幽门螺杆菌过氧化氢酶基因克隆及表达

Cloning and Expression of Catalase Gene of Helicobacter pylori

目的 克隆幽门螺杆菌(Helicobacter pylori,HP)过氧化氢酶(katA)基因并在大肠杆菌中进行表达。方法 采用PCR扩增幽门螺杆菌katA全长基因,将其克隆入pET-11c载体中,经测序证实后,在大肠杆菌中进行表达,产物用Western blot检测其抗原性并进行N末端氨基酸的测序。结果 幽门螺杆菌katA基因全长1 518bp,编码氨基酸505个。在BL21(DE3)中的表达量约占细菌总蛋白的24.9％,表达产物经SDS-PAGE显示其相对分子质量与软件预测结果 58 000相符,N末端5个氨基酸测序结果与Hp中天然的katA完全一致,经Western blot检测可被 Hp全菌抗血清识别。结论 katA能在大肠杆菌中进行高效表达,具有良好的免疫反应性,可望为研究 Hp的致病机理、实验诊断及亚单位疫苗等提供充足的katA原材料。

Objective To clone and express the catalase gene of Helicobacter pylori in E. coli. Methods Amplify the full length of H. pylori catalase (kat A) gene by PCR, clone into pET-1 Ic vector and transform to E. coli BL21(DE3) for expression. Detect the antigenicity of expressed product by Western blot,and sequence the amion acids at N-terminal. Results The full length of H. pylori catalase gene consisted of 1518bp encoding 505 amino acids. The expressed product contained about 24.9% of total somatic protein. SDS-PAGE showed a relative molecular weight of 58 000. The sequence of 5 amino acids at N-terminal was completely consistent with those of natural kat A in HP. Western blot proved that the expressed product was recognized by antiserum to H.pylori .Conclusion katA gene was highly expressed in E. coli, and the expressed product showed good immunoreactivity. It might be used as a material for the study of pathogenic mechanism, laboratory diagnosis and subunit vaccine of HP.