表达食血蝙蝠唾液纤溶酶原激活剂的毕赤酵母菌株的构建

Construction of Pichia pastoris strain expressing salivary plasminogen activator from vampire bat (Desmodus rotundus)

摘要:

食血蝙蝠(Desmodus rotundus)在吸食大型动物血液时能保持创口处血流的畅通。本研究根据食血蝙蝠唾液中发现的纤溶酶原激活剂(Bat-PA, 另称DSPAa1)的全长基因序列(GenBank Accession No. J05082), 首次在体外人工合成Bat-PA全长基因并亚克隆到巴斯德毕赤酵母(Pichia pastoris)表达载体pPIC9K上; 转化毕赤酵母菌株GS115后, 经甲醇诱导获得DSPAa1的分泌型表达, 表达条带为47 kD; 通过抗G418浓度梯度筛选、毕赤酵母中小量表达后SDS-PAGE电泳检测以及纤维平板法测活, 筛选出高表达的DSPAa1稳定菌株, 对电泳中的目的蛋白条带进行密度扫描测定, 产量达到约30 mg/L。目前, DSPAa1主要来自哺乳动物细胞, 如中国仓鼠卵巢细胞(CHO)、胚胎卵巢肾细胞(BHK)、非洲绿猴肾细胞(COS), 代价高昂。在毕赤酵母中生产DSPAa1可以降低成本, 增加产率, 为探索Bat-PA作为新一代溶栓剂奠定基础。

Abstract:

Vampire bat saliva contains a plasminogen activator that presumably assists these hematophagous animals during feeding. Bat-PA (H), the full-length form of Vampire Bat Salivary Plasminogen Activator (DSPAa1), is homologous and similar efficacy to tissue-type plasminogen activator (t-PA). The strict fibrin dependence of activity is a characteristic which could be desirable in the fibrinolytic therapy. It is a unique fibrinolytic enzyme that does not promote neurodegeneration. In this study, according to the reported gene sequence (GenBank Accession No. J05082) of Vampire bat (D. rotundus) plasminogen activator. It was the first time to synthesize the full sequence of DSPAa1 in vitro and clone it into the expression vector pPIC9K, the recombinant plasmid was linearized and transformed into Pichia pastoris GS115 strain. Secreted expression of recombinant DSPAa1 was attained by methanol induction and its molecular mass is 47 kD. To get recombinant GS115 with high amount of protein, hundreds of His+ transformants had been screened to isolate clones resistant to high levels G418 (2–4 mg/mL), the selected clones mini-expressed in Pichia pastoris, and test their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAa1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAa1 derived often from mammalian cells: Chinese hamster ovary (CHO) cells, Baby hamster kidney (BHK) cells, COS cells, which might be produced at high cost. In Pichia pastoris, it is expected to higher yield and lower cost, thus it might be able to serve as new thrombolytic candidate.