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

【摘要】：為實(shí)現(xiàn)番鴨呼腸孤病毒(MDRV)YB株NS非結(jié)構(gòu)基因的克隆分析及原核表達(dá),首先經(jīng)RT-PCR擴(kuò)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(cè)序后對(duì)獲得的NS基因進(jìn)行核苷酸及氨基酸序列分析。將重組質(zhì)粒轉(zhuǎn)化至E.coli BL21(DE3)感受態(tài)細(xì)胞中,進(jìn)行IPTG誘導(dǎo)表達(dá)及條件優(yōu)化。對(duì)表達(dá)產(chǎn)物進(jìn)行SDS-PAGE分析和Western-blot分析。測(cè)序結(jié)果顯示,成功構(gòu)建了包含MDRV-YB株NS基因的原核重組質(zhì)粒p ET-YB-NS,并獲取了NS基因序列。序列分析結(jié)果顯示,MDRV-YB NS基因的CDS大小為1 908 bp,編碼635個(gè)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(jìn)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(hào)肽序列,但是含有2個(gè)潛在的N-糖基化位點(diǎn)以及61個(gè)磷酸化位點(diǎn)。經(jīng)IPTG誘導(dǎo)表達(dá)后,SDS-PAGE分析顯示,成功高效表達(dá)出分子質(zhì)量約為88.7 ku的融合蛋白(p-NS),表達(dá)時(shí)IPTG最佳誘導(dǎo)時(shí)間、濃度和溫度分別為5 h、0.4 mmol/L和36℃。Western-blot結(jié)果顯示,表達(dá)的p-NS蛋白能特異性識(shí)別MDRV陽性血清,表明表達(dá)產(chǎn)物具備良好的反應(yīng)原性。上述結(jié)果表明,MDRV NS非結(jié)構(gòu)基因的克隆分析及其蛋白表達(dá)的實(shí)現(xiàn),為進(jìn)一步研究MDRV NS蛋白功能奠定了基礎(chǔ)。
[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(hào)：1777542