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目的原核表达重组金黄葡萄球菌肠毒素B(recombinant Staphylococcus aureus enterotoxin B,r SEB)及热休克蛋白65(heat shock protein 65,HSP65)融合蛋白r SEB-HSP65,并探讨其在小鼠体内的体液免疫效果。方法采用分子生物学方法将修改过TCR Vβ结合区的r SEB基因与HSP65基因融合,构建重组表达质粒p ET-28a-r SEB-HSP65,转化E.coli BL21(DE3),IPTG诱导表达,经铜柱亲和层析纯化。将纯化蛋白经小鼠背部皮下注射,分别于第0、14、28天各免疫1次,分别于第14、28、42 d经小鼠尾静脉采血,分离血清,间接ELISA法检测小鼠血清中抗-SEB的Ig G水平。结果重组表达质粒p ET-28a-r SEB-HSP65经双酶切及PCR鉴定,证明构建正确。表达的重组蛋白r SEB-HSP65的相对分子质量约85 000,主要以包涵体形式表达,表达量约占全菌总蛋白的40.81%,纯化蛋白纯度约为90%。各组免疫小鼠均可产生较高滴度抗体,且在第14、28及42天大部分含铝佐剂疫苗组的效价显著高于相同剂量的无铝佐剂疫苗组(P<0.05),第42天时无铝佐剂低剂量疫苗组与含铝佐剂低剂疫苗量组间差异无统计学意义(P>0.05)。结论成功在E.coli BL21(DE3)中表达了重组蛋白r SEB-HSP65,且可诱导小鼠产生较高的抗体水平。
Objective To express recombinant fusion protein SEB-HSP65 of recombinant Staphylococcus aureus enterotoxin B (r SEB) and heat shock protein 65 (HSP65) in prokaryotic cells and investigate its humoral immunity effect. Methods The SEB gene was fused with HSP65 gene by molecular biology method. The recombinant plasmid p ET-28a-r SEB-HSP65 was transformed into E.coli BL21 (DE3) and induced by IPTG. Copper column affinity chromatography purification. The purified protein was subcutaneously injected into the back of mice and immunized on the 0th, 14th and 28th day respectively. Blood was collected from the caudal vein on the 14th, 28th and 42th days, respectively. The serum was separated and the serum was detected by indirect ELISA Medium anti-SEB Ig G levels. Results The recombinant plasmid p ET-28a-r SEB-HSP65 was confirmed by double enzyme digestion and PCR. The expressed recombinant protein SEB-HSP65 has a relative molecular mass of about 85 000 and is mainly expressed in inclusion bodies. The expression level of the recombinant protein SEB-HSP65 is about 40.81% of the total bacterial total protein and the purity of the purified protein is about 90%. The titer of antibodies was higher in all immunized mice and the potency of most aluminum-containing adjuvanted vaccine groups on days 14, 28 and 42 was significantly higher than that of the same dose of aluminum-free adjuvanted vaccine (P <0.05 ), There was no significant difference between the low dose vaccine group without aluminum adjuvant and the low dose vaccine containing aluminum adjuvant on the 42nd day (P> 0.05). Conclusion The recombinant protein r SEB-HSP65 was successfully expressed in E. coli BL21 (DE3) and could induce higher antibody levels in mice.