利用基因工程技术在大肠杆菌BL21(DE3)中表达白喉毒素无毒突变体CRM197，经变性条件下亲和纯化后，作为蛋白载体与流感抗原M2e经BMPH偶联，制备CRM197?M2e结合物，用其免疫BALB/c小鼠，采用间接ELISA法测定血清中M2e特异性IgG抗体. 结果表明，重组CRM197在大肠杆菌中成功表达，优化后蛋白表达量约为250±30 mg/L，纯度达95%以上；所制CRM197?M2e结合物可有效免疫BALB/c小鼠，其刺激产生的M2e特异性IgG抗体滴度分别是健康组、M2e免疫对照组、CRM197免疫对照组及CRM197+M2e混合组的430.5, 32, 181和215倍.
Express the nontoxic mutant of diphtheria toxin (CRM197) in E. coli BL21 (DE3) by gene engineering technique and purify the protein by affinity chromatography under denaturation. As the carrier protein, recombinant CRM197 was conjugated with influenza antigen M2e via BMPH to prepare the conjugate vaccine to immune BALB/c mice. Determine the M2e specific IgG in sera by indirect ELISA. The results showed that rCRM197 was expressed in E. coli system successfully with optimized level (250±30 mg/L) and high purity (>95%). The conjugate vaccine CRM197?M2e prepared by chemical coupling in denaturation could effectively immunize BALB/c mice. And the M2e-specific IgG antibody titer elicited by CRM197?M2e conjugate vaccine was significantly higher than that in healthy, M2e, CRM197 and M2e+CRM197 mixed groups by 430.5, 32, 181 and 215 times.
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