猪DDX1基因的克

发布时间：2018-05-16 06:20

本文选题：猪 + DDX　；参考：《中国兽医学报》2017年11期

【摘要】：根据已知人、小鼠、牛、鸡、犬和恒河猴等物种的DEAD-box解旋酶1(DEAD-box helicase 1,DDX1)基因mRNA序列设计引物,从猪肾传代细胞系(PK-15)中克隆DDX1基因编码区(CDs)序列,对该基因进行生物信息学分析、组织表达谱分析。进一步将猪DDX1基因CDs序列克隆至真核表达载体pCMV-Tag2B,构建的重组真核表达质粒转染PK-15细胞后运用Western blot和间接免疫荧光试验检测DDX1基因的真核表达。结果显示:成功克隆了猪DDX1基因的cDNA序列(GenBank登录号为KU522467),其开放读码框全长为2 223bp,编码740个氨基酸;与牛、鸡、黑猩猩、犬、人、小鼠、大鼠和恒河猴的氨基酸序列同源性分别为98.8%、93.5%、97.6%、98.8%、97.7%、97.0%、97.6%和97.8%。表达谱分析表明,猪DDX1mRNA在不同组织中的表达水平存在差异,表现为脂肪、肝脏和脾脏中高表达,而在胃、心脏和肌肉中表达较低。成功构建了猪DDX1基因真核表达质粒,经证实目的基因可以在真核细胞中特异性表达,且表达的DDX1蛋白主要定位于细胞质中,少量定位于细胞核中。结果表明:本试验克隆了猪DDX1基因的序列,分析了其在不同组织的表达规律,并构建了DDX1基因重组表达质粒,为该基因下一步的功能学研究奠定了基础。
[Abstract]:According to the mRNA sequence of DEAD-box helicase 1(DEAD-box helicase 1 (DDX1) gene of known human, mouse, cattle, chicken, dog and rhesus monkey species, primers were designed to clone the sequence of DDX1 gene coding region from porcine kidney passage cell line PK-15, and to analyze the gene by bioinformatics. Tissue expression profile analysis. The CDs sequence of porcine DDX1 gene was further cloned into eukaryotic expression vector pCMV-Tag2B. the recombinant eukaryotic expression plasmid was transfected into PK-15 cells, and the eukaryotic expression of DDX1 gene was detected by Western blot and indirect immunofluorescence assay. The results showed that the cDNA sequence of porcine DDX1 gene was cloned successfully with the accession number of KU522467, and its open reading frame was 22 23 BP, encoding 740 amino acids with cattle, chicken, chimpanzee, dog, human, mouse, cattle, chicken, chimpanzee, dog, human and mouse. The amino acid sequence homology of rat and rhesus monkey was 98.8 and 93.50.The amino acid sequence of rat and rhesus monkey were 98.8% and 97.5% respectively. The amino acid sequence of rat and rhesus monkey were 97.6% and 97.8%, respectively. The expression level of porcine DDX1mRNA was different in different tissues, showing high expression in fat, liver and spleen, but low expression in stomach, heart and muscle. The eukaryotic expression plasmid of porcine DDX1 gene was successfully constructed. It was proved that the target gene could be specifically expressed in eukaryotic cells, and the expressed DDX1 protein was mainly located in the cytoplasm and a little in the nucleus. The results showed that the sequence of porcine DDX1 gene was cloned, the expression patterns of porcine DDX1 gene in different tissues were analyzed, and the recombinant expression plasmid of DDX1 gene was constructed, which laid a foundation for the further functional study of the gene.
【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;华中农业大学农业微生物学国家重点实验室;
【基金】：国家自然科学基金资助项目(31402181)
【分类号】：S828

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