载体描述:
Available as part of the BacPAK Baculovirus Expression System (Cat. No. 631402). pBacPAK8 is a transfer vector designed for high-level expression of a cloned gene driven by the strong AcMNPV polyhedrin promoter. Flanking AcMNPV sequences allow recombination with viral DNA to transfer the expression cassette to the polyhedrin locus of the viral DNA. The polyhedrin coding sequences have been replaced by a multiple cloning site with 18 unique sites that facilitate the insertion of foreign genes in the correct orientation for expression. The Pac I site at the end of the MCS region provides translational stop codons in all three reading frames for expression of truncated proteins. pBacPAK8 has a pUC origin of replication, an M13 origin for single-stranded DNA production, and an ampicillin resistance gene for selection in E. coli.
1 杆状病毒表达系统简介
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). AcMNPV can be propagated in certain insect cell lines; the virus enters the cells and replication begins approximately 6 hours post-infection (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, 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.
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. 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. 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%.
This User Manual contains directions for establishing insect cell cultures, as well as for isolating a recombinant baculovirus expression vector using the BacPAK system. More extensive protocols for using baculovirus expression systems are in the baculovirus laboratory manuals
2.实验流程
Obtain insect cell media and establish Sf21 cell line. This step will take3–4 weeks.
Maintain working stocks of Sf21 cells.
When the stock of cells has been passaged twice, freeze aliquots for long-term storage in liquid nitrogen. Aliquots of frozen cells provide a back-up in case the working stock dies or becomes contaminated. Frozen cells are also a source of fresh cells for replacing working stocks as they become old.
Isolating pure recombinant virus requires good viral plaques. Therefore, developing a good plaque assay technique before working with recombinant viruses is advisable.Practice assaying viral plaques.
Insert target gene into transfer vector and prepare plasmid DNA.
Produce a recombinant virus by cotransfecting Sf21 cells with BacPAK6 viral DNA and the transfer vector-target gene clone.
Perform plaque assays on the cotransfection supernatant to obtain individual viral plaques.
Test the putative recombinant viruses to confirm that they have incorporated the target gene and/or express the target protein.
Amplify recombinant viruses to obtain working stocks.
Titer amplified virus stock.
Perform small-scale infections to characterize gene expression and to determine the optimum harvest time and infection ratio that will give maximum protein yield.
Scale-up: produce target protein in large quantities by infecting larger batches of insect cells.
