pBacPAK8

pBacPAK8

pBacPAK8载体基本信息

pBacPAK8载体质粒图谱和多克隆位点信息

pBacPAK8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 The polyhedrin coding sequences have been replaced by a multiple cloning site with 18 unique sites that facilitate the insertion of foreign genes in the correct orientation for expression.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 take 3–4 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.

pBacPAK8载体序列

ORIGIN

    1 AACGGCTCCG CCCACTATTA ATGAAATTAA AAATTCCAAT TTTAAAAAAC GCAGCAAGAG

   61 AAACATTTGT ATGAAAGAAT GCGTAGAAGG AAAGAAAAAT GTCGTCGACA TGCTGAACAA

  121 CAAGATTAAT ATGCCTCCGT GTATAAAAAA AATATTGAAC GATTTGAAAG AAAACAATGT

  181 ACCGCGCGGC GGTATGTACA GGAAGAGGTT TATACTAAAC TGTTACATTG CAAACGTGGT

  241 TTCGTGTGCC AAGTGTGAAA ACCGATGTTT AATCAAGGCT CTGACGCATT TCTACAACCA

  301 CGACTCCAAG TGTGTGGGTG AAGTCATGCA TCTTTTAATC AAATCCCAAG ATGTGTATAA

  361 ACCACCAAAC TGCCAAAAAA TGAAAACTGT CGACAAGCTC TGTCCGTTTG CTGGCAACTG

  421 CAAGGGTCTC AATCCTATTT GTAATTATTG AATAATAAAA CAATTATAAA TGCTAAATTT

  481 GTTTTTTATT AACGATACAA ACCAAACGCA ACAAGAACAT TTGTAGTATT ATCTATAATT

  541 GAAAACGCGT AGTTATAATC GCTGAGGTAA TATTTAAAAT CATTTTCAAA TGATTCACAG

  601 TTAATTTGCG ACAATATAAT TTTATTTTCA CATAAACTAG ACGCCTTGTC GTCTTCTTCT

  661 TCGTATTCCT TCTCTTTTTC ATTTTTCTCC TCATAAAAAT TAACATAGTT ATTATCGTAT

  721 CCATATATGT ATCTATCGTA TAGAGTAAAT TTTTTGTTGT CATAAATATA TATGTCTTTT

  781 TTAATGGGGT GTATAGTACC GCTGCGCATA GTTTTTCTGT AATTTACAAC AGTGCTATTT

  841 TCTGGTAGTT CTTCGGAGTG TGTTGCTTTA ATTATTAAAT TTATATAATC AATGAATTTG

  901 GGATCGTCGG TTTTGTACAA TATGTTGCCG GCATAGTACG CAGCTTCTTC TAGTTCAATT

  961 ACACCATTTT TTAGCAGCAC CGGATTAACA TAACTTTCCA AAATGTTGTA CGAACCGTTA

 1021 AACAAAAACA GTTCACCTCC CTTTTCTATA CTATTGTCTG CGAGCAGTTG TTTGTTGTTA

 1081 AAAATAACAG CCATTGTAAT GAGACGCACA AACTAATATC ACAAACTGGA AATGTCTATC

 1141 AATATATAGT TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA

 1201 AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA CGGATCCCTG

 1261 CAGGCCTCGA GTTCGAATCT AGAAGATCTG GTACCGAGCT CGAATTCCCG GGCGGCCGCT

 1321 TAATTAATTG ATCCGGGTTA TTAGTACATT TATTAAGCGC TAGATTCTGT GCGTTGTTGA

 1381 TTTACAGACA ATTGTTGTAC GTATTTTAAT AATTCATTAA ATTTATAATC TTTAGGGTGG

 1441 TATGTTAGAG CGAAAATCAA ATGATTTTCA GCGTCTTTAT ATCTGAATTT AAATATTAAA

 1501 TCCTCAATAG ATTTGTAAAA TAGGTTTCGA TTAGTTTCAA ACAAGGGTTG TTTTTCCGAA

 1561 CCGATGGCTG GACTATCTAA TGGATTTTCG CTCAACGCCA CAAAACTTGC CAAATCTTGT

 1621 AGCAGCAATC TAGCTTTGTC GATATTCGTT TGTGTTTTGT TTTGTAATAA AGGTTCGACG

 1681 TCGTTCAAAA TATTATGCGC TTTTGTATTT CTTTCATCAC TGTCGTTAGT GTACAATTGA

 1741 CTCGACGTAA ACACGTTAAA TAAAGCTTGG ACATATTTAA CATCGGGCGT GTTAGCTTTA

 1801 TTAGGCCGAT TATCGTCGTC GTCCCAACCC TCGTCGTTAG AAGTTGCTTC CGAAGACGAT

 1861 TTTGCCATAG CCACACGACG CCTATTAATT GTGTCGGCTA ACACGTCCGC GATCAAATTT

 1921 GTAGTTGAGC TTTTTGGAAT TATTTCTGAT TGCGGGCGTT TTTGGGCGGG TTTCAATCTA

 1981 ACTGTGCCCG ATTTTAATTC AGACAACACG TTAGAAAGCG ATGGTGCAGG CGGTGGTAAC

 2041 ATTTCAGACG GCAAATCTAC TAATGGCGGC GGTGGTGGAG CTGATGATAA ATCTACCATC

 2101 GGTGGAGGCG CAGGCGGGGC TGGCGGCGGA GGCGGAGGCG GAGGTGGTGG CGGTGATGCA

 2161 GACGGCGGTT TAGGCTCAAA TGTCTCTTTA GGCAACACAG TCGGCACCTC AACTATTGTA

 2221 CTGGTTTCGG GCGCCGTTTT TGGTTTGACC GGTCTGAGAC GAGTGCGATT TTTTTCGTTT

 2281 CTAATAGCTT CCAACAATTG TTGTCTGTCG TCTAAAGGTG CAGCGGGTTG AGGTTCCGTC

 2341 GGCATTGGTG GAGCGGGCGG CAATTCAGAC ATCGATGGTG GTGGTGGTGG TGGAGGCGCT

 2401 GGAATGTTAG GCACGGGAGA AGGTGGTGGC GGCGGTGCCG CCGGTATAAT TTGTTCTGGT

 2461 TTAGTTTGTT CGCGCACGAT TGTGGGCACC GGCGCAGGCG CCGCTGGCTG CACAACGGAA

 2521 GGTCGTCTGC TTCGAGGCAG CGCTTGGGGT GGTGGCAATT CAATATTATA ATTGGAATAC

 2581 AAATCGTAAA AATCTGCTAT AAGCATTGTA ATTTCGCTAT CGTTTACCGT GCCGATATTT

 2641 AACAACCGCT CAATGTAAGC AATTGTATTG TAAAGAGATT GTCTCAAGCT CGGATCGATC

 2701 CCGCACGCCG ATAACAAGCC TTTTCATTTT TACTACAGCA TTGTAGTGGC GAGACACTTC

 2761 GCTGTCGTCG CCTGATGCGG TATTTTCTCC TTACGCATCT GTGCGGTATT TCACACCGCA

 2821 TACGTCAAAG CAACCATAGT ACGCGCCCTG TAGCGGCGCA TTAAGCGCGG CGGGTGTGGT

 2881 GGTTACGCGC AGCGTGACCG CTACACTTGC CAGCGCCCTA GCGCCCGCTC CTTTCGCTTT

 2941 CTTCCCTTCC TTTCTCGCCA CGTTCGCCGG CTTTCCCCGT CAAGCTCTAA ATCGGGGGCT

 3001 CCCTTTAGGG TTCCGATTTA GTGCTTTACG GCACCTCGAC CCCAAAAAAC TTGATTTGGG

 3061 TGATGGTTCA CGTAGTGGGC CATCGCCCTG ATAGACGGTT TTTCGCCCTT TGACGTTGGA

 3121 GTCCACGTTC TTTAATAGTG GACTCTTGTT CCAAACTGGA ACAACACTCA ACCCTATCTC

 3181 GGGCTATTCT TTTGATTTAT AAGGGATTTT GCCGATTTCG GCCTATTGGT TAAAAAATGA

 3241 GCTGATTTAA CAAAAATTTA ACGCGAATTT TAACAAAATA TTAACGTTTA CAATTTTATG

 3301 GTGCACTCTC AGTACAATCT GCTCTGATGC CGCATAGTTA AGCCAGCCCC GACACCCGCC

 3361 AACACCCGCT GACGCGCCCT GACGGGCTTG TCTGCTCCCG GCATCCGCTT ACAGACAAGC

 3421 TGTGACCGTC TCCGGGAGCT GCATGTGTCA GAGGTTTTCA CCGTCATCAC CGAAACGCGC

 3481 GAGACGAAAG GGCCTCGTGA TACGCCTATT TTTATAGGTT AATGTCATGA TAATAATGGT

 3541 TTCTTAGACG TCAGGTGGCA CTTTTCGGGG AAATGTGCGC GGAACCCCTA TTTGTTTATT

 3601 TTTCTAAATA CATTCAAATA TGTATCCGCT CATGAGACAA TAACCCTGAT AAATGCTTCA

 3661 ATAATATTGA AAAAGGAAGA GTATGAGTAT TCAACATTTC CGTGTCGCCC TTATTCCCTT

 3721 TTTTGCGGCA TTTTGCCTTC CTGTTTTTGC TCACCCAGAA ACGCTGGTGA AAGTAAAAGA

 3781 TGCTGAAGAT CAGTTGGGTG CACGAGTGGG TTACATCGAA CTGGATCTCA ACAGCGGTAA

 3841 GATCCTTGAG AGTTTTCGCC CCGAAGAACG TTTTCCAATG ATGAGCACTT TTAAAGTTCT

 3901 GCTATGTGGC GCGGTATTAT CCCGTATTGA CGCCGGGCAA GAGCAACTCG GTCGCCGCAT

 3961 ACACTATTCT CAGAATGACT TGGTTGAGTA CTCACCAGTC ACAGAAAAGC ATCTTACGGA

 4021 TGGCATGACA GTAAGAGAAT TATGCAGTGC TGCCATAACC ATGAGTGATA ACACTGCGGC

 4081 CAACTTACTT CTGACAACGA TCGGAGGACC GAAGGAGCTA ACCGCTTTTT TGCACAACAT

 4141 GGGGGATCAT GTAACTCGCC TTGATCGTTG GGAACCGGAG CTGAATGAAG CCATACCAAA

 4201 CGACGAGCGT GACACCACGA TGCCTGTAGC AATGGCAACA ACGTTGCGCA AACTATTAAC

 4261 TGGCGAACTA CTTACTCTAG CTTCCCGGCA ACAATTAATA GACTGGATGG AGGCGGATAA

 4321 AGTTGCAGGA CCACTTCTGC GCTCGGCCCT TCCGGCTGGC TGGTTTATTG CTGATAAATC

 4381 TGGAGCCGGT GAGCGTGGGT CTCGCGGTAT CATTGCAGCA CTGGGGCCAG ATGGTAAGCC

 4441 CTCCCGTATC GTAGTTATCT ACACGACGGG GAGTCAGGCA ACTATGGATG AACGAAATAG

 4501 ACAGATCGCT GAGATAGGTG CCTCACTGAT TAAGCATTGG TAACTGTCAG ACCAAGTTTA

 4561 CTCATATATA CTTTAGATTG ATTTAAAACT TCATTTTTAA TTTAAAAGGA TCTAGGTGAA

 4621 GATCCTTTTT GATAATCTCA TGACCAAAAT CCCTTAACGT GAGTTTTCGT TCCACTGAGC

 4681 GTCAGACCCC GTAGAAAAGA TCAAAGGATC TTCTTGAGAT CCTTTTTTTC TGCGCGTAAT

 4741 CTGCTGCTTG CAAACAAAAA AACCACCGCT ACCAGCGGTG GTTTGTTTGC CGGATCAAGA

 4801 GCTACCAACT CTTTTTCCGA AGGTAACTGG CTTCAGCAGA GCGCAGATAC CAAATACTGT

 4861 CCTTCTAGTG TAGCCGTAGT TAGGCCACCA CTTCAAGAAC TCTGTAGCAC CGCCTACATA

 4921 CCTCGCTCTG CTAATCCTGT TACCAGTGGC TGCTGCCAGT GGCGATAAGT CGTGTCTTAC

 4981 CGGGTTGGAC TCAAGACGAT AGTTACCGGA TAAGGCGCAG CGGTCGGGCT GAACGGGGGG

 5041 TTCGTGCACA CAGCCCAGCT TGGAGCGAAC GACCTACACC GAACTGAGAT ACCTACAGCG

 5101 TGAGCTATGA GAAAGCGCCA CGCTTCCCGA AGGGAGAAAG GCGGACAGGT ATCCGGTAAG

 5161 CGGCAGGGTC GGAACAGGAG AGCGCACGAG GGAGCTTCCA GGGGGAAACG CCTGGTATCT

 5221 TTATAGTCCT GTCGGGTTTC GCCACCTCTG ACTTGAGCGT CGATTTTTGT GATGCTCGTC

 5281 AGGGGGGCGG AGCCTATGGA AAAACGCCAG CAACGCGGCC TTTTTACGGT TCCTGGCCTT

 5341 TTGCTGGCCT TTTGCTCACA TGTTCTTTCC TGCGTTATCC CCTGATTCTG TGGATAACCG

 5401 TATTACCGCC TTTGAGTGAG CTGATACCGC TCGCCGCAGC CGAACGACCG AGCGCAGCGA

 5461 GTCAGTGAGC GAGGAAGCGG AAGAGCGCCC AATACGCAAA CCGCCTCTCC CCGCGCGTTG

 5521 GCCGATTCAT TAATGCAG

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pBacPAK8其他昆虫表达载体：