人SIRT2基因结构和功能的生物信息分析及原核表达

Bioinformatics Analysis on Structure and Function of Human Silent Information Regulator 2 Gene and its Prokaryotic Expression

目的:通过生物信息学方法预测和分析人沉默信息调节因子2(Silent Information Regulator 2,SIRT2)基因结构和功能,利用分子生物学方法构建SIRT2重组质粒并进行原核表达。方法:利用NCBI、STRING、ProtParam、Genecards和SWISS-MODEL等软件和数据库对SIRT2蛋白的理化性质、跨膜结构域、亚细胞定位、翻译后修饰、二级结构、三级结构及相互作用网络等生物学特性进行了预测分析,采用PCR扩增技术获得SIRT2基因的cDNA序列全长,通过同源重组的方法构建p ET-28aSIRT2重组表达质粒并进行原核表达。结果:人SIRT2蛋白由389个氨基酸组成,是一种呈酸性、不稳定的、亲水的胞内蛋白质。SIRT2主要分布在细胞质,具有38个潜在的磷酸化修饰位点,其二级结构主要是无规则卷曲。成功构建了重组质粒p ET-28aSIRT2,并通过IPTG诱导大肠杆菌BL21后成功获得SIRT2蛋白的表达,SIRT2主要存在于细菌裂解液离心之后的沉淀中。结论:本研究成功克隆了人SIRT2的编码区,结合多种生物信息软件对SIRT2的性质和功能进行了了解,为进一步研究SIRT2蛋白的生物学功能和肿瘤的靶向治疗作用机制提供思路。

Objective:The structure and function of the silent information regulator 2(SIRT2)gene are described by the bioinformatics method,as well as the use of molecular biology methods to construct a recombinant plasmid of SIRT2 and express it in prokaryotic cells.Methods:The SIRT2 protein’s physicochemical properties,transmembrane structure domains,subcellular localization,post-translational modifications,secondary and tertiary structures,and interaction networks are predicted and analyzed using software and databases such as NCBI,STRING,ProtParam,Genecards,and SWISS-MODEL.The full-length cDNA sequence of the SIRT2 gene is obtained using PCR amplification,and a homologous recombination method is employed to construct the pET-28a-SIRT2 recombinant expression plasmid,which is then expressed in prokaryotic cells.Results:The results show that the human SIRT2 protein consists of 389 amino acids and is an acidic,unstable,and hydrophilic intracellular protein.SIRT2 is mainly distributed in the cytoplasm and has 38 potential phosphorylation modification sites,with its secondary structure mainly being disordered coils.Furthermore,a recombinant plasmid pET-28a-SIRT2 is successfully constructed,and SIRT2 protein expression is induced in E.coli BL21 using IPTG.SIRT2 is mainly expressed in the bacterial precipitate of the cell lysate,laying the foundation for subsequent structural and functional analysis of SIRT2.Conclusion:this study successfully cloned the coding region of human SIRT2 and used multiple bioinformatics tools to investigate its properties and functions,providing insight into the biological functions of SIRT2 protein and its potential as a target for cancer therapy.