本文選題：番鴨呼腸孤病毒 + NS蛋白　； 參考：《中國獸醫(yī)科學》2017年12期

【摘要】：為實現(xiàn)番鴨呼腸孤病毒(MDRV)YB株NS非結構基因的克隆分析及原核表達,首先經(jīng)RT-PCR擴增NS基因的完整編碼序列(CDS),并將其克隆到p ET-32a(+)載體中。測序后對獲得的NS基因進行核苷酸及氨基酸序列分析。將重組質粒轉化至E.coli BL21(DE3)感受態(tài)細胞中,進行IPTG誘導表達及條件優(yōu)化。對表達產(chǎn)物進行SDS-PAGE分析和Western-blot分析。測序結果顯示,成功構建了包含MDRV-YB株NS基因的原核重組質粒p ET-YB-NS,并獲取了NS基因序列。序列分析結果顯示,MDRV-YB NS基因的CDS大小為1 908 bp,編碼635個氨基酸;該基因核苷酸序列與傳統(tǒng)MDRV的同源性為98.2%~99.4%。遺傳進化樹分析顯示,MDRV-YB株處于傳統(tǒng)型MDRV分支上。該蛋白氨基酸序列中并不含有潛在的信號肽序列,但是含有2個潛在的N-糖基化位點以及61個磷酸化位點。經(jīng)IPTG誘導表達后,SDS-PAGE分析顯示,成功高效表達出分子質量約為88.7 ku的融合蛋白(p-NS),表達時IPTG最佳誘導時間、濃度和溫度分別為5 h、0.4 mmol/L和36℃。Western-blot結果顯示,表達的p-NS蛋白能特異性識別MDRV陽性血清,表明表達產(chǎn)物具備良好的反應原性。上述結果表明,MDRV NS非結構基因的克隆分析及其蛋白表達的實現(xiàn),為進一步研究MDRV NS蛋白功能奠定了基礎。
[Abstract]:In order to clone and analyze the NS non-structural gene of muscovy duck reovirus strain MDRVV YB strain and prokaryotic expression, the complete coding sequence of NS gene was amplified by RT-PCR and cloned into pET-32a () vector. After sequencing, nucleotide and amino acid sequences of NS gene were analyzed. The recombinant plasmid was transformed into E.coli BL21 (DE3) competent cells for IPTG induced expression and optimized conditions. The expressed products were analyzed by SDS-PAGE and Western-blot. Sequencing results showed that the prokaryotic recombinant plasmid pET-YB-NScontaining NS gene of MDRV-YB strain was successfully constructed and the sequence of NS gene was obtained. The results of sequence analysis showed that the CDS of MDRV-YB NS gene was 1 908 BP, encoding 635 amino acids, and the nucleotide sequence of MDRV-YB NS gene was 98.299. 4% homology with traditional MDRV. Genetic phylogenetic tree analysis showed that the MDRV-YB strain was on the traditional MDRV branch. The amino acid sequence does not contain a potential signal peptide sequence, but contains two potential N-glycosylation sites and 61 phosphorylation sites. SDS-PAGE analysis showed that the fusion protein of about 88.7 ku was successfully expressed by SDS-PAGE. The optimal time, concentration and temperature of IPTG induction were 5 h, 0.4 mmol/L and 36 鈩，

本文編號：1777542