番鸭呼肠孤病毒YB株NS基因的序列分析及原核表达

Sequence analysis and prokaryotic expression of NS non-structural gene from Muscovy duck reovirus YB strain

为实现番鸭呼肠孤病毒(MDRV)YB株NS非结构基因的克隆分析及原核表达,首先经RT-PCR扩增NS基因的完整编码序列(CDS),并将其克隆到p ET-32a(+)载体中。测序后对获得的NS基因进行核苷酸及氨基酸序列分析。将重组质粒转化至E.coli BL21(DE3)感受态细胞中,进行IPTG诱导表达及条件优化。对表达产物进行SDS-PAGE分析和Western-blot分析。测序结果显示,成功构建了包含MDRV-YB株NS基因的原核重组质粒p ET-YB-NS,并获取了NS基因序列。序列分析结果显示,MDRV-YB NS基因的CDS大小为1 908 bp,编码635个氨基酸;该基因核苷酸序列与传统MDRV的同源性为98.2%~99.4%。遗传进化树分析显示,MDRV-YB株处于传统型MDRV分支上。该蛋白氨基酸序列中并不含有潜在的信号肽序列,但是含有2个潜在的N-糖基化位点以及61个磷酸化位点。经IPTG诱导表达后,SDS-PAGE分析显示,成功高效表达出分子质量约为88.7 ku的融合蛋白(p-NS),表达时IPTG最佳诱导时间、浓度和温度分别为5 h、0.4 mmol/L和36℃。Western-blot结果显示,表达的p-NS蛋白能特异性识别MDRV阳性血清,表明表达产物具备良好的反应原性。上述结果表明,MDRV NS非结构基因的克隆分析及其蛋白表达的实现,为进一步研究MDRV NS蛋白功能奠定了基础。

To realize the cloning,sequence analysis and prokaryotic expression of the pNS 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 DNA sequencing 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 635 amino acids. The nucleotide identity of NS gene between MDRV-YB and traditional MDRV strain was from 98.2%to 99.4%.The μNS-de-rived phylogenetic analysis showed that MDRV-YB strain 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 88.7 ku 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- μ NS protein could specifically recognize μDRV positive sera,indicating that it had good reactogenicity. The above-mentionged results showed that the sequencing analysis and prokaryotic expression of MI）RV μNS protein will lay a foundation for further MDRV μNS-associated bio-functional studies.