鲤疱疹病毒Ⅱ型ORF66基因DNA疫苗载体的构建及其免疫效果

鲤疱疹病毒Ⅱ型(CyHV-2)能够引起鲫大量死亡,严重威胁我国水产养殖业的健康发展,目前,针对CyHV-2尚无有效的商业化疫苗或治疗措施。为构建CyHV-2DNA疫苗,本研究将其衣壳蛋白ORF66编码基因克隆至pVAX1真核表达载体上,构建重组质粒pVAX-ORF66。此外,在BL21(DE3)pLysS中对ORF66蛋白进行了原核表达,表达蛋白经纯化后免疫新西兰白兔制备了ORF66特异性抗体。酶联免疫吸附(ELISA)检测显示,制备抗体效价达1∶20000以上。将pVAX-ORF66质粒转染金鱼脑细胞系(GFB),利用制备的ORF66抗体进行间接免疫荧光(IFA)检测,结果显示,ORF66蛋白可以在细胞中大量表达,且主要定位于细胞质中。将pVAX-ORF66质粒肌肉注射鲫后进行CyHV-2免疫保护实验,结果表明,其相对免疫保护率达55.6%。本研究针对CyHV-2构建了一种制备简单、成本低廉的DNA疫苗,为鲫造血器官坏死病的免疫预防及感染分子机制研究奠定了前期实验基础。

Construction and immune efficacy of a cyprinid herpesvirus 2 ORF66-containing DNA vaccine

Cyprinid herpesvirus 2 (CyHV-2) is a highly contagious pathogen that causes haematopoietic necrosis disease in goldfish (Carassius auratus auratus) and gibel carp (Carassius auratus gibelio). Currently, there are no licensed vaccines or therapeutics for CyHV-2. Nonetheless, pVAX1 is a eukaryotic plasmid designed for the development of animal DNA vaccines. This vector allows high-copy number replication in E. coli and contains a CMV promotor for high-level expression of foreign proteins. In this study, the coding region of the CyHV-2 major capsid protein ORF66 was amplified by PCR and cloned into the pVAX1 vector to construct a recombinant plasmid, pVAX-ORF66. Additionally, a recombinant prokaryotic expression plasmid, pRSET-ORF66, was constructed and transformed into E. coli BL21(DE3)pLysS. The ORF66 recombinant protein was then produced by induction with 1 mmol/L IPTG; subsequently, the polyclonal antibody was prepared by subcutaneously immunizing New Zealand rabbits with the purified protein. The titer and specificity of the prepared antibody were analyzed using an ELISA assay, Western blotting, and an indirect immunofluorescence assay (IFA). Next, the recombinant plasmid pVAX-ORF66 was transfected into goldfish brain (GFB) cells, and the expression of ORF66 protein was detected by IFA and Western blotting. Finally, pVAX-ORF66 was intramuscularly injected into gibel carp at the anterior-to-dorsal fin region, before being infected with CyHV-2. The results demonstrated that the full nucleotide length of ORF66 was 1200 bp and the molecular weight of the recombinant protein was approximately 45 kDa, which were both consistent with their predicted sizes. The prepared rabbit polyclonal antibody was highly specific to CyHV-2, with a minimum titer of 1:20000. ORF66 expression could be detected in both infected and pVAX-ORF66 transfected GFB cells and was mainly distributed in the cytoplasm. Finally, the relative survival rate of immunized gibel carp was determined to 55.6%. Collectively, in this study, a eukaryotic expression plasmid, pVAX-ORF66, was constructed that could serve as a DNA vaccine for the prevention and control of CyHV-2. These results also provide a basis for further studies regarding the molecular function of ORF66 during CyHV-2 infection.