急性病毒性坏死病毒魁蚶株 IAP-86基因全长cDNA 克隆和生物信息学分析

摘要

为深入探讨急性病毒性坏死病毒(Acute Viral Necrosis Virus, AVNV)魁蚶株的致病机理以及IAP-86蛋白的功能，从感染 AVNV 的濒死魁蚶外套膜中提取总 RNA，反转录获得 cDNA。根据 NCBI公布的 AVNV 全基因组序列中 ORF86序列设计两对反向套式引物，通过 cDNA 末端快速扩增(RACE)技术获得 ORF865¢端和3¢端的非编码区，拼接获得全长 cDNA 序列。Blast 序列比对显示，该基因与牡蛎疱疹病毒的同源性为100%，与栉孔扇贝 AVNV 的同源性为99%，并存在重叠基因。生物信息学分析预测，该蛋白不含信号肽，不存在跨膜区，最大疏水指数为1.800，最小疏水指数为–3.456。该蛋白存在8个潜在的磷酸化位点(包括5个丝氨酸、1个苏氨酸和2个酪氨酸)，存在 1个潜在的 O-糖基化位点，不存在潜在的 N-糖基化位点；其抗原表位主要集中在8−11、14−16、28−39、75−76、88−95、97−100和147−158位氨基酸。推测该株病毒可能为牡蛎疱疹病毒的变异株，并且基因重叠在该类病毒进化过程中可能扮演重要角色。%Acute viral necrosis virus (AVNV) was reported as the causative agent for summer mass mortality of adult Zhikong scallop (Chlamys farreri) that has been widely cultured along northern China coast. To understand the pathogenesis and the function of IAP-86, a strain of acute viral necrosis virus (AVNV) was isolated from Anadara uropygimelana. RNA was extracted from the mantle of moribund A. uropygimelana that was infected with AVNV, and cDNA was obtained by reverse transcription. Two pairs of nested reverse primer were designed according to the ORF86 sequence of AVNV complete genome sequence that registered in NCBI. The non-coding region of 5' and 3' end of the ORF86 were amplified using the designed primers by rapid amplification of cDNA ends (RACE) technique, and the full-length cDNA sequences were spliced. Blast sequence alignment illustrated that this gene has 100% homology with oyster herpetovirus and 99% with the AVNV. Moreover, overlapping genes were found in the cDNA sequence. Bioinformatics analysis indicated that the protein contains neither a signal peptide nor a transmembrane region. The maximum hydrophobic index was 1.800 and the minimum hydrophobic index was -3.456. There are eight potential phosphorylation sites (including five serine sites, two tyrosine sites and one threonine site), a potential O-glycosylation site, but no potential N-glycosylation site. The epitope were mainly located on the amino acids of 8-11, 14-16, 28-39, 75-76, 88-95, 97-100 and 147-158. Results suggest that the virus may be a strain of oyster herpetovirus, and gene overlapping may play an important role in the virus evolution.