中国兽医科学2018，Vol.48Issue(1)：53-61,9.DOI:10.16656/j.issn.1673-4696.2018.0009
番鸭呼肠孤病毒YB株μA基因的克隆分析与原核表达
Cloning,sequence analysis and prokaryotic expression of μA gene from muscovy duck reovirus YB strain
摘要
Abstract
In order to clone and express the μA gene of muscovy duck reovirus(MDRV),we designed a pair of specific primers and amplified μA gene of MDRV-YB strain with RT-PCR,and the obtained target gene was cloned into expression vector pET-32a(+) after the same double digestion.The resultant recombinant plasmid was further identified by DNA sequencing and then transformed into E.coli BL21 (DE3)competent cells for protein expression and condition optimization of IPTG induction.Finally,the expressed protein was identified by SDS-PAGE and Western-blot analysis.The results showed that the combinant plasmid pET-YB μA containing the μA gene of MDRV-YB strain was successfully constructed and the μA gene sequence was obtained.The CDS size of MDRV-YB μA gene was 2199 bp,which was responsible for coding 732amino acids.The μA gene of MDRV-YB shared 81.7％-99.7％ nucleotide identity with traditional MDRV strains,and 82.5％ homology with novel duck-origin reovirus(NDRV),and 80.9％ homology with goose-origin reovirus(GRV) and only 73.1％homologywith avian reovirus(ARV).μA-derivedphylogenetic analysis showed that the MDRV-YB strain was located at the branch of traditional MDRV.The acid sequence of μA protein contained no potential signal peptides,but contained 6 potential N-glycosylation sites and 45 potential phosphorylation sites.SDS-PAGE analysis showed that a fusion protein(μA) with a molecular weight of about 99.7 ku was successfully expressed,and the optimal time,concentration and temperature of IPTG induction were 6 h,0.8mmol/L and 33 ℃,respectively.Western-blot analysis showed that the expressed μ A protein could specifically recognize MDRV positive sera,indicating that it had good reactogenicity.This study would lay a foundation for further exploration of biological functions of MDRV μA protein.关键词
番鸭呼肠孤病毒/μA蛋白/序列分析/原核表达Key words
muscovy duck reovirus/μA protein/sequence analysis/prokaryotic expression分类
农业科技引用本文复制引用
张英扬,李烨,吴异健,蔡栋灵,朱二鹏,陈强,李鸿文,林绍青,骆钰,刘珍妮,林琳..番鸭呼肠孤病毒YB株μA基因的克隆分析与原核表达[J].中国兽医科学,2018,48(1):53-61,9.基金项目
福建省农业良种选育及集约化种养技术研究与示范项目(2014NZ0002) （2014NZ0002）
福建农林大学创新专项基金——猴头菇多糖对MDRV感染雏番鸭肠黏膜淋巴细胞归巢过程的影响及其机制研究(CXZX2016016) （CXZX2016016）
