pBacPAK8-GUS 昆虫杆状病毒表达系统质粒载体 BioVector NTCC质粒载体菌种细胞基因保藏中心
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| Baculovirus gene expression is a popular method for producing large quantities of recombinant proteins in insect host cells. In most cases, posttranslational processing of eukaryotic proteins expressed in insect cells is similar to protein processing in mammalian cells. As a result, insect cell-processed proteins have comparable biological activities and immunological reactivities to proteins expressed in mammalian cells. Protein yields from baculovirus systems are higher, and costs are significantly lower than in mammalian expression systems. The baculovirus expression system can express genes from bacteria, viruses, plants, and mammals at levels from 1–500 mg/liter; most proteins are expressed in the 10–100 mg/liter range, although making predictions is difficult. The baculovirus most commonly used to express foreign proteins is Autographa californica nuclear polyhedrosis virus (AcMNPV; V. A. Luckow 1991; Vlak, J. M., Keus 1990; Bishop, D. H. L. & Possee 1990; Miller 1988; Verne A. Luckow and Summers 1988; O’Reilly, D. R., Miller, L. K., Luckow 1992). AcMNPV can be propagated in certain insect cell lines; the virus enters the cells and replication begins approximately 6 hours postinfection (h.p.i.). At approximately 20–48 h.p.i., transcription of nearly all genes ceases. The viral polyhedrin and p10 genes, however, are transcribed at high rates. The polyhedrin protein is essential for propagation of the virus in its natural habitat; however, in cell culture, polyhedrin is not needed, and its coding sequence can be replaced with a sequence for a target protein. Hence, the powerful polyhedrin promoter can drive high-level transcription of the insert, resulting in expression of a recombinant protein that can account for over 30% of total cellular protein. The large 134 kb-size of the AcMNPV genome (Ayres et al. 1994), makes direct manipulation of it difficult, so recombinant baculovirus expression vectors are constructed in two steps (Figure 1). First, a target gene is cloned into a modified polyhedrin locus contained in a relatively small transfer vector (<10 kb). The polyhedrin coding sequence has been deleted and replaced with a multiple cloning site (MCS). A target gene is inserted into this MCS, between the polyhedrin promoter and polyadenylation signals. Transfer vectors also contain a plasmid origin of replication and an antibiotic resistance gene for propagation in E. coli, but they are unable to replicate in insect cells. In the second step, the transfer vector and a viral expression vector are cotransfected into insect cells. Double recombination between viral sequences in the transfer vector and the corresponding sequences in the viral DNA transfers the target gene to the viral genome.</br>The BacPAK Baculovirus Expression System uses BacPAK6, a specially engineered virus that facilitates construction and selection of recombinant expression vectors. BacPAK6 has an essential gene adjacent to the polyhedrin locus that provides selection for recombinant viruses (P A Kitts and Possee 1993) (Figure 1). Sites for Bsu36 I, which does not cut wild-type AcMNPV DNA, were introduced into the genes flanking the polyhedrin expression locus of BacPAK6. Digesting BacPAK6 with Bsu36 I releases two fragments. The first carries part of a downstream gene, ORF1629, that is essential for viral replication (Possee, R. D., Sun, T.-P., Howard, S. C., Ayres, M. D., Hill-Perkins, M., Gearing 185AD). If the second large DNA fragment recircularizes by itself, the resulting viral DNA will lack an essential part of the genome and be unable to produce viable viruses. However, the transfer vector carries the missing ORF1629 sequence, and if the large fragment recombines with it, the resulting circular DNA will contain all the genes necessary for viral replication. This double recombination event restores the essential gene and transfers the target gene from the transfer vector to the viral genome. Cotransfections using Bsu36 I-digested BacPAK6 viral DNA produce recombinant viruses at frequencies approaching 100%. |
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