猪DDX21基因的克

发布时间：2018-01-22 06:33

本文关键词： 猪 DDX基因克隆序列分析原核表达　出处：《华北农学报》2017年03期 　论文类型：期刊论文

【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。
[Abstract]:DEAD-box unwindase 21 (DEx helicase 21 D-box, DDX21) is one of the RNA helicase family member with DEAD-box, not only in the process of synthesis and processing of RNA, the natural immune response is also involved in the regulation of the body, the replication of the virus. In order to study the structure and function of DDX21 gene in pig, pig kidney cell (PK-15) cDNA as a template, according to the published sequences of GenBank (XM_005657387.2) amplification of porcine DDX21 gene primers were designed using molecular biology software to its amino acid sequence encoding the bioinformatics analysis. The results showed that the first cloned cDNA sequence of porcine DDX21 gene (GenBank accession No. KX396051), the open reading frame the total length of 2355 BP, encoding 784 amino acids; and bovine, zebrafish, human, mouse, rat and Xenopus amino acid sequence homology were 91.7%, 57.5%, 88%, 82.3%, 83.8% and 48.9%; the gene encoding Protein domain and code, cattle, mice, rats, the C terminal has a conserved GUCT domain; phylogenetic tree analysis indicated that the closest relationship between porcine DDX21 and bovine DDX21. Further to construct the prokaryotic expression plasmid P ET28a-DDX21, identified by sequencing after the transformation of competent cells BL21 (DE3), for prokaryotic expression of DDX21 gene. SDS-PAGE analysis showed that the expression of molecular weight of 90 K Da recombinant fusion protein, in line with expectations; in the condition of a certain concentration induced by IPTG, the best induction time for expression of recombinant bacteria for cloning and prokaryotic DDX21 base 5 h. pigs, laid the foundation for further study on the structure and biological function of DDX21 protein.

【作者单位】：武汉生物工程学院应用生物技术研究中心;湖北省病毒载体工程技术研究中心;河北北方学院医学检验学院;华中农业大学农业微生物学国家重点实验室;
【基金】：国家自然科学基金项目(31402181) 湖北省教育厅科学技术研究项目(B2016303)
【分类号】：Q78;S828
【正文快照】： DEAD-box(DEAD-box proteins/DEAD-box heli-cases)家族蛋白是一类兼具有ATP酶活性和RNA解旋酶活性的RNA解旋酶,属于细胞RNA解旋酶超家族2型(SF2)。该家族蛋白广泛存在于从细菌到哺乳动物的许多物种中,通常含有9个保守的氨基酸基序,分别为基序Ⅰ、Ⅰa、Ⅰb、Ⅱ、Ⅲ、Ⅳ、Ⅴ、

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