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

为了对番鸭呼肠孤病毒（MDRV）μA基因进行克隆分析和原核表达,本研究设计了1对特异性引物,对MDRV-YB株的μA基因进行RT-PCR扩增,扩增产物经过双酶切后克隆到p ET-32a（＋）载体中,经测序验证成功后,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。随后对表达产物进行SDS-PAGE以及W estern-blot分析。结果显示,成功构建了包含MDRV-YB株A基因的原核重组质粒,并获取了A蛋白的基因序列。序列分析结果显示,MDRV-YB株μA基因CDS大小为2 199 bp,负责编码732个氨基酸,该基因核苷酸序列与传统MDRV的同源性为83.8%~99.7%,与新型鸭源呼肠孤病毒（NDRV）的同源性约为82.5%,与鹅源呼肠孤病毒（GRV）同源性为80.9%,而与禽（鸡源）呼肠孤病毒（ARV）的同源性仅为73.1%;进化树分析μA基因的结果显示,MDRV-YB株属于传统型呼肠孤病毒,该蛋白氨基酸序列中无潜在的信号肽序列,6个潜在的N-糖基化位点以及45个磷酸化位点;SDS-PAGE分析表明,目的蛋白为分子质量约为99.7 ku的融合蛋白,主要以包涵体形式存在,其IPTG最佳诱导温度为33℃,浓度为0.8 mmol/L,时间为6 h;Western-blot结果显示,表达的目的蛋白能与抗MDRV阳性血清发生特异性反应,表明其具备良好的反应原性。本研究为进一步探索MDRV μA蛋白功能奠定了基础。

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 genewas 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 μ A gene was 2199 bp,which was responsible for coding 732 amino acids. The gA gene of MDRV-YB shared 81.7%-99.7% nucleotide identity with tra- ditional 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%homology with avian reovirus（ARV）. μA-derived phyloge- netic 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-glycosyla- tion 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 MDRVpositive sera,indicating that it had good reactogenicity, rhis study would lay a foundation for further exploration of biological functions-of MDRV μA protein.