摘要
采用RT-PCR方法从鸭源呼肠孤病毒DRV-GZ株中扩增出了S2基因片段,将其克隆到表达载体pET-28a(+)中,测序验证后转化入表达宿主菌RosettaTM2(DE3)plysS,进行IPTG诱导表达。结果表明,重组菌可表达出相对分子质量约为50 000的重组融合蛋白,在浓度为0.6 mmol/L的IPTG诱导4 h的情况下表达效果最好。表达的蛋白以包涵体的形式存在于菌体中。表达产物经Ni柱纯化后可得到纯度较高的目的蛋白。经Western-blot分析,所纯化的蛋白能与抗呼肠孤病毒DRV-GZ株阳性血清进行特异性的免疫印迹反应,证实表达的蛋白具有较好的反应原性。
S2 gene of reovirus strain DRV-GZ originated from duck was amplified by RT-PCR and cloned into the pET-28a （＋） expression vector. After sequencing, the recombinant plasmid was transformed into Rosetta^TM 2 （DE3）plysS. The transformed bacteria were induced by IPTG and produced a recombinant protein of 50 ku in mass. The results showed that 0.6 mmol/L IPTG and 4 h of induction time were the optimal conditions for protein production and more pure proteins would be produced after purification with Ni^＋-column. The purified protein could react with the positive serum against strain DRV-GZ in a Western-blot test,indicating that the expressed protein had strong reactogenicity.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2008年第3期224-228,共5页
Chinese Veterinary Science
基金
广东省科技计划项目(2006A20301001)
教育部新世纪优秀人才支持计划项目(NCET-06-0752)
广东省动物防疫检疫专项(粤农[2006]264号)
关键词
鸭源呼肠孤病毒
S2基因
克隆
原核表达
纯化
免疫印迹
reovirus-originated duck
S2 gene
cloning
prokaryotic expression
purification
Western-blot
