DSPAα1缺失突变体在毕赤酵母中的表达及活性研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.033

摘要/Abstract

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（mDSPAα1）,分别构建mDSPAα1/pPIC9K、DSPAβ/pPIC9K和DSPAγ/pPIC9K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2.64×10⁵U/mg,DSPAβ的活性为1.32×10⁴U/mg,mDSPAα1活性为151.52 U/mg;同时使用DSPAα1、mDSPAα1、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。mDSPAα1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。

关键词: DSPAα, 1, SOE-PCR, 缺失突变体, 毕赤酵母, 纤维平板法

Abstract: There are 4 types of desmodus salivary plasminogen activators（DSPAs）,i.e.,DSPAα1,DSPAα2,DSPAβ and DSPAγ. 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 contains E,K and P domains;and DSPAγ contains K and P. The effect of DSPAα1’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 mDSPAα1/pPIC9K,DSPAβ/pPIC9K and DSPAγ/pPIC9K were constructed and transformed into Pichia pastoris GS115. Fibrinolytic activity was determined by fibrin plate assay. Results showed that the expression of DSPAγ was not detected,the activities of DSPAα1,DSPAβ,and mDSPAα1 were 2.64 × 10⁵ U/mg,1.32 × 10⁴ U/mg and 151.52 U/mg respectively. The total activity hardly changed when using DSPAα1,mDSPAα1 and DSPAβ together,but there led a 2-3folds of reduction by using either two of them. The mDSPAα1 exhibited almost no fibrinolytic activity,whilst DSPAβ retained comparable catalytic activity to the wild-type DSPAα1. Deletion mutant studies illustrated that N-terminal region of DSPAα1 greatly affected the PA activity,and it without E domain nearly lost its activity,indicating that the E domain of DSPAα1 plays a key role during fibrinolytic process.

Key words: DSPAα1, SOE-PCR, deletion mutants, Pichia pastoris, fibrin plate assay

陈韵, 牛纯青, 宋小双, 苏畅, 华子春, 刘堰. DSPAα1缺失突变体在毕赤酵母中的表达及活性研究[J]. 生物技术通报, 2016, 32(9): 246-252.

CHEN Yun¹, NIU Chun-qing¹, SONG Xiao-shuang¹, SU Chang¹, HUA Zi-chun², LIU Yan¹. Expression and Activity Analysis of DSPAα1 Deletion Mutants in Pichia pastoris[J]. Biotechnology Bulletin, 2016, 32(9): 246-252.

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