摘要
新型番鸭呼肠孤病毒是近年来新发现的一种引起鸭严重疾病的病原,该病毒与经典MDRV主要在病毒宿主范围、体外培养特性、临床致病性和免疫保护特性上存在显著的差异。通过RT-PCR扩增新型呼肠孤病毒σC基因,经NcoⅠ和XhoⅠ双酶切处理,插入到p ET-28a(+)原核表达载体,获得重组质粒p ET-28a(+)-σC。将重组阳性质粒转化至BL21(DE3)感受态细胞,诱导表达后经SDS-PAGE电泳对σC蛋白进行初步分析,蛋白分子量约为36 k Da。重组蛋白经纯化后免疫实验兔,获得抗σC蛋白的高效免疫血清。通过Western Blot与间接免疫荧光实验初步表明,新型鸭呼肠孤病毒σC蛋白具有良好的反应活性。
The new Muscovy duck reovirus（ N-DRV） was detected in recently years which could result in serious disease of Muscovy duck. It was different from classical MDRV in host range,characteristics in vitro,clinical pathogenicity and characterization of autoimmunity etc. The σC gene of N-DRV was amplified by RT-PCR,digested by NcoⅠ and XhoⅠ,and inserted into prokaryotic expression vector p ET-28a（ ＋）. The expression plasmid p ET-28a-σC was transformed into the competent BL21（ DE3）. The fusion protein was expressed after induction and presented mainly in inclusion body. SDS-PAGE analysis showed that the fusion protein（ 36 k Da） was expressed correctly. The specific antiserum against σC was produced in rabbit immunized three times by the purified fusion protein σC. The protein σC had high reaction activity through Western Blot and IFA assay.
出处
《浙江农业学报》
CSCD
北大核心
2014年第6期1448-1452,共5页
Acta Agriculturae Zhejiangensis
基金
公益性行业(农业)科研专项经费(201003012)
国家自然科学基金(31302121)
浙江省自然科学基金(LY13C180002)
浙江省公益性项目(2012C22074)
浙江省重大动物传染病防治科技创新团队项目(2012R1003104)
关键词
新型呼肠孤病毒
σC基因
原核表达
兔抗血清
new Muscovy duck reovirus
σC gene
prokaryotic expression
rabbit anti serum
