中介蝮蛇毒纤溶酶基因克隆及生物信息学分析

目的：克隆中介蝮蛇(Gloydius intermedius)纤维溶解酶（Fibrinolytic enzyme，FLE）基因，分析其基因序列并对该基因的功能进行分析。材料及方法：从中介蝮蛇毒腺细胞中提取总RNA，通过RT-PCR法扩增纤溶酶基因，与pMD18-T载体连接进行TA克隆，再进行序列的测定并且对该序列进行分析。结果：扩增得到完整开放读码框在内的长为777bp的纤溶酶基因序列。结论：成功克隆了编码中介蝮蛇纤溶酶的基因序列，推导其编码的氨基酸序列。开放读码框编码258个氨基酸，含有大量疏水氨基酸。其中，前1-19位氨基酸编码信号肽；根据同源性推测，以His65、Asp110和Ser204组成纤溶酶的活性中心并高度保守；序列内含12个Cys，两两配对结合成的6个二硫键对纤溶酶的高级构型和稳定性至关重要。成功克隆中介蝮蛇毒纤溶酶基因为进一步研究该基因的遗传特征以及为该酶在原核及真核生物中的表达研究提供依据。

Cloning and bioinformatic analysis of fibrinolytic enzyme from Gloydius intermedius

Objective: Fibrinolytic enzyme gene of Gloydius intermedius was cloned,then analysis the gene sequence and function. Methods:Total RNA was extracted from venom cells from Gloydius intermedius,FLE gene was amplified by RT-PCR.Then it was connected to pMD18-T vector by TA cloning.At last,sequencing and analysis the gene sequence. Results:FLE gene was successfully cloned.Its length was 777bp including whole open reading frame(ORF). Conclusion:Gene sequence of FLE of Gloydius intermedius was cloned successfully and deduced the animo acid sequence.The ORF codes 258 animo acids,including lots of hydrophobic animo acids.19 animo acids is a secretory signal peptide.Based on the animo acid sequences of other fibrinolytic enzyme,the His65、Asp110 and Ser204 constitute the catalytic resduces and the frame is high identity;the whole sequence includes 12 cys what matching 6 disulfide bridges are essential to the tertiary structure and stability of FLE .Cloning the gene of FLE successfully affords basis for the research of genetic character of this gene and prokaryotic expression or eukaryotic expression.