Wu Y C, Chi S C
Institute of Zoology and Department of Life Science, National Taiwan University, 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan, ROC.
Fish Shellfish Immunol. 2007 Jul;23(1):97-108. doi: 10.1016/j.fsi.2006.09.008. Epub 2006 Oct 4.
We obtained a full-length cDNA clone for the Mx gene of barramundi (Lates calcarifer), using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) amplification of RNA extracted from a barramundi brain cell line cBB. The Mx cDNA of 2.2kb contains an open reading frame (ORF) of 1875 nucleotides encoding a protein of 624 amino acids. The predicted barramundi Mx protein is 71.4 kDa and contains a tripartite guanosinetriphosphate (GTP)-binding motif at the amino terminal and a leucine zipper at the carboxyl terminal, characteristic of all known Mx proteins. Poly I:C-transfection induced the expression of Mx gene in cBB cells, and the induction level at 28 degrees C was higher than that at 20 degrees C. Moreover, Mx gene expression was also induced by viral infection, including fish nodavirus, birnavirus, and iridovirus. Among these, nodavirus was a stronger inducer than the other two viruses. Using an antiviral activity assay, we revealed that poly I:C-transfected cBB cells had antiviral activity against fish nodavirus and birnavirus, but not iridovirus. Furthermore, the replication of nodavirus and birnavirus could be restored after the expression of Mx gene was down-regulated by siRNA. Therefore, these results indicated that the expression of barramundi Mx gene was able to inhibit the proliferation of fish nodavirus and birnavirus.
我们利用从尖吻鲈脑细胞系cBB中提取的RNA，通过cDNA末端快速扩增（RACE）聚合酶链反应（PCR）扩增，获得了尖吻鲈（Lates calcarifer）Mx基因的全长cDNA克隆。2.2kb的Mx cDNA包含一个1875个核苷酸的开放阅读框（ORF），编码一个624个氨基酸的蛋白质。预测的尖吻鲈Mx蛋白为71.4 kDa，在氨基末端含有一个三联鸟苷三磷酸（GTP）结合基序，在羧基末端含有一个亮氨酸拉链，这是所有已知Mx蛋白的特征。聚肌胞苷酸（Poly I:C）转染诱导了cBB细胞中Mx基因的表达，28℃时的诱导水平高于20℃时。此外，病毒感染，包括鱼类诺达病毒、双RNA病毒和虹彩病毒，也诱导了Mx基因的表达。其中，诺达病毒是比其他两种病毒更强的诱导剂。通过抗病毒活性测定，我们发现聚肌胞苷酸转染的cBB细胞对鱼类诺达病毒和双RNA病毒具有抗病毒活性，但对虹彩病毒没有。此外，在用小干扰RNA（siRNA）下调Mx基因表达后，诺达病毒和双RNA病毒的复制得以恢复。因此，这些结果表明尖吻鲈Mx基因的表达能够抑制鱼类诺达病毒和双RNA病毒的增殖。
