目的 研究细粒棘球绦虫cAMP依赖性蛋白激酶A（EgPKA）基因的生物信息学特征,分析其重组蛋白的免疫反应性。 方法 利用细粒棘球绦虫全基因组测序数据设计特异性引物扩增EgPKA基因的编码区序列,并通过生物信息学方法综合分析EgPKA蛋白的理化性质、亚细胞定位、三维结构。构建GS115/EgPKA-pPIC9K重组酵母工程菌并用甲醇诱导表达,抗His标签镍柱亲和层析柱纯化EgPKA重组蛋白,用细粒棘球蚴病患者血清、细粒棘球蚴感染的BALB/c小鼠血清作为一抗,蛋白质免疫印迹（Western blotting）分析纯化的EgPKA重组蛋白的免疫反应性。 结果 EgPKA基因编码区全长1 053 bp,编码350个氨基酸残基,发生了48 bp的缺失突变（417~464 bp）,突变后EgPKA编码区碱基A⁴¹⁵、G⁴¹⁶和T⁴⁶⁵重新结合形成新的氨基酸Ser¹⁵⁴。生物信息学分析结果显示,EgPKA理论等电点为8.63,不稳定性系数为29.04,脂肪系数为82.49,总平均亲水性为-0.374,不含信号肽,无跨膜区。Cell-Ploc 2.0软件分析结果显示,EgPKA蛋白可能定位于细胞膜、细胞质、线粒体和细胞核。Western blotting分析结果显示,1%甲醇诱导表达72 h时EgPKA重组蛋白的产量最高,纯化的EgPKA重组蛋白能与细粒棘球蚴病患者血清,细粒棘球蚴感染后4、8、12和16个月的小鼠血清发生特异性反应,出现两条分别为M_r 40 000和35 000蛋白条带,而与健康小鼠及健康人血清无特异性反应,无条带出现。 结论 EgPKA编码区序列存在基因缺失突变,EgPKA重组蛋白具有较好的免疫反应性,有望作为细粒棘球蚴病的早期诊断抗原。

Objective To investigate the bioinformatics characteristics of protein kinase A gene in Echinococcus granulosus (EgPKA), and analyze the immunoreactivity of its recombinant protein. Methods The coding region of EgPKA gene was amplified using primers designed based on the whole-genome sequencing data of Echinococcus granulosus, and the physicochemical properties, subcellular localization, and tertiary structure of EgPKA were comprehensively analyzed using bioinformatics methods. The recombinant plasmid of GS115/EgPKA-pPIC9K was constructed and induced by methanol in Pichia pastoris eukaryotic system. The recombinant EgPKA protein was purified by nickel-affinity chromatography with anti-His label, and the immunoreactivity of purified EgPKA was detected by Western blotting with serum of a patient with cystic echinococcosis and sera from mice 4, 8, 12 and 16 months after infected with E. granulosus protoscolex. Results The EgPKA gene had a full length of 1 053 bp and encoded 350 amino acid residues. A 48-bp deletion mutation (417-464 bp) occurred in the coding region. After mutation, the bases A⁴¹⁵, G⁴¹⁶ and T⁴⁶⁵ in the EgPKA coding region formed a new amino acid Ser¹⁵⁴. Bioinformatics analysis showed that EgPKA had theoretical isoelectric point of 8.63, instability coefficient of 29.04, fat coefficient of 82.49, and a total average hydrophilicity of -0.374, with no signal peptide and no transmembrane region. Cell-ploc 2.0 analysis showed that the EgPKA protein may be localized to the cell membrane, cytoplasm, mitochondria and nucleus. Western blotting analysis showed that the yield of recombinant protein EgPKA was highest after induction for 72 h in 1% methanol. The purified recombinant protein EgPKA could react specifically with sera of infected mice at 4, 8, 12 and 16 months post-infection, resulting in two protein bands at M_r 40 000 and 35 000, while no specific reaction occurred in sera from healthy mice and healthy humans. Conclusion The coding region of EgPKA has a gene deletion mutation, and the recombinant EgPKA protein has good immunoreactivity, which could be a potential diagnostic antigen for early diagnosis of echinococcosis.