Cyprinid Herpesvirus 3 (CyHV-3), also known as Koi Herpesvirus (KHV), causes Koi Herpesvirus Disease (KHVD) which leads to serious economic losses worldwide. To exploit DNA/subunit vaccine candidates, CyHV-3 ORF131 gene and cDNA was cloned and analyzed in the present study. Major B cell epitopes of deduced CyHV-3 pORF131 was also predicted. Then the complete CDS of CyHV-3 ORF131 was inserted into pEGFP-N1 vector and a modified pYD1/EBY100 system to construct the DNA and subunit vaccine, respectively. Subsequently, carp were immunized with homologous and heterologous prime-boost regimens relying on the constructed DNA and oral subunit vaccines. Then the protective immunity generated from different vaccines and regimens as well as the capacity of yeast (Saccharomyces cerevisiae) as an oral vaccine vehicle was evaluated. Our study confirmed that CyHV-3 ORF131 gene consisted of 2 introns and 3 exons encoding a 428 amino acids peptide. Further analysis indicated that four fragments of CyHV-3 pORF131 contained the major B cell epitopes (Cys20~Val140, Ser169~Tyr245, Thr258~Pro390, Phe414~Gln428), which could be linked and expressed in E. coli (BL21) as a truncated pORF131. The expression of full-length CyHV-3 pORF131 by pEGFP-N1 and yeast surface display was verified by In vitro assays before vaccination. Immunization of carp with CyHV-3 ORF131 DNA and subunit vaccines could evoke the activation of immune-related genes such as CXCa, CXCR1, IL-1β, TNF-α, INF-a1, Mx-1, IgM, IgT1 and production of specific serum IgM measured by ELISA. RPS (relative percent of survival) ranging from 53.33% to 66.67% was acquired post challenge test. Moreover, flow cytometry analysis illustrated the delivery of surface-displayed CyHV-3 pORF131 to midgut after oral gavage. Thus, our findings suggest that CyHV-3 ORF131 can serve as DNA/subunit vaccines candidate and the yeast as an ideal oral vaccine vehicle.

中文翻译：

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通过使用基于CyHV-3 ORF131的DNA /蛋白质亚基疫苗在鲤鱼Cyprinus carpio var中通过不同的初免-加强疫苗接种方案对CyHV-3感染的保护性免疫。健。

塞浦路斯疱疹病毒3（CyHV-3），也称为锦鲤疱疹病毒（KHV），可导致锦鲤疱疹病毒病（KHVD），从而导致全球范围内的严重经济损失。为了利用DNA /亚基疫苗候选物，本研究中克隆并分析了CyHV-3 ORF131基因和cDNA。还预测了推导的CyHV-3 pORF131的主要B细胞表位。然后将完整的CyHV-3 ORF131 CDS插入pEGFP-N1载体和经过修饰的pYD1 / EBY100系统中，分别构建DNA疫苗和亚单位疫苗。随后，依赖于构建的DNA和口服亚单位疫苗，用同源和异源的初免-加强方案免疫鲤鱼。然后，评估了从不同疫苗和方案产生的保护性免疫力以及酵母（酿酒酵母）作为口服疫苗载体的能力。我们的研究证实，CyHV-3 ORF131基因由2个内含子和3个外显子组成，编码428个氨基酸。进一步的分析表明，CyHV-3 pORF131的四个片段含有主要的B细胞表位（Cys20〜Val140，Ser169〜Tyr245，Thr258〜Pro390，Phe414〜Gln428），可以在大肠杆菌（BL21）中连接并表达。截短的pORF131。疫苗接种前通过体外试验验证了pEGFP-N1全长CyHV-3 pORF131的表达和酵母表面展示。用CyHV-3 ORF131 DNA和亚基疫苗免疫鲤鱼可激活诸如CXCa，CXCR1，IL-1β，TNF-α，INF-a1，Mx-1，IgM，IgT1等免疫相关基因的激活并产生特异性通过ELISA测量的血清IgM。激发试验后获得的RPS（相对生存百分比）为53.33％至66.67％。此外，流式细胞仪分析表明，经口管饲法将表面展示的CyHV-3 pORF131递送至中肠。因此，我们的发现表明，CyHV-3 ORF131可作为DNA /亚单位疫苗的候选者，而酵母则可作为理想的口服疫苗载体。