摘要目的 表达纯化肠出血性大肠杆菌(EHEC) O15∶H7的紧密黏附素(intimin)及突变体intiminN916Y,并构建其转位紧密黏附素受体(translocated intimin receptor,Tir)结合片段(intimin binding domain,Tir-IBD),通过BIACore技术检测紧密黏附素及突变体intiminN916Y与Tir-IBD蛋白质相互作用特性的变化,探索特定氨基酸的突变对其黏附结合功能的影响.方法 设计引物采用PCR法自EHEC O157∶H7基因组扩增Tir-IBD的编码基因tir-ibd,TA克隆后构建原核表达质粒pET-21a(+)-tir-ibd,经测序鉴定后转化E.coli BL21( DE3),IPTG诱导表达,PAGE检测.目的蛋白经Ni-NTA亲和纯化后,再用阴离子交换柱Resource Q和分子筛柱Superdex 200进行纯化.同时按包涵体复性后纯化的方案制备紧密黏附素及突变体intiminN916Y,将所得高纯度目的蛋白Tir-IBD适当稀释后耦联BIACore3000配套的NTA芯片,在25℃和37℃条件下分别以紧密黏附素及突变体intiminN916Y作为流动相进行BIACore检测.结果 EHEC O15∶H7基因组扩增出了约270 bp的目的片段；原核表达质粒pET-21a(+)-tir-ibd经酶切及测序鉴定与设计序列一致.转化E.coli BL21( DE3)后IPTG诱导目的蛋白表达率约15％；PAGE初步测定目的蛋白的相对分子质量(Mr)约10×103,破菌后电泳证实目的蛋白为可溶表达.Ni-NTA亲和纯化和阴离子交换纯化效果明显,高纯度的Tir-IBD蛋白耦联NTA芯片,在25℃和37℃条件下对紧密黏附素及突变体intiminN916Y与Tir-IBD相互作用的BIACore检测结果显示,intiminN916Y特定氨基酸的突变对其结合能力有明显影响并呈温度依赖性.结论 EHEC O157∶H7的紧密黏附素、突变体intiminN916Y及Tir-IBD经基因克隆后获得了较好的表达,纯化后获得高纯度的目的蛋白.在此基础上建立了蛋白相互作用的BIACore检测体系,证明intiminN916Y特定氨基酸的突变对其结合能力有明显影响并呈温度依赖性.

abstractsObjective To analyze the combination characteristics between Tir-IBD( intimin binding domain) and its ligand intimin or mutant intiminN916Y of EHEC O157 ∶H7.Methods The gene of TirIBD (tir-ibd) from EHEC O157 ∶H7 chromosome was cloned into pMD18-T vector.Thereafter,the amplified gene was cloned into prokaryotic expression plasmid pET-21 a (+).The recombinant pasmid pET-21 a( +)-tir-ibd was transformed into E.coli BL21 (DE3).After inducement,the protein Tir-IBD was successfully expressed and analyzed with SDS-PAGE and Western blot.It was purified by affinity chromatography and ionexchange chromatography and was coupled on the Ni-NTA chip of BIACore 3000.Then the ligand intimin and mutant intiminN916Y were flow through the chip and their combination characteristics were detected.Results In the present study,the gene of Tir-IBD(tir-ibd) was successfully cloned into pET-21a(+).The results of SDS-PAGE and Western blot assay showed that the protein was successfully expressed,which accounts for 15％ of total expression products,and its molecular weight was about 10×103.The purity was above 95％ after purification.After coupled on the Ni-NTA chip of BIACore 3000,their combination characteristics with ligand intimin and mutant intiminN916Y were successfully detected.The equilibrium binding constants Ka was obtained by fitting the data with the BIACore evaluation program ( Version 4.1 ).The result showed that the combination characteristics between Tir-IBD and intimin have some difference compared with that of mutant intiminN916Y and the difference is temperature dependent.Conclusion Tir-IBD of EHEC O157 ∶H7 was successfully constructed and purified.The method to analyze the combination characteristics between Tir-IBD and its ligand intimin or mutant was established.The combination characteristics between Tir-IBD and intimin or mutant intiminN916Y have some temperature dependent difference and the mutated amino acids residue is crucial for their receptor-ligand binding.