Cloning and expression of ORF134 in Cyprinid herpesvirus 3 | 锦鲤疱疹病毒ORF134基因的克隆与表达

Chinese. 以感染锦鲤疱疹病毒的患病鱼为材料，通过RT-PCR克隆ORF134的编码序列，在细胞水平分析其表达模式，并在原核表达系统中表达，获得可溶性的ORF134重组蛋白。结果表明ORF134在锦鲤疱疹病毒裂解感染中转录，证实ORF134为非结构功能基因。ORF134全长为624bp，含有1个内含子，编码179个氨基酸，序列分析显示ORF134同已报道的高等脊椎动物病毒编码的vIL-10具有较低的序列同源性，但具有较高的结构相似性。体外表达分析显示ORF134呈分泌性表达，但不能形成寡聚体，与其他病毒编码的vIL-10具有差异。进一步通过原核表达获得可溶性的ORF134重组蛋白。

English. Cyprinid herpesvirus 3 (CyHV-3), also known as koi herpesvirus (KHV), is the causative agent of an emerging and mortal disease in common (Cyprinus carpio carpio) and koi (Cyprinus carpio koi) carp. Since its emergence in the late 1990s, KHV has caused severe financial and economic losses in both koi and common carp culture industries worldwide. CyHV-3 contains an interleukin-10 like open reading frame (ORF134), which has been identified from multiple members of Herpesvirales and Poxviridae. Here in this study, ORF134 of CyHV-3 was amplified by RT-PCR. It is 624 bp in full-length with an intron and encodes a peptide of 179 amino acids. Multiple sequence alignment indicated that ORF134 shared 23.5% amino acid sequence similarity with carp IL-10. Homology modeling suggested that ORF134 had a similar three-dimensional structure with vIL-10 encoded by EBV, but shared low sequence similarity with other vIL-10. To analysis the expression profile of ORF134, the coding region of ORF134 was cloned into pcDNA3.1 vector and expressed in 293T cells. The results suggested that ORF134 could secrete from expressed cells, but different from other identified viral IL-10 homologs, ORF134 could not form homodimer under non-reduced condition. Further study showed that soluble fusing protein of ORF134 was also expressed in E. coli.