Liu Yan, Su Chang, Song Xiaoshuang, Tang Yalan, Bao Zhenhong
School of Life Science, Southwest University, Chongqing 400715, China.
Sheng Wu Gong Cheng Xue Bao. 2009 Apr;25(4):566-74.
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 (DSPAalpha1), 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 DSPAalpha1 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 DSPAalpha1 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 tested their fibrinolytic activities and expressed protein bands by fibrin plate assay and SDS-PAGE. DSPAalpha1 was determined by optical density after SDS-PAGE, the yield is about 30 mg per liter of fermentation culture. DSPAalpha1 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.
吸血蝙蝠的唾液中含有一种纤溶酶原激活剂，推测其在吸血过程中帮助这些吸血动物。蝙蝠纤溶酶原激活剂（H），即吸血蝙蝠唾液纤溶酶原激活剂（DSPAα1）的全长形式，与组织型纤溶酶原激活剂（t-PA）同源且具有相似的功效。活性对纤维蛋白的严格依赖性是纤维蛋白溶解疗法中可能需要的一个特性。它是一种独特的纤溶酶，不会促进神经退行性变。在本研究中, 根据已报道的吸血蝙蝠（圆叶吸血蝠）纤溶酶原激活剂的基因序列（GenBank登录号J05082），首次在体外合成了DSPAα1的全序列，并将其克隆到表达载体pPIC9K中，将重组质粒线性化后转化到毕赤酵母GS115菌株中。通过甲醇诱导实现重组DSPAα1的分泌表达, 其分子量为47kD。为了获得高表达量蛋白质的重组GS115，筛选了数百个His+转化子以分离对高水平G418（2-4mg/mL）具有抗性的克隆，所选克隆在毕赤酵母中进行小量表达，并通过纤维蛋白平板试验和SDS-PAGE检测其纤溶活性和表达的蛋白条带。通过SDS-PAGE后的光密度测定DSPAα1，每升发酵培养物的产量约为30mg。DSPAα1通常来源于哺乳动物细胞：中国仓鼠卵巢（CHO）细胞、幼仓鼠肾（BHK）细胞、COS细胞，其生产成本可能很高。在毕赤酵母中，有望实现更高的产量和更低的成本，因此它可能能够作为新的溶栓候选药物。
