摘要： DEAD-box解旋酶21（DExD-box helicase 21，DDX21）是含DEAD-box的RNA解旋酶家族成员之一，不仅参与RNA的合成和加工过程，同时还参与机体的天然免疫应答，调控病毒的复制。为了研究猪DDX21基因的结构和功能，以猪肾传代细胞（PK-15）总cDNA为模板，根据GenBank公布的预测序列（XM_005657387.2）设计引物扩增猪源DDX21基因，并采用分子生物学软件将其编码的氨基酸序列进行生物信息学分析。结果显示，首次成功克隆了猪DDX21基因的cDNA序列（GenBank登录号为KX396051），其开放读码框全长为2 355 bp，编码784个氨基酸；与牛、斑马鱼、人、小鼠、大鼠和非洲爪蟾的氨基酸序列同源性分别为91.7%，57.5%，88.0%，82.3%，83.8%和48.9%；该基因编码的蛋白结构域和人、牛、小鼠、大鼠一样，其C端都具有保守的GUCT结构域；系统进化树分析表明猪DDX21与牛DDX21的亲缘关系最近。进一步构建原核表达质粒pET28a-DDX21，经测序鉴定正确后，将其转化感受态细胞BL21（DE3），对DDX21基因进行原核表达。经SDS-PAGE分析显示重组菌可表达分子量约为90 kDa的融合蛋白，与预期相符；在IPTG诱导浓度一定的条件下，重组菌的最佳诱导时间为5 h。猪DDX21基因的克隆和原核表达，为进一步研究DDX21蛋白的结构和生物学功能奠定了基础。

关键词: 猪, DDX21基因, 克隆, 序列分析, 原核表达

Abstract: DDX21 (DExD-box helicase 21) is a RNA helicase,which belongs to the DEAD-box family of RNA helicases.Previous reports showed DDX21 not only take part in the generation and processing of RNA,but also have functional roles in virus's replication.This experiment was conducted to obtain sequence of swine DEAD-box helicase 21 gene,and to study its prokaryotic expression.According to the predicted Sus scrofa DDX21 gene mRNA sequence in GenBank(XM_005657387.2),proper primers were designed.Total RNA was then extracted from porcine kidney passage cells(PK-15),and CDs sequence of the gene was amplified by RT-PCR using the primers.The sequence analysis results showed that the sequence of porcine DDX21 CDs was in length of 2 355 bp which encoded 784 amino acids (GenBank Accession No.KX396051); Compared with Bos taurus,Danio rerio,Homo sapiens,Mus musculus,Rattus norgicus and Xenopus tropicalis,homology of porcine DDX21 was 91.7%,57.5%,88.0%,82.3%,83.8%,and 48.9% at the amino acid level,respectively; Structural analysis with the SMART program indicated that porcine DDX21 contained a putative C-terminal GUCT domain.Similar GUCT domain domains had been identified in cattle,human,mouse and rat DDX21; Phylogenetic tree analysis showed that porcine DDX21 had the closest relationship with cattle DDX21.Additionally,prokaryotic expression vector pET28a-DDX21 was constructed for further study.After sequencing,the recombinant was transformed into Escherichia coli BL21 (DE3) competent cells.The SDS-PAGE experiment demonstrated that the recombinant objective protein appeared a molecular mass of approximately 90 kDa which was consistent with the anticipated size.When IPTG concentration was kept constant,5 h induction was optimal.Cloning of porcine DDX21 gene and expression in E.coli laid a foundation for the subsequent structural analysis and function research of this gene.

Key words: Porcine, DDX21 gene, Cloning, Sequence analysis, Prokaryotic expression

中图分类号: 

• Q78

• S828