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根据G en Bank上已登录的鲤疱疹病毒2型的O RF5基因序列(登录号:AFJ20457.1)设计1对引物,采用PCR扩增技术获得目的基因。序列分析表明,目的基因长357 bp,是一个完整的开放阅读框,编码119个氨基酸,理论分子质量为13 059.70 u,无信号肽,无跨膜区,含有4个抗原表位。将扩增片段克隆至原核表达载体p ET-32a中,并将获得的重组质粒转化至大肠杆菌BL21中进行诱导表达。对表达蛋白进行复性纯化后免疫小鼠和异育银鲫,SD S-PAGE和W estern-blot检测分析表明,重组蛋白的分子质量约为33 ku,与预期相符,表达形式为包涵体,该蛋白能够与抗H is标签的抗体发生特异性反应,也能识别病毒粒子上的ORF5蛋白。对免疫的异育银鲫进行攻毒,结果显示注射生理盐水组的银鲫保护率为0,注射15、25、50 g重组蛋白组的保护率分别为20%、30%、35%,表明免疫重组蛋白组的保护率高于对照组。本研究证实了表达的融合蛋白具有一定的免疫原性,丰富了鲤疱疹病毒2型ORF5基因及基因组的研究,为研发制备新型鲤疱疹病毒2型的疫苗和诊断试剂盒奠定了基础。
One pair of primers was designed based on O RF5 gene sequence of Cyprinid herpesvirus type 2 (Accession No .: AFJ20457.1) registered on G en Bank, and the target gene was obtained by PCR amplification. Sequence analysis showed that the target gene was 357 bp in length and contained a complete open reading frame (ORF) encoding 119 amino acids with a theoretical molecular mass of 13 059.70 u, without signal peptide, no transmembrane region and 4 epitopes. The amplified fragment was cloned into the prokaryotic expression vector p ET-32a, and the recombinant plasmid obtained was transformed into E. coli BL21 for induction of expression. The results of SD S-PAGE and Western-blot showed that the molecular weight of the recombinant protein was about 33 ku, which accorded with the expectation. The expressed protein was expressed in the form of inclusion bodies, This protein specifically reacts with anti-His-tagged antibodies and also recognizes the ORF5 protein on virions. The results showed that the protection rate of silver crucian carp injected with saline group was 0 and the protection rates of 15, 25 and 50 g recombinant protein groups were 20%, 30% and 35% respectively Immunoprotective proteomics protection rate was higher than the control group. This study confirmed that the expressed fusion protein has some immunogenicity and enriched the herpes simplex virus 2 type ORF5 gene and genome research, which laid the foundation for the development of a new type 2 carp herpes virus 2 vaccine and diagnostic kit.