单 位：
武汉生物工程学院应用生物技术研究中心;河北北方学院医学检验学院;湖北省病毒载体工程技术研究中心;华中农业大学农业微生物学国家重点实验室
关键词：
猪;DDX21基因;克隆;序列分析;原核表达
摘 要：
DEAD-box解旋酶21(DEx D-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p ET28a-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 k Da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。
译 名：
Cloning,Sequence Analysis and Prokaryotic Expression of Porcine DDX21
作 者：
XIE Lilan;AN Kang;CHEN Li;SUN Zide;FANG Liurong;Center of Applied Biotechnology,Wuhan Institute of Bioengineering;Hubei Engineering Research Center of Viral Vector;College of Lab Medicine,Hebei North University;State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University;
关键词：
Porcine;;DDX21 gene;;Cloning;;Sequence analysis;;Prokaryotic expression
摘 要：
DDX21( DEx D-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 encoded784 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 p ET28a-DDX21 was construc-ted 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 k Da 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.
