关键词: 肠出血性大肠杆菌O157:H7')">肠出血性大肠杆菌O157:H7, 转位紧密素受体, 表达, 粘附, A/E损伤, 免疫保护

Abstract: 【Objective】 The objective of the experiment is to clone, express and study the antigenicity of translocation intimin receptor (tir) gene from EHEC O157：H7. 【Method】 The vector pET28 and BL21(DE3) were used to construct and overexpress the recombinant protein of tir gene by prokaryotic expression. The recombinant Tir protein was used to immunize rabbits to obtain high-titer polyclonal antibodies,which was used to analyze the antigenicity of Tir by Western blot. HEp-2 cell was selected for EHEC adhesion and adhesion inhibition of to EHEC by light microscopy, electron microscopy and fluorescence microscopy observation. Balb/c mice was inoculated with purified 200µg Tir protein subcutaneously and the protection rate was analyzed. 【Result】 We obtain successfully high-level expression of recombinant protein Tir was successfully obtained and the polyclonal antibodies that have good antigenicity from rabbits were successfully prepared. Tir antibody was able to inhibit EHEC O157:H7 adhesion and attaching and effacing lesion(A/E) to HEp-2 cells. Balb/c mice immunized twice showed protection rate up to 87.5%. 【Conclusion】 Tir gene was cloned and expressed successfully in Escherichia coli, and Tir recombinant protein have good antigenicity. The above-mentioned results indicated that tir gene could be used for EHEC O157 genetic engineering vaccines.

Key words: EHEC O157：H7')">EHEC O157：H7, translocation intimin receptor, expression, adhesion, attaching and effacing lesion, immunization