pBacPAK8-GUS Vector桿狀病毒表達系統對照質粒載體

15 μg pBacPAK8 Transfer Vector (500 ng/μl)
? 15 μg pBacPAK9 Transfer Vector (500 ng/μl)
? 2.5 μg pBacPAK8-GUS Vector (100 ng/μl)
? 20 μl Bac1 Primer (20 μM)
? 20 μl Bac2 Primer (20 μM)
Note: The following kit component is also available separately:
? BacPAK6 DNA (Bsu36 I digest) Cat No. BioVector631401

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.

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