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目的 构建表达肠出血性大肠杆菌 (EHEC)O1 57∶H7志贺毒素Ⅱ结合亚单位 (Stx2B)与紧密黏附素C 端免疫保护性片段 (IntiminC30 0 )的融合蛋白 (Stx2B IntiminC30 0 ) ,并观察其免疫保护作用。方法 设计引物采用PCR法自EHECO1 57∶H7基因组扩增Stx2B的编码基因stx2b ,T A克隆后在前期已构建原核表达质粒 pET 2 8a( + ) eaeC30 0的基础上构建融合基因表达质粒 pET 2 8a( + ) stx2b eaeC30 0 ,经测序鉴定后转化E .coliBL2 1 (DE3) ,IPTG诱导表达 ,PAGE检测 ;通过包涵体洗涤与阴离子柱层析纯化 ,获得目的蛋白Stx2B IntiminC30 0 ,以Al(OH ) 3为佐剂皮下注射免疫BALB/c小鼠 ,再以EHECO1 57∶H7超声破菌液腹腔注射免疫后的小鼠 ,观察攻毒保护效果。结果 PCR法自EHECO1 57∶H7基因组扩增出了约 2 90bp的目的片段 ;原核表达质粒 pET 2 8a( + ) stx2b eaeC30 0经酶切及测序鉴定与设计序列一致。转化E .coliBL2 1 (DE3)后IPTG诱导目的蛋白表达率约 2 5% ;PAGE初步测定目的蛋白的相对分子质量 (Mr)约 4 3× 1 0 3,破菌后电泳证实目的蛋白以包涵体形式表达。纯化后目的蛋白Stx2B IntiminC30 0的纯度达 75% ,免疫BALB/c小鼠后血清特异性抗体阳转率及抗体效价均显著增高 ,能够抵御致死剂量EHECO1 57∶H7超声破菌?
Objective To construct a fusion protein (Stx2B IntiminC30 0) expressing the O1 57:H7 Shiga toxin Ⅱ binding subunit (EhEC) and the C-terminal immunoprotective fragment (IntiminC30 0) of enterohemorrhagic Escherichia coli (EHEC) Its immune protection. Methods Primer was designed to amplify stx2b gene of Stx2B gene from EHECO1 57:H7 genome by PCR method. After TA cloning, the prokaryotic expression plasmid pET 2 8a (+) eaeC30 0 was constructed and the fusion gene expression plasmid pET 2 8a ( +) stx2b eaeC30 0, transformed into E.coli BL21 (DE3) after induced by IPTG, induced by IPTG and detected by PAGE. Purified by inclusion body washing and anion column chromatography, the target protein Stx2B IntiminC30 0 was obtained. BALB / c mice were immunized subcutaneously with adjuvant, and the immunized mice were injected intraperitoneally with EHECO1 57: H7 ultrasonic disruption liquid to observe the protective effect of challenge. Results The target fragment of about 290bp was amplified from the genome of EHECO1 57:H7 by PCR. The prokaryotic expression plasmid pET 2 8a (+) stx2b eaeC30 0 was identified and sequenced according to the designed sequence. The relative molecular mass (Mr) of the target protein was about 43 × 10 3 after IPTG induction by E. coli BL21 (DE3). The result of electrophoresis showed that the target protein was expressed in inclusion bodies Form of expression. After purification, the purity of the target protein Stx2B IntiminC30 0 reached 75%. After immunization of BALB / c mice, the serum specific antibody positive rate and antibody titer were significantly increased, which could resist the lethal dose of EHECO1 57:H7 sonication.