肠出血性大肠杆菌紧密黏附素保护性多肽的表达与免疫原性分析  

Prokaryotic expression and immunogenicity analysis of intimin polypeptides of enterohemorrhagic Escherichia coli

作　　者：高翔[1] 包士中[1] 史晶[1] 蔡昆[1] 刘昊[1] 侯晓军[1] 王慧[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学科学院院刊》2008年第4期316-318,共3页Bulletin of the Academy of Military Medical Sciences

摘　　要：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 express intimin polypeptides of enterohemorrhagic Escherichia coli （EHEC） in E. coli BL21, and analvze its immunogenicity. Methods：The sequence of int281 was cloned from EHEC O157：H7 EDL 933 by PCR. The product of PCR was inserted into pMD18-T cloning vector. The gene of int281 in cloning plasmid was sequenced and was cut down by Nde I and Not I endonucleases. Then it was recombined with the same cut pET-22b（ ＋ ）. The expression plasmid certified by sequencing was transformed into E. coli BL21 （ DE3 ） , and induced by IPTG to express the recombinant intimin fragment（ rlnt281 ）. The rlnt281 protein was detected by SDS-PAGE, and its conformation was validated by Western blotting. The BALB/c mice were immunized with the antigen. Titers of mice serum total IgG against rInt281 protein were detected by ELISA. Results：The 843 bp DNA was gained. The plasmid pET-22b（ ＋ ）-int281 was identified with our design. The rInt proteins were expressed as inclusion bodies, which were denatured with 8 mol/L urea, renatured by several grades of urea, and dialyzed into PB solution. The renatured proteins were purified by Ni^2＋ affinity chromatograghy. The result of Western blotting suggested that purified rInt proteins could bind with antibodies of lysed O157： HT, which indicated that it had the same or similar conformation as the native proteins. In indirect ELISA, the serum IgG specific for rInt reached the peak point at 1：106 ten days after the third immunization. Three months later,the antibodies level kept almost a constant level. Conclusion：The study shows that Int281 has an excellent immunogenicity that deserves further studies on evaluating its protection against intestinal colonization by EHEC O157： H7.

关 键 词：肠出血性大肠杆菌 紧密黏附素 免疫原性 

分 类 号：R378.2[医药卫生—病原生物学]