Hua Y, Hui D Y, Rong Z H, Cheng T
College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan, China.
College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan, China
Genet Mol Res. 2014 Jan 21;13(1):386-95. doi: 10.4238/2014.January.21.6.
The full-length cDNA sequence of a novel expressed sequence tag (GenBank accession No. HQ184338) that was differentially expressed during Newcastle disease virus (NDV) infection in chickens was cloned from the chicken spleen by a rapid amplification of cDNA ends assay. This gene was further analyzed using bioinformatic methods and named grni. The full-length cDNA sequence was 1698 bp without introns, locating between 104,691,934 and 104,693,618 in galGal4 on chromosome 2. The open reading frame (ORF) contained 261 bp and encoded a deduced protein of 86 amino acid residues. Furthermore, the encoded protein contained two transmembrane regions without signal peptides, indicating that this protein is located in the mitochondrial membrane. Moreover, its homologous protein was not identified. Real-time polymerase chain reaction was used to detect the dynamic mRNA expression of this gene in the spleen, thymus, bursa of Fabricius, and trachea of NDV-infected chickens. Results suggested that the gene was involved in the transcriptional response of chicken to NDV infection. To obtain a fusion protein and prepare rabbit anti-serum, the predicted ORF of this gene was expressed in Escherichia coli. The expression of this gene at the protein level was further confirmed in the spleen, thymus, and bursa of Fabricius of NDV-infected chickens using Western blot analysis. In conclusion, a novel protein-coding gene named grni was successfully cloned and identified in chickens. Furthermore, this gene was found to be involved in the response of chickens to NDV infection.
通过cDNA末端快速扩增法，从鸡脾脏中克隆出一个在新城疫病毒（NDV）感染鸡的过程中差异表达的新型表达序列标签（GenBank登录号：HQ184338）的全长cDNA序列。利用生物信息学方法对该基因进行进一步分析，并将其命名为grni。全长cDNA序列为1698 bp，无内含子，位于2号染色体galGal4上的104,691,934至104,693,618之间。开放阅读框（ORF）包含261 bp，编码一个由86个氨基酸残基组成的推导蛋白。此外，该编码蛋白含有两个无信号肽的跨膜区域，表明该蛋白位于线粒体膜上。而且，未鉴定出其同源蛋白。采用实时聚合酶链反应检测该基因在NDV感染鸡的脾脏、胸腺、法氏囊和气管中的动态mRNA表达。结果表明该基因参与了鸡对NDV感染的转录反应。为获得融合蛋白并制备兔抗血清，该基因的预测ORF在大肠杆菌中表达。利用蛋白质印迹分析进一步证实了该基因在NDV感染鸡的脾脏、胸腺和法氏囊中的蛋白水平表达。总之，在鸡中成功克隆并鉴定出一个名为grni的新型蛋白质编码基因。此外，发现该基因参与了鸡对NDV感染的反应。
