微小隐孢子虫转录共激活因子CpADA2的生物信息学分析及其编码基因的真核表达

Bioinformatics Analysis and Eukaryotic Expression of Cryptosporidium parvum Transcriptional Coactivator CpADA2

本研究旨在对微小隐孢子虫转录共激活因子ADA2的结构和功能进行生物信息学分析,对其编码基因CpADA2进行真核表达。利用在线生物信息学软件对CpADA2进行生物信息学分析,结果显示,CpADA2含有ZnF和SANT结构域,全长673个氨基酸残基,分子质量为76 kDa,属于可溶性蛋白;不含跨膜区和信号肽,但存在多样化的激酶特异性磷酸化位点;二级结构由α螺旋(36.70%)、β折叠(11.74%)和无规卷曲(51.56%)构成;三级结构中SANT结构域呈典型的α螺旋串联重复构象;互作网络模型和功能预测表明,该蛋白具有锌离子结合特性,并可能参与组蛋白乙酰化过程。以微小隐孢子虫cDNA为模板,PCR扩增得到CpADA2基因,成功构建了真核表达质粒p3×FLAG-CMV14-CpADA2,转染至293T细胞后,Western blot分析显示,转染重组质粒的细胞成功表达重组CpADA2蛋白。本研究为后续开展CpADA2的功能研究提供了基础。

The aim of this study was to predict the structure and function of the Cryptosporidium parvum transcriptional coactivator CpADA2 and express eukaryotically its encoding gene.The structure and function of CpADA2 protein were analyzed by online bioinformatics software.The results showed that CpADA2 protein contained ZnF and SANT domains with a total length of 673 amino acid residues and the soluble protein had a molecular weight of 76 kDa.There were no transmembrane region and signal peptide in CpADA2 protein while there were a variety of kinase-specifi c phosphorylation sites.The secondary structure was mainly composed ofαhelix(36.70%),βsheets(11.74%)and random coil(51.56%).The SANT domain in the tertiary structure showed a typical tandem alpha helical repeats conformation.Interaction network model and functional prediction suggested that this protein had zinc ion binding properties and might participate in histone acetylation process.CpADA2 gene was amplifi ed by PCR using C.parvum cDNA as template and the p3×FLAG-CMV14-CpADA2 plasmid was constructed for eukaryotic expression in 293T cells.Western blot analysis showed that the recombinant CpADA2 protein was successfully expressed in the transfected cells.This study laid a foundation for the subsequent functional studies of CpADA2 protein.