中国兽医科学2017，Vol.47Issue(12)：1539-1548,10.DOI:10.16656/j.issn.1673-4696.2017.12.012
番鸭呼肠孤病毒YB株μNS基因的序列分析及原核表达
Sequence analysis and prokaryotic expression of μNS non-structural gene from Muscovy duck reovirus YB strain
摘要
Abstract
To realize the cloning,sequence analysis and prokaryotic expression of the μNS non-structural gene of Muscovy duck reovirus YB strain,the complete coding sequence(CDS)of the μNS gene was amplified by RT-PCR and then cloned into expression vector pET-32a(+).The obtained sequence of MDRV-YB μNS gene by DNAsequencing was subjected to nucleotide and amino acid sequence analysis.The recombinant plasmid was transformed into E.coli BL21(DE3)competent cells for protein expression and condition optimization of IPTG induction,and the expressed protein was then identified by SDS-PAGE and Westernblot analyses.In result,the recombinant plasmid pET-YB-μNS containing the μNS gene of MDRV-YB strain was successfully constructed and the μNS gene sequence was obtained.Sequence analysis showed that CDS of MDRV-YB μNS gene was 1908 bp in size,encoding 635amino acids.The nucleotide identity of μNS gene between MDRY-YB and traditional MDRV strain was from 98.2％to 99.4％.The μ NS-de-rived phylogenetic analysis showed that MDRV-YBstrain was located at the branch of traditional MDRV.The amino acid sequence of μNS protein contained no potential signal peptide sequences,but contained two potential N-glycosylation sites and 61 potential phosphorylation sites.SDS-PAGE analysis showed that the expressed fusion protein(p-μNS)with a molecular weight of about 88.7ku was successfully expressed by IPTG induction,and the optimal time,concentration and temperature of IPTG induction were 5 h,0.4 mmol/L and 36 ℃,respectively.Western-blot results showed that the expressed p-μNS protein could specifically recognize MDRV positive sera,indicating that it had good reactogenicity.The above-mentionged results showed that the sequencing analysis and prokaryotic expression of MDRY μNS protein will lay a foundation for further MDRV μNS-associated bio-functional studies.关键词
番鸭呼肠孤病毒/μNS蛋白/序列分析/原核表达Key words
Muscovy duck reovirus/μNS protein/sequence analysis/prokaryotic expression分类
农业科技引用本文复制引用
蔡栋灵,吴异健,张英扬,陈强,朱二鹏,林琳,刘珍妮,骆钰,黄烨敏,李烨..番鸭呼肠孤病毒YB株μNS基因的序列分析及原核表达[J].中国兽医科学,2017,47(12):1539-1548,10.基金项目
福建省科技重大专项:农业良种选育及集约化种养技术研究与示范(2014NZ0002) （2014NZ0002）
福建农林大学科技创新专项基金: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）
