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当前位置: 首页 > 文章 > 鲤疱疹病毒2型ORF5截短基因的克隆表达及免疫原性研究 中国兽医科学 2016 (11) 1394-1400
Position: Home > Articles > Cloning, expression and identification of immunogenicity of ORF5 truncation genes of cyprinid herpesvirus 2 Chinese Veterinary Science 2016 (11) 1394-1400

鲤疱疹病毒2型ORF5截短基因的克隆表达及免疫原性研究

作  者：
廖红;林华;郝中香;佘容;陈世界
单  位：
四川农业大学动物医学院;四川出入境检验检疫局;四川农业大学动物医学院四川出入境检验检疫局
关键词：
鲤疱疹病毒2型;ORF5截短基因;序列分析;克隆表达;攻毒保护试验
摘  要：
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。
译  名：
Cloning, expression and identification of immunogenicity of ORF5 truncation genes of cyprinid herpesvirus 2
作  者：
LIAO Hong;LIN Hua;HAO Zhong-xiang;SHE Rong;CHEN Shi-jie;YANG Miao;CHEN Zhen-rong;XUE Chang-hua;ZHAO Shan;HAN Guo-quan;CAO San-jie;YAN Qi-guil;College of Veterinary Medicine,Sichuan Agricultural University;Technology Center,Sichuan Enter-Exit Inspection and Quarantine Bureau;Inspection Center,Tongwei Co.,Ltd.;College of Food Science,Sichuan Agricultural University;
关键词：
cyprinid herpesvirus 2(CyHV-2);;ORF5 truncation gene;;sequence analysis;;cloning and expression;;protection test
摘  要：
A pair of primers was designed based on the nucleotide sequence(AFJ20457.1) of ORF5 retrieved from Gen Bank. The result showed that we successfully amplified the sequence of ORF5 truncation gene(357 bp),which encodes 119 amino acids and contains 4 epitopes,the theoretical molecular mass is13 059.70 u.It was indicated that there was no signal peptide and transmembrane region in the ORF5 truncation gene.The expression vector p ET-32a-ORF5(truncation) was constructed and transformed into Escherichia coli BL21.The recombinant protein was 33 ku detected by SDS-PAGE and Western-blot,confirmedwith expected size,and the expression protein was an insoluble inclusion body.The expression protein could specifically react with antibody of His tag and also recognize protein ORF5 which was in virus particles. The results of immunity protective tests showed that protection ratio of Carassius auratus gibelio were 20%,30% and 35%,respectively,the immune recombinant protein,so it was higher than the control group.The study confirmed that the expressed protein had immunogenicity.It enriched the studies of Cy HV-2 ORF5 gene and membrane cyprinid herpesvirus 2 genome,and laid the foundation for development of the novel vaccine and diagnostic kits.

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