中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2012年
6期
525-531
,共7页
易勇%肖敏%罗萍%樊峥%贾莉萍%魏平%陈兴明%李丹%刘春雷%高峰%王贵华%司少艳%毛旭虎%邹全明%敬华
易勇%肖敏%囉萍%樊崢%賈莉萍%魏平%陳興明%李丹%劉春雷%高峰%王貴華%司少豔%毛旭虎%鄒全明%敬華
역용%초민%라평%번쟁%가리평%위평%진흥명%리단%류춘뢰%고봉%왕귀화%사소염%모욱호%추전명%경화
EHECO157∶H7%蛋白质相互作用%紧密黏附素%intiminN916Y%Tir-IBD
EHECO157∶H7%蛋白質相互作用%緊密黏附素%intiminN916Y%Tir-IBD
EHECO157∶H7%단백질상호작용%긴밀점부소%intiminN916Y%Tir-IBD
EHEC O157∶H7%Protein-protein interaction%Intimin%IntiminN916Y%Tir-IBD
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.
目的 錶達純化腸齣血性大腸桿菌(EHEC) O15∶H7的緊密黏附素(intimin)及突變體intiminN916Y,併構建其轉位緊密黏附素受體(translocated intimin receptor,Tir)結閤片段(intimin binding domain,Tir-IBD),通過BIACore技術檢測緊密黏附素及突變體intiminN916Y與Tir-IBD蛋白質相互作用特性的變化,探索特定氨基痠的突變對其黏附結閤功能的影響.方法 設計引物採用PCR法自EHEC O157∶H7基因組擴增Tir-IBD的編碼基因tir-ibd,TA剋隆後構建原覈錶達質粒pET-21a(+)-tir-ibd,經測序鑒定後轉化E.coli BL21( DE3),IPTG誘導錶達,PAGE檢測.目的蛋白經Ni-NTA親和純化後,再用陰離子交換柱Resource Q和分子篩柱Superdex 200進行純化.同時按包涵體複性後純化的方案製備緊密黏附素及突變體intiminN916Y,將所得高純度目的蛋白Tir-IBD適噹稀釋後耦聯BIACore3000配套的NTA芯片,在25℃和37℃條件下分彆以緊密黏附素及突變體intiminN916Y作為流動相進行BIACore檢測.結果 EHEC O15∶H7基因組擴增齣瞭約270 bp的目的片段；原覈錶達質粒pET-21a(+)-tir-ibd經酶切及測序鑒定與設計序列一緻.轉化E.coli BL21( DE3)後IPTG誘導目的蛋白錶達率約15％；PAGE初步測定目的蛋白的相對分子質量(Mr)約10×103,破菌後電泳證實目的蛋白為可溶錶達.Ni-NTA親和純化和陰離子交換純化效果明顯,高純度的Tir-IBD蛋白耦聯NTA芯片,在25℃和37℃條件下對緊密黏附素及突變體intiminN916Y與Tir-IBD相互作用的BIACore檢測結果顯示,intiminN916Y特定氨基痠的突變對其結閤能力有明顯影響併呈溫度依賴性.結論 EHEC O157∶H7的緊密黏附素、突變體intiminN916Y及Tir-IBD經基因剋隆後穫得瞭較好的錶達,純化後穫得高純度的目的蛋白.在此基礎上建立瞭蛋白相互作用的BIACore檢測體繫,證明intiminN916Y特定氨基痠的突變對其結閤能力有明顯影響併呈溫度依賴性.
목적 표체순화장출혈성대장간균(EHEC) O15∶H7적긴밀점부소(intimin)급돌변체intiminN916Y,병구건기전위긴밀점부소수체(translocated intimin receptor,Tir)결합편단(intimin binding domain,Tir-IBD),통과BIACore기술검측긴밀점부소급돌변체intiminN916Y여Tir-IBD단백질상호작용특성적변화,탐색특정안기산적돌변대기점부결합공능적영향.방법 설계인물채용PCR법자EHEC O157∶H7기인조확증Tir-IBD적편마기인tir-ibd,TA극륭후구건원핵표체질립pET-21a(+)-tir-ibd,경측서감정후전화E.coli BL21( DE3),IPTG유도표체,PAGE검측.목적단백경Ni-NTA친화순화후,재용음리자교환주Resource Q화분자사주Superdex 200진행순화.동시안포함체복성후순화적방안제비긴밀점부소급돌변체intiminN916Y,장소득고순도목적단백Tir-IBD괄당희석후우련BIACore3000배투적NTA심편,재25℃화37℃조건하분별이긴밀점부소급돌변체intiminN916Y작위류동상진행BIACore검측.결과 EHEC O15∶H7기인조확증출료약270 bp적목적편단；원핵표체질립pET-21a(+)-tir-ibd경매절급측서감정여설계서렬일치.전화E.coli BL21( DE3)후IPTG유도목적단백표체솔약15％；PAGE초보측정목적단백적상대분자질량(Mr)약10×103,파균후전영증실목적단백위가용표체.Ni-NTA친화순화화음리자교환순화효과명현,고순도적Tir-IBD단백우련NTA심편,재25℃화37℃조건하대긴밀점부소급돌변체intiminN916Y여Tir-IBD상호작용적BIACore검측결과현시,intiminN916Y특정안기산적돌변대기결합능력유명현영향병정온도의뢰성.결론 EHEC O157∶H7적긴밀점부소、돌변체intiminN916Y급Tir-IBD경기인극륭후획득료교호적표체,순화후획득고순도적목적단백.재차기출상건립료단백상호작용적BIACore검측체계,증명intiminN916Y특정안기산적돌변대기결합능력유명현영향병정온도의뢰성.
Objective 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.
