番鸭源呼肠孤病毒σC基因的表达与分析

EXPRESSION AND ANALYSIS OF σC GENE OF MUSCOVY DUCK REOVIRUS

为开展番鸭源呼肠孤病毒(Muscovy duck reovirus,MDRV)σC基因编码蛋白的相关研究,本研究采用RT-PCR技术从MDRV全基因组中扩增σC基因片段,与p ET24a载体连接,构建原核表达重组质粒p ET24a-C,转化BL21(DE3)感受态细胞后,IPTG诱导表达σC重组蛋白;并利用SDS-PAGE电泳和Western blot检测其表达情况和免疫原性。结果显示,扩增的σC基因序列与Gen Bank所发表的序列同源性为100%;σC重组蛋白能在大肠杆菌中大量表达,其分子量约为30 kDa,且能被MDRV阳性血清识别,具有良好的抗原结合活性。σC重组蛋白的成功表达,将对后续围绕该蛋白的功能鉴定、诊断用抗原制备或新型疫苗研制工作有所帮助。

In the present study,theσC gene was amplified from the whole genome of Muscovy duck reovirus(MDRV)and connected to the plasmid pET24 a for construction of the plasmid pET24 a-C.The plasmid pET24 a-C was then transformed into BL21(DE3)cells forσC protein expression with IPTG induction.Recombinant protein was examined in SDS-PAGE electrophoresis and Western blot.The results showed that the amplifiedσC gene sequence was 100%homology with the published sequence and the recombinant protein was expressed in E.coli with a molecular weight of about 30 kDa.The recombinant protein reacted with MDRV positive serum in Western blot.The prokaryotic expression ofσC gene of MDRV would be beneficial to future research on functional characterization,preparation of diagnostic antigens and development of new vaccines.