人NIRF基因真核表达质粒的构建及其蛋白产物的生物信息学分析

目的克隆人NIRF基因,构建其真核表达质粒,并运用生物信息学方法对NIRF蛋白进行分析。方法应用RT-PCR技术,从HeLa细胞中扩增NIRF基因,插入到真核表达载体pcDNA3.1-Flag中,双酶切鉴定并测序。在NCBI蛋白质结构数据库中寻找与NIRF具有较高同源性的1WY8和1Z6U,并利用Insight II结构生物信息学分析软件分析NIRF蛋白的结构和功能。结果扩增出约2400bp的人NIRF全长cDNA;重组真核表达质粒pcDNA3.1-Flag-NIRF酶切后,可见约2400bp的目的片段;测序结果表明NIRF全长cDNA与GenBank中NIRF序列完全一致。1WY8片段有3个赖氨酸残基,主要位于蛋白空间构象的表面;1Z6U片段含有Ring finger结构域,具有ligase活性。结论已成功克隆了人NIRF基因全长cDNA,构建了其真核表达质粒,并利用生物信息学方法,初步分析了NIRF蛋白泛素化作用的机理。

Construction of Eukaryotic Expression Vector for Human NIRF Gene and Bioinformatics of Deduced Expressed Protein

Objective To clone human NIRF gene,construct its eukaryotic expression vector and analyze the bioinformatics of deduced expressed protein.Methods NIRF gene was amplified from HeLa cells by RT-PCR and inserted into eukaryotic expres-sion vector pcDNA3.1-Flag.The constructed recombinant plasmid pcDNA3.1-Flag-NIRF was identified by restriction analysis and se-quencing.Fragments 1WY8 and 1Z6U highly homologous to NIRF were screened from NCBI protein structure data bank for analyzing structure and function of NIRF protein by Insight Ⅱ software.Results The NIRF cDNA fragment at a full length of about 2 400 bp was amplified.The target gene fragment at a length of about 2 400 bp was recovered from recombinant plasmid pcDNA3.1-Flag-NIRF digested with restriction endonuclease,and its sequence was completely identical to that of NIRF reported in GenBank.1WY8 frag-ment contained 3 lysine residues which was mainly located on the surface of spatial conformation of protein.1Z6U fragment contained a ring finger structural domain and showed the activity of ligase.Conclusion The full-length cDNA of human NIRF was successfully cloned,and its eukaryotic expression vector was constructed.The mechanism of ubiquitination of NIRF protein was preliminarily ana-lyzed by bioinformatical method.