摘要
根据禽腺联病毒Rep基因序列设计2对引物,分别扩增出Rep78和Rep52基因,将其克隆至杆状病毒双表达载体pFastBacDual,获得重组穿梭质粒pFastBacDual-Rep,将其转化到E.coli DH10Bac感受态细胞中,经抗性和蓝白斑筛选,获得重组病毒表达质粒rBacmid-Rep。在脂质体的介导下转染昆虫细胞Sf9,获得重组杆状病毒rBac-Rep。SDS-PAGE结果分析表明,Rep78、Rep52均获得了表达,Western blot结果显示表达的重组蛋白能够与Rep52多抗反应,具有良好的反应原性。结论:禽腺联病毒的非结构蛋白Rep78、Rep52基因在昆虫细胞中获得了成功表达。
In order to express the non-structural Rep proteins of an avian adeno-associated virus in insect cells, two pairs of specific primers were designed according to the published genome sequences of AAAV to amplify Rep78 and Rep52 genes by PCR, and the amplified fragments were cloned into Baculovirus expression vector pFastBacDual. Then the recombinant vector pFastBacDual-Rep was transformed into Escherichia coli DHIOBac, and the positive recombinant bacmid rBacmid-Rep screened according to the resistant and the blue-white plague screening, rBacmid-Rep was transfected into the Sf9 insect cells by liposome. Once the cytopathic effect was found,the rBac-Rep could be aquired. SDS-PAGE analysis showed Rep78 and Rep52 proteins were expressed in Sf9 with the right molecular weight. The result of Western blot indicated that the recombinant proteins could be recognized by antisera of Rep52, indicating that the protein had good reactiongenicity. These results suggested that the two non-structural proteins Rep78 and Rep52 gene of avian adeno-associated virus had successfully expressed in insect cells.
出处
《南京农业大学学报》
CAS
CSCD
北大核心
2014年第2期111-115,共5页
Journal of Nanjing Agricultural University
基金
江苏省自然科学基金项目(BK2011536)
国家自然科学基金项目(31302096)
关键词
禽腺联病毒
Rep基因
昆虫细胞
表达
avian adeno-associated virus
Rep gene
insect cells
express
