大黄鱼虹彩病毒ORF 121R蛋白的表达及亚细胞定位

吴彩霞; 刘晓东; 池洪树; 谢岸桦; 陈秀霞; 许斌福; 郑在予; 潘滢; 龚晖

大黄鱼虹彩病毒ORF 121R蛋白的表达及亚细胞定位

Expression and Subcellular Localization of ORF 121R Protein of Large Yellow Croaker Iridovirus

摘要

摘要:
目的解析大黄鱼虹彩病毒（large yellow croaker iridovirus, LYCIV）开放阅读框（Open reading frame, ORF） 121R蛋白的免疫原性与亚细胞定位，探究其在病毒致病机制中的潜在作用。
方法以LYCIV ORF 121R蛋白为研究对象，通过生物信息学分析手段，进行同源蛋白之间的序列对比，并预测其亚细胞定位；将目的基因通过同源重组方法分别克隆至pET32a及pEGFP-N1载体中，构建重组质粒ORF 121R-His和ORF 121R-EGFP；诱导表达并纯化ORF121R-His重组蛋白，将其免疫小鼠制备多克隆抗体，并通过ELISA、Western-blotting和细胞间接免疫荧光等手段评估抗体效价及特异性；以ORF 121R-EGFP质粒转染EPC细胞后，利用激光共聚焦显微镜观察ORF121R-EGFP的亚细胞定位。
结果 LYCIV ORF 121R蛋白与传染性脾肾坏死病毒（Infectious spleen and kidney necrosis virus, ISKNV）中具有高免疫原性的ORF 117R蛋白氨基酸序列一致性为79.96%，且预测定位于细胞质。orf 121R全长1 377 bp，将其分别克隆至pET32a及pEGFP-N1载体中，成功构建重组质粒ORF 121R-His和ORF 121R-EGFP。经亲和纯化获得可溶性ORF 121R-His重组蛋白，其分子量为72.6 kDa，用该重组蛋白制备的多克隆抗体效价为1∶128 000，可特异性识别天然病毒蛋白；ORF 121R-EGFP在鲤上皮瘤（Epithelioma papulosum cyprini, EPC）细胞中呈现胞质特异性分布，荧光信号均匀富集于核周区域。
结论 LYCIV ORF 121R主要定位于细胞质中，该蛋白良好的免疫原性与特异性，使其成为潜在的疫苗候选抗原和诊断靶标，为开发针对LYCIV的特异性防控策略提供了新的可能性。

Abstract:
Objective Immunogenicity and subcellular localization of ORF 121R of large yellow croaker iridovirus (LYCIV) were analyzed to understand the pathogenesis role of the protein.
Method Sequence of LYCIV ORF 121R was compared with those of the homologous proteins, and subcellular localization predicted by Cell-PLoc 2.0. The gene sequence was cloned into pET32a and pEGFP-N1 vectors to construct recombinant plasmids, ORF 121R-His and ORF 121R-EGFP. ORF121R-His was expressed and purified to prepare polyclonal antibodies from mice. Titer and specificity of the antibody were measured using ELISA, western-blotting, and indirect immunofluorescence techniques. After transfecting EPC cells with the ORF 121R-EGFP plasmid, subcellular location of ORF 121R-EGFP was observed under a confocal microscope.
Result LYCIV ORF 121R was 79.96% homologous in sequence with the immunogenic ISKNV ORF 117R proteins and predicted to localize in the cytoplasm. Successfully constructed from the 1 377 bp orf 121R, the recombinant plasmids were further analyzed. The purified soluble ORF 121R-His had a molecular weight of 72.6 kDa with a polyclonal antibody specifically recognizing natural LYCIV viral protein with a titer of 1:128 000. Whereas ORF 121R-EGFP exhibited a cytoplasmic distribution in EPC cells with the fluorescence signal evenly enriched in the perinuclear region.
Conclusion LYCIV ORF 121R was mainly found in the cytoplasm. The immunogenicity and specificity of the protein as demonstrated in this study lent it a potential candidate for vaccine production and disease diagnosis on yellow croakers.

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