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目的:在基因工程菌中实现肠出血性大肠杆菌(EHEC)O157∶H7紧密黏附素(intimin)保护性片段(Int281)的高效表达,并进行抗原性的初步分析。方法:应用PCR从EHEC O157∶H7基因组中钓取int281基因,插入pMD18-T克隆载体。克隆质粒测序鉴定后,采用NdeⅠ、NotⅠ限制性核酸内切酶双酶切pMD18-T-int281质粒获得int281基因,连接同样经过双酶切的pET-22b(+)。表达质粒测序鉴定后转化E.coliBL21(DE3),IPTG诱导表达,经SDS-PAGE检测相对分子质量和Western印迹验证免疫学活性后,重组Int281蛋白免疫BALB/c小鼠,ELISA检测鼠血清抗体效价。结果:PCR扩增得到843 bp的目的片段。构建的原核表达质粒pET-22b(+)-int281经酶切鉴定及测序与预期序列一致。目的蛋白以包涵体形式表达,表达量约25%。变性复性后经镍柱纯化后纯度达95%以上。免疫印迹检测显示,重组Int281片段可以特异性地识别抗O157∶H7多抗。免疫小鼠抗体效价达1∶106。结论:重组Int281具有良好的免疫原性,为制备相应的抗体及研究其对EHEC O157∶H7黏附定植的被动保护效果奠定了基础。
OBJECTIVE: To efficiently express the intimin protective fragment (O157: H7) of enterohemorrhagic Escherichia coli (EHEC) O157: H7 in genetically engineered bacteria and perform a preliminary antigenicity analysis. Methods: The int281 gene was amplified by PCR from the genome of EHEC O157:H7 and inserted into pMD18-T cloning vector. After sequencing and identification of the cloned plasmid, the int281 gene was obtained by digesting the plasmid pMD18-T-int281 with Nde I and Not I restriction endonucleases to ligate the same double-digested pET-22b (+). The expression plasmids were identified by sequencing and transformed into E.coli BL21 (DE3) for expression under IPTG. After the relative molecular mass was confirmed by SDS-PAGE and the immunological activity was verified by Western blotting, BALB / c mice were immunized with recombinant Int281 protein. price. Results: The target fragment of 843 bp was amplified by PCR. The constructed prokaryotic expression plasmid pET-22b (+) - int281 was identified by restriction enzyme digestion and sequencing was consistent with the expected sequence. The target protein is expressed in the form of inclusion body, the expression amount is about 25%. After denaturation and renaturation purification by nickel column purity of more than 95%. Immunoblotting showed that the recombinant Int281 fragment could specifically recognize anti-O157: H7 polyclonal antibody. Anti-mouse antibody titer of 1:106. Conclusion: Recombinant Int281 has good immunogenicity, which lays the foundation for the preparation of the corresponding antibody and its passive protection on EHEC O157:H7 adhesion colonization.