To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into ...To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.展开更多
目的对肠出血性大肠埃希菌O157：H7紧密黏附素C300与黏膜佐剂不耐热肠毒素（LTB）融合蛋白二级结构及B细胞识别表位进行预测,为开发新型疫苗提供理论依据.方法采用DNA star软件中的Protean预测软件对Intim in C300与LTB融合蛋白的亲水...目的对肠出血性大肠埃希菌O157：H7紧密黏附素C300与黏膜佐剂不耐热肠毒素（LTB）融合蛋白二级结构及B细胞识别表位进行预测,为开发新型疫苗提供理论依据.方法采用DNA star软件中的Protean预测软件对Intim in C300与LTB融合蛋白的亲水性指数、β-转角、柔韧性、表面可及性和抗原指数进行预测.结果在融合蛋白N端第1-10、20-30、110-120、210-220、300-310、350-360、380-390和415-420区段可能是α-螺旋中心,在N端第195-205、300-320、335-350和390-410区段形成4个大的β-折叠中心区域,在各β-折叠区间存在均匀且较丰富的转角区域.融合蛋白的蛋白柔性区域可能位于N端第20-35、52-65、75-85、105-115、150-160、172-188、260-275和372-388区域,这些区域有一定幅度的折叠或摆动,可形成较复杂的三级结构.亲水性区域位于第50-60、70-80、155-165、190-200、240-250、260-270、330-340和375-385区段,这8个区域作为抗原表位可能性大.结论通过生物信息学技术分析,可有效地预测融合蛋白二级结构和B细胞表位,为人工合成优势肽段进行肠出血性大肠埃希菌O157：H7的重组疫苗研制提供理论依据.展开更多
文摘To construct and express the fusion protein Stx2B-IntiminC300 of EHEC O157 ： H7, and to further investigate its immunoprophylactic potential, the gene of Stx2B （stx2b） from EHEC O157：H7 chromosome was cloned into pMD18-T vector. Thereafter, the amplified gene was cloned into prokary- otic expression plasmid pET-28a （ ＋ ）-eaeC300, which was constructed previously. The recombinant pasmid pET-28a（ ＋ ）-stx2b-eaeC300 was transformed into E. coli BL21 （DE3）. After inducement, the protein Stx2B-IntiminC300 was successfully expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis （SDS-PAGE）, Western blotting and N-terminal amino acid residual sequencing. To evaluate its immunoprophylactic potential, it was primarily purified by ion-exchange chromatography and injected into 30 BALB/c mice with AI（OH）3 in the subscapular region. Ten days after the last booster vaccination, 20 mice were attacked with EHEC O157：H7 lysate and the protective efficacy was observed. In the present study, the gene of Stx2B-intiminC300 was successfully cloned into pET-28a （ ＋ ） vector. The results of SDS-PAGE and Western blotting assay showed that the fusion protein was successfully expressed in the inclusion body form, accounting for 25 % of total expression products, and its molecular weight was about 43 kDa. The result of the N-terminal amino acid residual sequencing showed that it was identical to that of the molecular designed. The purity was about 75 % after primary purification. Animal tests revealed that the fusion protein Stx2B-intiminC300 has elicited high titer of protective antibody relatively. These results demonstrate that the fusion protein Stx2B-IntiminC300 is successfully expressed in prokaryotic expression system and shows certain immunoprophylactic potential.
文摘目的对肠出血性大肠埃希菌O157：H7紧密黏附素C300与黏膜佐剂不耐热肠毒素（LTB）融合蛋白二级结构及B细胞识别表位进行预测,为开发新型疫苗提供理论依据.方法采用DNA star软件中的Protean预测软件对Intim in C300与LTB融合蛋白的亲水性指数、β-转角、柔韧性、表面可及性和抗原指数进行预测.结果在融合蛋白N端第1-10、20-30、110-120、210-220、300-310、350-360、380-390和415-420区段可能是α-螺旋中心,在N端第195-205、300-320、335-350和390-410区段形成4个大的β-折叠中心区域,在各β-折叠区间存在均匀且较丰富的转角区域.融合蛋白的蛋白柔性区域可能位于N端第20-35、52-65、75-85、105-115、150-160、172-188、260-275和372-388区域,这些区域有一定幅度的折叠或摆动,可形成较复杂的三级结构.亲水性区域位于第50-60、70-80、155-165、190-200、240-250、260-270、330-340和375-385区段,这8个区域作为抗原表位可能性大.结论通过生物信息学技术分析,可有效地预测融合蛋白二级结构和B细胞表位,为人工合成优势肽段进行肠出血性大肠埃希菌O157：H7的重组疫苗研制提供理论依据.
