豆豉纤溶酶基因的克隆及其在毕赤酵母中的表达

[目的]为豆豉纤溶酶的进一步研究与应用奠定基础。[方法]以从芽孢杆菌中提取的总DNA为模板,根据GenBank的豆豉纤溶酶基因(AY720895.2)DNA序列设计1对引物,克隆豆豉纤溶酶基因并进行序列测定。构建毕赤酵母表达载体pL3,在毕赤酵母中表达豆豉纤溶酶基因。[结果]经PCR扩增可获得约1.1 kb的DNA片段。序列分析表明所克隆DNA片段包含1个1089 bp的开放阅读框,编码363个氨基酸。该克隆基因与所发表的豆豉纤溶酶基因序列的核苷酸序列同源性为98%,而氨基酸序列同源性达100%。[结论]所克隆的豆豉溶纤酶基因在毕赤酵母中成功表达,且表达产物具有正常的生物学活性。

Cloning of Douchi Fibrinolytic Enzyme Gene and Its Expression in Pichia pastoris

[Objective] The aim of the research was to lay the foundation for further research and application of Douchi fibrinolytic enzyme(DFE).[Method] With the total DNA extracted from Bacillaceae as template,1 pair of primers were designed according to DNA sequence of DFE gene(AY720895.2) on Genbank website and DFE gene was cloned and its sequence was determined.The expression vector pL3 of Pichia pastoris was constructed and DFE gene was expressed in P.pastoris.[Result] About 1.1 kb DNA fragment was obtain from PCR amplification.The sequence analysis showed that the cloned DNA fragment contained an open reading frame with the length of 1 089 bp and coded 363 amino acids. The nucleotide homology between the cloned gene and the reprted DFE gene sequence was 98% and the homology of amino acid sequences reached 100%.[Conclusion] The cloned Douchi fibrinolytic enzyme gene was successfully expressed in P.pastoris and its expression products had normal biological activities.