肠杆菌中利用外源性多糖O-糖基化修饰靶标蛋白奠定基础。方法：PCR扩增大肠杆菌O157、甲型副伤寒沙门菌CMCC50973和铜绿假单胞杆菌CMCC10110的O-多糖合成基因簇，将多糖基因簇与细菌人工染色体pCC1BAC连接后，分别转化O-多糖合成缺陷的大肠杆菌W3110，并用相应多糖抗血清ELISA检测重组大肠杆菌是否利用外源O-多糖生成脂多糖（LPS），从而验证外源多糖基因簇克隆载体在大肠杆菌内是否能够生成相应的O-多糖；在此基础上，将构建的3种外源多糖基因簇克隆载体分别转化表达O-寡糖转移酶和蛋白底物菌毛蛋白PilE的糖基工程大肠杆菌，用相应的抗血清进行Western印迹检测，以验证克隆的O-多糖能否修饰蛋白底物PilE。结果：与阴性对照菌相比，带有大肠杆菌O157的O-多糖合成基因簇克隆载体和带有甲型副伤寒沙门菌CMCC50973的O-多糖合成基因簇克隆载体的重组菌ELISA呈阳性，提示大肠杆菌O157和甲型副伤寒沙门菌CMCC50973的O-多糖合成基因簇在大肠杆菌中被利用生成了相应的LPS；而带有铜绿假单胞杆菌CMCC10110的O-多糖合成基因簇克隆载体的重组菌W3110/BAC-10110则ELISA呈阴性。West？ern印迹结果显示，只有带有O157型大肠杆菌O-多糖合成基因簇克隆载体的糖基工程大肠杆菌CLM24/pMMB66EH-pilE-his/pETtac28-pglL/BAC-O157在相对分子质量40×103~58×103处出现了特异条带，表明菌毛蛋白PilE被大肠杆菌O157型O-多糖O-糖基化修饰。结论：建立了大肠杆菌O157、甲型副伤寒沙门菌CMCC50973的O-多糖合成基因簇大片段的克隆载体，克隆的O157型O-多糖合成基因簇可实现O157型多糖对菌毛蛋白PilE的修饰，从而为在大肠杆菌中建立稳定的利用外源病原菌多糖修饰靶标蛋白的糖基工程大肠杆菌提供了技术基础。Objective： To establish stable exogenous pathogen polysaccharide gene cluster clones in E.coli, for the use of exogenous polysaccharide O-glycosylation in glycosylation engineering E.coli modification of target proteins to lay the foundation. Methods： The O-polysaccharide gene clusters of E.coli O157, Salmonella paratyphi-A CMCC50973 and Pseudomonas aeruginosa CMCC10110 were amplified by PCR and connected to pCC1BAC. The polysaccharide gene cluster cloning vectors were transferred into E.coli W3110. Using the corresponding antiserum to test whether the exogenous O-polysaccharide could generate lipopolysaccharide（LPS） by ELISA, which proved that exogenous polysaccharide gene cluster cloning vectors were able to generate the corresponding O-polysaccha？ride. Meanwhile, transfer the three recombinant into glycosylation engineering E.coli, then detected the modified PilE with corresponding antiserum by Western blot. Results： ELISA results showed that, compared with the nega？tive control, cloning vectors of recombinant with E.coli O157, S.paratyphi-A CMCC50973 O-polysaccharide gene clusters had obvious color reaction, pointed the O-polysaccharide of E.coli O157 and S.paratyphi-A could generate LPS; the recombinant W3110/BAC-10110 showed negative. The CLM24/pMMB66EH-pilE-his/pETtac28-pglL/BAC-O157 had a ladder-like band between 40~58 kD by Western blot with anti-E.coli O157 polyclonal antibody,＆amp;nbsp;while the control, CLM24/pMMB66EH-pilE-his/pETtac28-pglL/BAC-50973, CLM24/pMMB66EH-pilE-his/pETtac28-pglL/BAC-10110 without specific band appearing. This system can modify the PilE by exogenous O157 type O-polysaccharides. Conclusion： E.coli O157, S.paratyphi-A CMCC50973 O-polysaccharide gene cluster were success？fully constructed. For the O-linked protein glycosylation system, heterogenic O157 type O-polysaccharide can be modified the PilE, so as to provide a technical basis for establishing a stable exogenous polysaccharides modified target protein glycosylation engineering E.coli.

关 键 词：细菌人工染色体 O-157型大肠杆菌O-多糖合成基因簇 甲型副伤寒沙门菌O-多糖合成基因簇 铜绿假单胞杆菌O-多糖合成基因簇 糖基工程大肠杆菌 

分 类 号：Q78[生物学—分子生物学]