肠出血性大肠杆菌O157：H7志贺样毒素2B亚基的表达
发布时间：2019-05-16 21:35
【摘要】： 目的克隆表达肠出血性大肠杆菌(Enterohemorrhagic Escherichia coli,EHEC)0157:H7志贺样毒素2B亚单位(Shiga-like toxin,Stx2B),并对其初步纯化。 方法设计寡核苷酸引物,利用PCR法自EHEC 0157:H7基因组扩增Stx2B的编码基因stx2b,TA克隆后连接到表达载体pET-28a以构建重组质粒,经测序鉴定后转入表达宿主菌E.coli BL-21(DE3)感受态细胞,IPTG诱导表达,以SDS-PAGE和Western-blot分析重组表达产物,固相亲和层析纯化重组蛋白。 结果PCR扩增stx2b基因约210bp,成功构建重组质粒pET28a-stx2b,并在E.coli BL21(DE3)中得到高效表达,目的蛋白的相对分子质量约为7.5kD,约占总蛋白量的40%;免疫印迹显示目的蛋白可与特异性抗体发生为特异性阳性反应。 结论成功克隆EHEC 0157:H7 Stx2b基因,在原核系统获得目的蛋白的高效表达,为0157:H7感染机制以及疾病的诊断和疫苗研究奠定基础。
[Abstract]:Objective to clone and express Shiga-like toxin 2B subunit (Shiga-like toxin,Stx2B) of enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli,EHEC) 0157:H7 and purify it. Methods oligodeoxynucleotides primers were designed and cloned from EHEC 0157:H7 genome to amplify Stx2B coding gene stx2b,TA by PCR and ligated to expression vector pET-28a to construct recombinant plasmid. After sequencing, the host strain E.coli BL-21 (DE3) receptive cells were transferred to express them. The expression was induced by IPTG. The recombinant expression products were analyzed by SDS-PAGE and Western-blot, and the recombinant proteins were purified by solid phase affinity chromatography. Results the stx2b gene was about 210 BP amplified by PCR, and the recombinant plasmid pET28a-stx2b, was successfully constructed and highly expressed in E.coli BL21 (DE3). The relative molecular weight of the target protein was about 7.5 KD, accounting for 40% of the total protein. Western imprinting showed that the target protein could react specifically with the specific antibody. Conclusion EHEC 0157:H7 Stx2b gene was successfully cloned and the high expression of target protein was obtained in prokaryotic system, which laid a foundation for the mechanism of 0157:H7 infection, disease diagnosis and vaccine research.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378
本文编号：2478571
[Abstract]:Objective to clone and express Shiga-like toxin 2B subunit (Shiga-like toxin,Stx2B) of enterohemorrhagic Escherichia coli (Enterohemorrhagic Escherichia coli,EHEC) 0157:H7 and purify it. Methods oligodeoxynucleotides primers were designed and cloned from EHEC 0157:H7 genome to amplify Stx2B coding gene stx2b,TA by PCR and ligated to expression vector pET-28a to construct recombinant plasmid. After sequencing, the host strain E.coli BL-21 (DE3) receptive cells were transferred to express them. The expression was induced by IPTG. The recombinant expression products were analyzed by SDS-PAGE and Western-blot, and the recombinant proteins were purified by solid phase affinity chromatography. Results the stx2b gene was about 210 BP amplified by PCR, and the recombinant plasmid pET28a-stx2b, was successfully constructed and highly expressed in E.coli BL21 (DE3). The relative molecular weight of the target protein was about 7.5 KD, accounting for 40% of the total protein. Western imprinting showed that the target protein could react specifically with the specific antibody. Conclusion EHEC 0157:H7 Stx2b gene was successfully cloned and the high expression of target protein was obtained in prokaryotic system, which laid a foundation for the mechanism of 0157:H7 infection, disease diagnosis and vaccine research.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378
【参考文献】
中国期刊全文数据库 前1条
1 汪华,史智扬;O157:H7大肠杆菌流行病学研究概况[J];江苏卫生保健;2001年01期
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