细粒棘球蚴AgB亚单位抗原基因的克隆和表达系统的优化

目的从我国细粒棘球绦虫分离株（新疆源和甘肃源）中克隆AgB1和AgB2两个亚单位基因,并试用不同的表达载体对两个AgB亚单位基因的表达情况进行比较观察。方法设计特异性引物,扩增AgB1和AgB2亚单位抗原基因,分别用pET28a（＋）、pET32a（＋）和pGEX4T-13种表达载体构建重组质粒,对AgB亚单位基因在不同载体中的表达情况进行比较。结果从我国细粒棘球绦虫分离株中克隆的AgB1和AgB2抗原基因与GenBank中的序列比对,AgB1与德国人源、巴西羊源、意大利牛源均有一定的核苷酸和氨基酸差异;AgB2与乌拉圭羊源序列完全一致。在3种载体中表达重组蛋白的结果显示,不同载体和表达系统对重组表达蛋白的生物活性和可溶性有较大的影响。结论成功地克隆了细粒棘球蚴AgB亚单位基因,AgB1和AgB2基因在3种不同表达载体中的表达情况及对融合标签蛋白的血清基础反应性的比较分析表明,两个重组抗原在pET32a载体中的表达优于pET28a和pGEX4T-1载体。

Cloning and Expression of Antigen B Subnit 1 and 2(AgB1 and AgB2)of Echinococcus granulosus

Objective Isolates of Echinococcus granulosus from Xinjiang and Gansu Province were used in the cloning of the antigen B subnit B1 and B2 genes(AgB1 and AgB2).Their expression levels in three different expression systems were compared.Method Specific primers were designed to amplify the two antigen B subunit genes,AgB1 and AgB2.These 2 genes were cloned into three different vectors,pET28a( ),pET32a( )and pGEX4T-1 for the expression in E.coli.The expression levels of AgB1 and AgB2 gene in each of the vectors were compared.Result The sequences of AgB1 and AgB2 gene were compared with the gene sequences in GenBank.The nucleotide and amino acids sequences of AgB1 are differ from the sequence of AgB1 of the parasite isolated from human of German origin,isolated from sheep of Brazil origin,and isolated from cattle of Italy origin.AgB2 had the same sequence with the AgB2 of the parasite isolated from sheep of Uruguay origin.The expression of AgB1 and AgB2 in these 3 expression vectors showed differences in the expression level,biological activity and solubility of the recombinant antigens.Conclusion The antigen B subnit AgB1 and AgB2 were successfully cloned and expressed in three different vectors.Results from the studies on the expression levels and the reactivity of the fused tag proteins with the pooled sera from healthy persons and echinococcosis patients suggested that the two genes was expressed better in pET32a vector than in the pET28a and pGEX4T-1 vectors.