本文报道了从被AcNPV感染的大蜡螟中提纯到AcNPV DNA,以质粒pBR325作为载体,从AcNPV基因组DNA EeoRI片段克隆中得到含AcNPV多角体蛋白基因片段的重组质粒。经原位杂交,酶切鉴定,DNA顺序分析,并与Hooft Van Iddekinge等人测定的结果比较...本文报道了从被AcNPV感染的大蜡螟中提纯到AcNPV DNA,以质粒pBR325作为载体,从AcNPV基因组DNA EeoRI片段克隆中得到含AcNPV多角体蛋白基因片段的重组质粒。经原位杂交,酶切鉴定,DNA顺序分析,并与Hooft Van Iddekinge等人测定的结果比较,证明筛选到的7.3kb EcoRI片段包含了AcNPV多角体蛋白结构基因及其启动基因。核多角体病毒多角体蛋白启动基因是迄今为止在真核细胞中所发现的最强启动基因之一,是理想的表达外源基因的启动子。展开更多
AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insect-baculovirus expression systems, each having different characteristics. AcNPV has a w...AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insect-baculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect, A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm, Bombyx mori, which is well-known for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for large-scale industrial ex-pression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV. The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an appli-cation example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in common-use cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the produc-tion efficiency of the bFGF recombinant protein.展开更多
文摘AcNPV (Autographa californica nuclear polyhedrosis virus) and BmNPV(Bombyx mori nuclear polyhedrosis virus) are two principal insect-baculovirus expression systems, each having different characteristics. AcNPV has a wider host range and can infect a series of cell lines thus making it suitable for cell suspension culture expression, but the small size of the host insect, A. californica, makes AcNPV less suitable for large scale protein synthesis. In contrast, BmNPV can only infect the silkworm, Bombyx mori, which is well-known for its easy rearing and large size. These characteristics make the BmNPV system especially suitable for large-scale industrial ex-pression. To utilize the advantages of both AcNPV and BmNPV, we tried to expand their host range through homologous recombination and successfully constructed a hybrid baculovirus of AcNPV and BmNPV, designated as HyNPV. The hybrid baculovirus can infect the hosts of both AcNPV and BmNPV. Taking the human basic fibroblast growth factor (bFGF) gene as an appli-cation example, we constructed a recombinant, HyNPV-bFGF. This construct is able to express the bFGF protein both in silkworm larvae and in common-use cell lines, sf21, sf9 and High-five. Moreover, to reduce the loss of recombinant protein due to degradation by proteases that are simultaneously expressed by the baculovirus, we knocked out the cysteinase gene coding for one of the most important baculovirus proteases. This knockout mutation improves the produc-tion efficiency of the bFGF recombinant protein.
