DSPAα1缺失突变体在毕赤酵母中的表达及活性研究  

Expression and Activity Analysis of DSPAα1 Deletion Mutants in Pichia pastoris

作　　者：陈韵[1] 牛纯青[1] 宋小双[1] 苏畅[1] 华子春[2] 刘堰[1] 

机构地区：[1]重庆西南大学生命科学学院,重庆400715 [2]南京大学医药生物技术国家重点实验室,南京210093

出　　处：《生物技术通报》2016年第9期246-252,共7页Biotechnology Bulletin

摘　　要：吸血蝙蝠唾液纤溶酶原激活剂(Desmodus salivary plasminogen activators,DSPAs)共有4种:DSPAα1、α2、β及γ。其中,DSPAα含有指形区(F)、表皮生长因子区(E)、kringle区(K)和丝氨酸蛋白酶区(P),DSPAβ含E、K、P区,DSPAγ含K和P区。以DSPAα1为基础,研究其结构对纤溶活性的影响。采用重叠延伸PCR技术(splicing overlap extension PCR,SOE-PCR)获得缺失E区的DSPAα1突变体(m DSPAα1),分别构建m DSPAα1/p PIC9K、DSPAβ/p PIC9K和DSPAγ/p PIC9K重组质粒并转化到巴斯德毕赤酵母(Pichia pastoris)菌株GS115中表达,纤维平板法测活。结果显示未检测到DSPAγ的表达,DSPAα1活性为2.64×105 U/mg,DSPAβ的活性为1.32×104 U/mg,m DSPAα1活性为151.52 U/mg;同时使用DSPAα1、m DSPAα1、DSPAβ时,总活性几乎不受影响,单独使用任意两者时,发现活性均降低2-3倍。m DSPAα1纤溶活性几乎没有,同时DSPAβ与野生型DSPAα1相比保留了相当的催化活性。DSPAα1的N端区域可以影响其溶栓活性,而缺失E区后几乎丧失活性,说明E区在DSPAα1溶栓过程中起着至关重要的作用。There are 4 types of desmodus salivary plasminogen activators （ DSPAs ）, i.e., DSPAα1, DSPAα2, DSPAβ and DSPAy. DSPAα1 and DSPAα2 contain a finger domain （ F ）, an epidermal growth factor domain （ E ）, a kringle domain （ K ） and a serine proteinase domain （ P ） ; DSPAβ cbntains E, K and P domains ; and DSPAy contains K and P. The effect of DSPAetl＇ s structure on fibrinolytic activity was also investigated. A deletion mutant of DSPAα1 （ mDSPAα1 ） lacking the E domain was synthesized by splicing overlap extension PCR （ SOE-PCR ） method, and the recombinant plasmids of mDSPActl/pPIC9K, DSPAβ/pPIC9K and DSPAy/pPIC9K were constructed and transformed into Pichia pastoris GS 115. Fibrinolytic activity was determined by fibrin plate assay. Results showed that the expression of DSPAT was not detected, the activities of DSPAα1, DSPAβ, and mDSPAal were 2.64 × 10s U/mg, 1,32× 104 U/mg and 151.52 U/mg respectively. The total activity hardly changed when using DSPAeα1, mDSPAal and DSPAα1 together, but there led a 2-3 folds of reduction by using either two of them. The mDSPAIxl exhibited almost no fibrinolytic activity, whilst DSPAβ retained comparable catalytic activity to the wild-type DSPActl. Deletion mutant studies illustrated that N-terminal region of DSPActl greatly affected the PA activity, and it without E domain nearly lost its activity, indicating that the E domain of DSPActl plays a key role during fibrinolytic procesi.

关 键 词：DSPAα1 SOE-PCR 缺失突变体 毕赤酵母 纤维平板法 

分 类 号：Q78[生物学—分子生物学]