蔡铭升;程安春;汪铭书;朱德康;罗启慧;招丽婵;贾仁勇;刘菲;陈孝跃;

• 1:四川农业大学动物医学院禽病防治研究中心
• 2:动物疫病与人类健康四川省重点实验室

摘要(Abstract)：

根据本实验室获得的鸭瘟病毒(DPV)UL35基因序列(GenBank登录号:EF643558),利用Oligo6.0和Prim-er5.0软件设计一对引物,PCR扩增出DPV CHv强毒株UL35基因,随后将其克隆至pMD18-T构建克隆质粒pMD18-T-UL35,经PCR和酶切鉴定后亚克隆至大肠杆菌原核表达载体pET-32a(+),获得表达质粒pET-32a(+)-UL35后将其转化至大肠杆菌BL21(DE3),经1.0mmol/L的IPTG在34℃诱导5h获得最佳表达。SDS-PAGE分析表明UL35基因表达的重组蛋白(VP26)分子量约为33kD,薄层扫描分析结果显示VP26占菌体总蛋白的32.3%,主要以包涵体的形式存在。经Ni+-NTA琼脂糖凝胶亲和层析纯化后免疫家兔制备抗VP26重组蛋白血清,血清琼脂扩散试验抗体滴度检测结果达1:32。用High-Q阴离子交换层析纯化抗血清获得兔抗VP26重组蛋白IgG,经Western blot检测显示抗血清可与VP26发生特异性反应。通过免疫荧光技术进行DPVVP26亚细胞定位检测,结果表明DPV感染DEF后2～8h细胞核内荧光的量相对较少,12～36h逐渐增加,48～72h达到最多,并且在细胞核内的斑点区域聚集呈颗粒状分布;而在细胞质内12h才开始出现少量荧光,荧光量在DPV感染后24～48h期间随感染时间延长而增加,在感染后72h最多。以上结果为阐明和进一步开展DPVUL35基因的功能研究提供了重要数据和材料。

关键词(KeyWords)： 鸭瘟病毒;UL35基因;克隆;表达;亚细胞定位

Abstract:

Keywords:

基金项目(Foundation): 现代农业产业技术体系建设专项资金(nycytx-45-12);; 教育部“长江学者和创新团队发展计划”创新团队项目(IRT0848)

作者(Authors): 蔡铭升;程安春;汪铭书;朱德康;罗启慧;招丽婵;贾仁勇;刘菲;陈孝跃;

DOI: 10.13242/j.cnki.bingduxuebao.002065

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