Liu Xingjian, Wei Yonglong, Li Yinü, Li Haoyang, Yang Xin, Yi Yongzhu, Zhang Zhifang
Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China.
State Key Laboratory of Biomembrane and Membrane Biotechnology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China.
PLoS One. 2016 Mar 23;11(3):e0152140. doi: 10.1371/journal.pone.0152140. eCollection 2016.
The silkworm baculovirus expression system is widely used to produce recombinant proteins. Several strategies for constructing recombinant viruses that contain foreign genes have been reported. Here, we developed a novel defective-rescue BmNPV Bacmid (reBmBac) expression system. A CopyControl origin of replication was introduced into the viral genome to facilitate its genetic manipulation in Escherichia coli and to ensure the preparation of large amounts of high quality reBmBac DNA as well as high quality recombinant baculoviruses. The ORF1629, cathepsin and chitinase genes were partially deleted or rendered defective to improve the efficiency of recombinant baculovirus generation and the expression of foreign genes. The system was validated by the successful expression of luciferase reporter gene and porcine interferon γ. This system can be used to produce batches of recombinant baculoviruses and target proteins rapidly and efficiently in silkworms.
家蚕杆状病毒表达系统被广泛用于生产重组蛋白。已经报道了几种构建含有外源基因的重组病毒的策略。在此，我们开发了一种新型的缺陷拯救型家蚕核型多角体病毒杆粒（reBmBac）表达系统。将一个复制控制复制起点引入病毒基因组，以促进其在大肠杆菌中的遗传操作，并确保制备大量高质量的reBmBac DNA以及高质量的重组杆状病毒。ORF1629、组织蛋白酶和几丁质酶基因被部分缺失或使其功能缺陷，以提高重组杆状病毒的产生效率和外源基因的表达。通过成功表达荧光素酶报告基因和猪干扰素γ验证了该系统。该系统可用于在家蚕中快速、高效地批量生产重组杆状病毒和靶蛋白。
