摘要
以质粒pMD18T-env为模板,利用PCR技术扩增禽网状内皮组织增生病病毒的gp90基因,酶切、纯化后,克隆至载体pFastBacHTA中,构建了重组供体载体pFgp90,并将其转化大肠杆菌DH10Bac感受态细胞,使gp90基因整合到Bacmid穿梭载体中,获得重组穿梭载体Bacmidgp90。通过脂质体介导将其转染sf9昆虫细胞,获得重组杆状病毒rBacgp90。抗gp90蛋白小鼠超免疫血清介导的Western-blot分析和间接免疫荧光试验(IFA)结果显示,gp90蛋白被正确表达,分子质量约为45 ku;gp90蛋白在变性和自然状态下均能够被识别,具有良好的生物活性。
gp90 gene encoding gp90 protein of reticuloendotheliosis virus was amplified from the plasmid pMD18T-env and then was cloned into pFastBacHTA to generate recombinant donor plasmid pFgp90.Being transformed into Escherichia coli DH10Bac competent cell,gp90 gene was integrated into bacmid shuttle vector in DH10Bac cells to generate a recombinant bacmid plasmid Bacmidgp90.The recombinant baculovirus rBacgp90 was obtained after Bacmidgp90 was transfected into sf9 cells.Western-blot analysis and indirect immunofluorescence assay showed that the gp90 protein expressed successfully was 45ku in molecular weight,possessed good reactionogenicity and could be recognized by mouse hyperimmune sera against the gp90 protein under natural or denatured state.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2009年第11期973-977,共5页
Chinese Veterinary Science
基金
现代农业肉鸡产业技术体系建设项目(nycytx-42-G3-01)
