苦参双功能核酸酶1的体外表达及结构性质分析

Experssion and structure analysis of bifunctional nuclease 1 in Sophora flavescens Ait.

植物双功能核酸酶1影响植株生长、发育及衰老,且研究表明其具有抗肿瘤活性。基于苦参转录组数据克隆了苦参(Sophora flavescens Ait.)中双功能核酸酶1基因的开放读码框(SfBFN1),分析其编码蛋白的理化性质和结构特点。采用RT-PCR方法扩增SfBFN1基因片段,纯化后与pET22b (+)载体连接并转化到大肠杆菌感受态细胞BL21,并以不同的诱导条件,筛选其体外表达的最适宜条件。结果获得了SfBFN1的开放读码框片段,长度为984 bp,编码327个氨基酸,SfBFN1蛋白分子质量约为36.2 ku;利用PHYRE2在线软件预测SfBFN1蛋白的部分三级结构,其富含α螺旋与β折叠,5个α螺旋包裹着7个β折叠,构成了蛋白质立体结构的框架。研究为SfBFN1蛋白开发成为抗癌蛋白质药物制剂奠定了实验基础。

Plant bifunctional nuclease 1 enzymes are responsible for plant growth and developmental processes,including senescence,and they also have anti-tumor effects in vitro.Based on the unigene sequences of the Sophora flavescens transcriptome data,the open reading frame(ORF)of bifunctional nuclease 1 gene(SfBFN1)from Sophora flavescens was cloned,and the physicochemical properties and structural characteristics of the encoded protein were analyzed.SfBFN1 was amplified by RT-PCR and further ligated into pET22b(+)vector,the recombinant plasmids were then transformed into BL21 competent cells.Optimization of the expression conditions for the SfBFN1 gene was performed at different temperatures and IPTG concentrations.The ORF fragment of SfBFN1 was 984 bp encoding SfBFN1 with 327 amino acids,which molecular weight was about 36.2 ku.SfBFN1 and the bifunctional nuclease 1 from Lupinus angustifolius showed the smallest genetic distance by the evolutionary analysis.The tertiary structure of SfBFN1 was predicted by PHYRE2 online software.The structure is rich inαhelix andβfold,with fiveαhelices wrapping sevenβfolds,forming the frame of the third dimension structure of the protein.The physicochemical properties and structural characteristics of SfBFN1 protein may lay the foundation for further studies on the protein as a potential anti-cancer drug.