大鲵蛙病毒编码96L蛋白(ADRV-96L)的腺苷三磷酸酶活性和促进细胞生长作用

Adenosine triphosphatase activity and cell growth promotion of Andrias davidianus ranavirus 96L-encoded protein (ADRV-96L)

摘要:

【背景】腺苷三磷酸酶(ATPase，ATP酶)是控制DNA复制起始及宿主对病原微生物的应答开关。近期关于水生动物虹彩病毒基因组学的研究表明，它们共享编码ATPase的基因。【目的】大鲵蛙病毒(Andrias davidianus ranavirus，ADRV)属于虹彩病毒科，是世界现存最大两栖动物——中国大鲵的致死性病毒病原体。为了鉴定病毒基因及其表达产物对病毒复制和宿主细胞的影响，对ADRV编码ATPase的基因ADRV-96L进行了克隆、表达及功能分析，阐明这个虹彩病毒核心基因的功能。【方法】采用PredicProtein软件分析序列，构建重组原核表达质粒pET32a/His ADRV-96L，经IPTG (Isopropyl β-D-1-thiogalactopyranoside)诱导在大肠杆菌DE3中表达蛋白，用镍树脂纯化和咪唑液洗脱。钼蓝分光光度法检测产生的无机磷(Pi)以确定纯化ADRV-96L的ATPase活性。建立表达蛋白的稳定转染细胞系并进行鉴定，再分别通过绘制病毒的一步生长曲线和评估转染细胞的生长速率来测定该蛋白对病毒复制及细胞生长的影响。【结果】多重序列比对显示ADRV-96L存在含Walker A和Walker B基序的AAA-ATPase (ATPases associated with a variety of cellular activities，与各种细胞活性相关的ATPase)结构域(20?159位氨基酸)及两个高度保守的精氨酸。重组原核质粒表达了含ADRV-96L的52 kD融合蛋白，该蛋白质具有ATPase活性(酶的比活性平均为4.68 U/mg)。结果未检出ADRV-96L对病毒的复制影响，但显示该蛋白可促细胞生长。【结论】大鲵蛙病毒96L基因(ADRV-96L)编码一个促细胞增殖和生长的ATPase。

Abstract:

[Background] Adenosine triphosphatase (ATPase) controls DNA replication initiation and host response switch to pathogenic microbes. Recent genomics studies on aquatic animal iridoviruses have suggested that they share a gene encoding ATPase. Here, the function of the core gene in iridovirus will be first elucidated. [Objective] Andrias davidianus ranavirus (ADRV), which belong to the family Iridoviridae, is the viral pathogen of mass mortality in Chinese giant salamander—the world’s largest living amphibians. In order to identify virus gene and test the gene expression effects on virus replication and host cell, an ADRV gene, ADRV-96L was cloned, expressed and function analyzed. [Methods] Sequence data were analyzed using PredicProtein software. pET32a/His ADRV-96L recombinant prokaryotic expression plasmid was constructed, and the protein was expressed in Escherichia coli DE3 using IPTG induction, purified by Nickel Resin and eluted with imidazole. ATPase activity of the purified ADRV-96L recombinant protein was measured by the production of inorganic phosphorus (Pi) using molybdenum blue spectrophotometry method. Stable transfected cells were constructed and characterized. Then, the effects of ADRV-96L were analyzed by one-step multiplication curve for virus replication and estimating the growth rates of transferred cell, respectively. [Results] The multiple sequence alignments provided that ADRV-96L was characterized by the presence of the conserved AAA-ATPase (The ATPase associated with a variety of cellular activities) domains (20–159 residues) that contain Walker A and Walker B motifs, and two highly conserved arginines. The recombinant prokaryotic plasmid expressed a 52 kD fusion protein containing ADRV-96L, and the protein has ATPase activity 4.68 U/mg. The results show that ADRV-96L has no measurable impact on the ADRV replication, but it has been shown to promote cell growth. [Conclusion] Andrias davidianus ranavirus 96L gene (ADRV-96L) encodes an ATPase that is involved in cell proliferation and growth.