摘要
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。
Based on the duck plague virus （DPV） UL35 gene sequence that our laboratory obtained （GenBank accession number EF643558）,a pair of primers was designed using Oligo6.0 and primer5.0,then the UL35 gene was amplified from DPV CHv strain genomic DNA and cloned into the pMD18-T to construct a clone plasmid pMD18-T-UL35. After identification of the pMD18-T-UL35 by PCR amplification and restriction digestion,the fragment of the UL35 gene was subcloned into the prokaryotic expression vector pET-32a（＋）. The resultant recombinant plasmid pET-32a（＋）-UL35 was then transformed into E.coli BL21 （DE3） strain and optimally-expressed under the induction of 1.0mmol/L IPTG at 34℃ for 5 hours. SDS-PAGE analysis showed the recombinant protein （VP26） had a molecular weight of about 33KDa and accounted for 32.3% of total bacterial protein by gel scanning. The protein was then purified by Ni^2＋-affinity chromatography and used to immunize rabbit for producing the VP26 anti-serum and its antibody titer was up to 1：32 detected by agar diffusion reaction. After the IgG of the polyclonal antibodies was purified by High-Q anion-exchange chromatography,Western blot analysis indicated that the IgG had specific reaction with the VP26. Moreover,the subcellular localization detection was observed using immunofluorescence technique. The results showed that the specific fluorescences appeared relatively few in nucleus in 2 to 8 hours and increased gradually in 12 to 36 hours and eventually reached to the maximum,which aggregated in the spot region of the nucleus after the duck embryo fibroblast （DEF） were infected by DPV. However,there were only a small amount of specific fluorescences in the cytoplasm in 12 hours and increased with the extension of infection time in 24 to 48 hours. The specific fluorescences finally reached to the maximum in the cytoplasm in 72 hours. The results provided significant data for furthering the study on the function of DPV UL35 gene.
出处
《病毒学报》
CAS
CSCD
北大核心
2010年第2期143-149,共7页
Chinese Journal of Virology
基金
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)
关键词
鸭瘟病毒
UL35基因
克隆
表达
亚细胞定位
duck plague virus
UL35 gene
clone
expression
subcellular localization
