pBacPAK8昆虫表达载体说明

pBacPAK8编号 载体名称北京华越洋生物KCVECT129 pBacPAK8pBacPAK8载体基本信息载体名称: pBacPAK8质粒类型: 昆虫表达载体；杆状病毒表达载体克隆方法: 限制酶，多克隆位点启动子: Polyhedrin载体大小: 5538 bp5' 测序引物及序列: Bac1 Forward3' 测序引物及序列: Bac2 Reverse载体标签: 无标签载体抗性: 氨苄青霉素真核筛选标记: 无克隆菌株: TOP10宿主细胞（系）: 果蝇细胞系 IPLB-Sf21备注: --瞬表达/稳表达: 稳表达组成型/诱导型: 组成型病毒/非病毒: 杆状病毒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 The polyhedrin coding sequences have been replaced by a multiple cloning s ite w ith 18 u nique s ites t hat f acilitate t he i nsertion o f f oreign g enes i n t he c orrect orientation for expression. The Pac I site at the end of the MCS region provides translational s top c odons i n a ll t hree r eading f rames f or e xpression o f t runcated p roteins. pBacPAK8 has a pUC origin of replication, an M13 origin for single-­‐stranded DNA production, a nd a n a mpicillin r esistance g ene f or s election i n E. c oli.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 y ields f rom b aculovirus s ystems a re h igher, a nd c osts a re s ignificantly l ower t han in m ammalian e xpression s ystems. T he b aculovirus e xpression s ystem c an e xpress g enes from b acteria, v iruses, p lants, a nd m ammals a t l evels f rom 1–500 m g/liter; m ost p roteins are e xpressed i n t he 10–100 m g/liter r ange, a lthough m aking p redictions i s d ifficult.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 p rotein i s e ssential f or p ropagation o f t he v irus i n i ts n atural h abitat; h owever, 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 drivehigh-­‐level transcription of the insert, resulting in expression of a recombinant protein that c an a ccount f or o ver 30% o f t otal c ellular p rotein.The l arge 134 k b-­‐size o f t he A cMNPV g enome, m akes d irect m anipulation o f i t d ifficult, s o recombinant b aculovirus e xpression v ectors a re c onstructed i n t wo s teps (Figure 1). F irst, 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 p romoter a nd p olyadenylation s ignals. T ransfer v ectors a lso c ontain a p lasmid 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 v ector a re c otransfected i nto i nsect c ells. D ouble r ecombination b etween v iral sequences in the transfer vector and the corresponding sequences in the viral DNA transfers t he t arget g ene t o t he v iral g enome.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 i nto t he g enes f lanking t he p olyhedrin e xpression l ocus o f B acPAK6. D igesting 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 b e u nable t o p roduce v iable v iruses. H owever, t he t ransfer v ector c arries t he m issing 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 r estores t he e ssential g ene a nd t ransfers t he t arget g ene f rom t he t ransfer v ector t o the viral genome. Cotransfections using Bsu36 I-­‐digested BacPAK6 viral DNA produce recombinant v iruses a t f requencies a pproaching 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 m anuals2.实验流程Obtain i nsect c ell m edia a nd e stablish S f21 c ell l ine. T his s tep w ill t ake 3–4 w eeks.Maintain w orking s tocks o f S f21 c ells.When t he s tock o f c ells h as b een p assaged t wice, f reeze a liquots f or l ong-­‐term s torage i n 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 s tocks a s t hey b ecome o ld.Isolating pure recombinant virus requires good viral plaques. Therefore, developing a good plaque assay technique before working with recombinant viruses is advisable. Practice a ssaying v iral p laques.Insert t arget g ene i nto t ransfer v ector a nd p repare p lasmid D NA.Produce a recombinant virus by cotransfecting Sf21 cells with BacPAK6 viral DNA and the t ransfer v ector-­‐target g ene c lone.Perform plaque assays on the cotransfection supernatant to obtain individual viral plaques.Test t he p utative r ecombinant v iruses t o c onfirm t hat t hey h ave i ncorporated t he t arget gene a nd/or e xpress t he t arget p rotein.Amplify r ecombinant v iruses t o o btain w orking s tocks.Titer a mplified v irus s tock.Perform small-­‐scale infections to characterize gene expression and to determine the optimum h arvest t ime a nd i nfection r atio t hat w ill g ive m aximum p rotein y ield.Scale-­‐up: produce target protein in large quantities by infecting larger batches of insect cells.pBacPAK8载体序列ORIGIN1 AACGGCTCCG CCCACTATTA ATGAAATTAA AAATTCCAAT TTTAAAAAAC GCAGCAAGAG61 AAACATTTGT ATGAAAGAAT GCGTAGAAGG AAAGAAAAAT GTCGTCGACA TGCTGAACAA121 CAAGATTAAT ATGCCTCCGT GTATAAAAAA AATATTGAAC GATTTGAAAG AAAACAATGT181 ACCGCGCGGC GGTATGTACA GGAAGAGGTT TATACTAAAC TGTTACATTG CAAACGTGGT241 TTCGTGTGCC AAGTGTGAAA ACCGATGTTT AATCAAGGCT CTGACGCATT TCTACAACCA301 CGACTCCAAG TGTGTGGGTG AAGTCATGCA TCTTTTAATC AAATCCCAAG ATGTGTATAA361 ACCACCAAAC TGCCAAAAAA TGAAAACTGT CGACAAGCTC TGTCCGTTTG CTGGCAACTG421 CAAGGGTCTC AATCCTATTT GTAATTATTG AATAATAAAA CAATTATAAA TGCTAAATTT481 GTTTTTTATT AACGATACAA ACCAAACGCA ACAAGAACAT TTGTAGTATT ATCTATAATT541 GAAAACGCGT AGTTATAATC GCTGAGGTAA TATTTAAAAT CATTTTCAAA TGATTCACAG601 TTAATTTGCG ACAATATAAT TTTATTTTCA CATAAACTAG ACGCCTTGTC GTCTTCTTCT661 TCGTATTCCT TCTCTTTTTC ATTTTTCTCC TCATAAAAAT TAACATAGTT ATTATCGTAT721 CCATATATGT ATCTATCGTA TAGAGTAAAT TTTTTGTTGT CATAAATATA TATGTCTTTT781 TTAATGGGGT GTATAGTACC GCTGCGCATA GTTTTTCTGT AATTTACAAC AGTGCTATTT841 TCTGGTAGTT CTTCGGAGTG TGTTGCTTTA ATTATTAAAT TTATATAATC AATGAATTTG901 GGATCGTCGG TTTTGTACAA TATGTTGCCG GCATAGTACG CAGCTTCTTC TAGTTCAATT961 ACACCATTTT TTAGCAGCAC CGGATTAACA TAACTTTCCA AAATGTTGTA CGAACCGTTA1021 AACAAAAACA GTTCACCTCC CTTTTCTATA CTATTGTCTG CGAGCAGTTG TTTGTTGTTA1081 AAAATAACAG CCATTGTAAT GAGACGCACA AACTAATATC ACAAACTGGA AATGTCTATC1141 AATATATAGT TGCTGATATC ATGGAGATAA TTAAAATGAT AACCATCTCG CAAATAAATA1201 AGTATTTTAC TGTTTTCGTA ACAGTTTTGT AATAAAAAAA CCTATAAATA CGGATCCCTG1261 CAGGCCTCGA GTTCGAATCT AGAAGATCTG GTACCGAGCT CGAATTCCCG GGCGGCCGCT1321 TAATTAATTG ATCCGGGTTA TTAGTACATT TATTAAGCGC TAGATTCTGT GCGTTGTTGA1381 TTTACAGACA ATTGTTGTAC GTATTTTAAT AATTCATTAA ATTTATAATC TTTAGGGTGG1441 TATGTTAGAG CGAAAATCAA ATGATTTTCA GCGTCTTTAT ATCTGAATTT AAATATTAAA1501 TCCTCAATAG ATTTGTAAAA TAGGTTTCGA TTAGTTTCAA ACAAGGGTTG TTTTTCCGAA1561 CCGATGGCTG GACTATCTAA TGGATTTTCG CTCAACGCCA CAAAACTTGC CAAATCTTGT1621 AGCAGCAATC TAGCTTTGTC GATATTCGTT TGTGTTTTGT TTTGTAATAA AGGTTCGACG1681 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 AGGCGGATAA4321 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//pBacPAK8其他昆虫表达载体：pMT/BioEase-DEST pVL1392 pVL1393 pXINSECT-DEST39 pXINSECT-DEST38 pBacPAK9 pBacPAK8 pIB/V5-His-TOPO pIEX/Bac-1 pFastBacHT B pFastBacHT A pFastBac1pMT/V5-His-TOPO pMT/BiP/V5-His A pCoHygro pAc5.1/V5-His C pAc5.1-V5-His A pAc5/V5-HisA pAc5-V5-HisB pAc5-V5-HisC pCoBlast pAc5.1/V5-His B pIZ/V5-His pIB/V5-HispIZT/V5-His pMIB/V5-His C pMIB/V5-His A pMT/BiP/V5-His C pMT/BiP/V5-His B pIEx/Bac-4 pIEx/Bac-3 pIEXBac-c-EGFP-3 pIEXBac-c-EGFP-1 pIEXBac-c-EGFP-4 pAc5.1B-EGFP pBlueBacHis2 A pBlueBacHis2 C pBlueBacHis2 B pFastBacHT C pMT-Bip-V5-HisA pFBDM pUCDM pFastBac Dual pMIB/V5-His B pMT/V5-His C pMT/V5-His B pMT/V5-His A pAcGP67ApAc5.1b pAc5.1a pVL1392-XyIE control vector pFastBac-c-His-TEV pFastBac-N-GST-TEV pFastBacI-Gus pFastBacHT-CAT pBADZ-His6Cre。