肠出血性大肠杆菌O157:H7Tir基因的克隆、表达及生物学活性研究

Cloning,Expression,Biological Activities of Recombinant Tir gene from Enterohemorrhagic Escherichia coli O157:H7

【目的】克隆、表达肠出血性大肠杆菌(EHEC)O157:H7转位紧密素受体(Tir)基因,并研究其抗原性。【方法】选用pET28原核表达载体,体外构建Tir原核表达重组菌,并在大肠杆菌中获得高效表达。选用家兔制备高滴度多克隆抗体,Western blot分析其免疫原性。选用HEp-2细胞进行粘附和粘附抑制试验,通过光镜、电镜和荧光显微镜进行观察。选用Balb/c小鼠进行免疫试验。【结果】成功获得高效表达重组Tir蛋白,并制备了兔源Tir多克隆抗体,Western blot分析此抗体能与Tir发生特异性抗原抗体反应。表达Tir蛋白能够抑制O157:H7对HEp-2细胞的粘附和A/E损伤。二免后Balb/c小鼠保护率高达75%以上。【结论】在大肠杆菌中成功克隆表达了Tir基因,所获重组Tir蛋白具有良好的抗原性,可能用于肠出血性大肠杆菌O157基因工程疫苗的研究。

【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.