枯草芽孢杆菌表达载体pHT01质粒图谱和序列

摘 要:

目前,对枯草芽孢杆菌的遗传背景和生理特性的了解仅次于大肠杆菌。在所有革兰氏阳性菌中,枯草芽孢杆菌载体尤为引人瞩目。基于大肠杆菌-枯草芽孢杆菌穿梭质粒的表达载体pHT01可以在枯草芽孢杆菌中高效表达重组外源蛋白。pHT01质粒全长7955bp,载体基于强σA-依赖性启动子的枯草杆菌groE操纵子,通过lac操纵子改造成为一种高效可控的(IPTG诱导)启动子。

关键词: , , ,

革兰氏阳性菌因其在农业、医疗和食品生物技术和重组蛋白表达等方面的贡献而广为人知。枯草芽孢杆菌作为一种重要的工业微生物,其遗传学和生理学已得到深入研究,人们对其遗传背景和生理特性的了解仅次于大肠杆菌。在所有革兰氏阳性菌中,枯草芽孢杆菌载体因下列原因尤为引人瞩目。(一)无致病性、安全;(二)无明显的密码子偏嗜性;(三)可直接将表达蛋白分泌到培养基中(目前,大约60%的市售酶由芽孢杆菌表达生产),表达产物可溶,表达产物与胞内蛋白分离,无需破碎细胞,有利于分离纯化;(四)对枯草芽孢杆菌的转录、翻译、蛋白质折叠、分泌等机制背景清楚,有利于遗传操作和大规模发酵。

虽然枯草芽孢杆菌是极具潜在应用前景的重组蛋白表达宿主,但是枯草芽孢杆菌作为表达宿主也有两个主要缺陷:(一)产生一些胞内蛋白酶,会识别并降解表达的外源蛋白;(二)载体质粒稳定性。第一个障碍已因构建了枯草芽孢杆菌蛋白酶缺失株而基本解决,如WB800N菌株 。第二个障碍因引入使用θ-复制模式质粒被完全克服。

基于大肠杆菌-枯草芽孢杆菌穿梭质粒的表达载体pHT01可以在枯草芽孢杆菌中高效表达重组外源蛋白。pHT01质粒全长7955bp,载体基于强σA-依赖性启动子的枯草杆菌groE操纵子,通过添加lac操纵子改造成为一种高效可控的(IPTG诱导的)启动子。

一、pHT01质粒图谱

枯草芽孢杆菌表达载体pHT01质粒图谱和序列

枯草芽孢杆菌表达载体pHT01质粒图谱和序列

二、pHT01质粒序列 (7955 bp):

http://www.mobitec.com/cms/download/sequence/pHT01.txt

    1  ttaagttatt ggtatgactg gttttaagcg caaaaaaagt tgctttttcg tacctattaa
   61  tgtatcgttt tagaaaaccg actgtaaaaa gtacagtcgg cattatctca tattataaaa
  121  gccagtcatt aggcctatct gacaattcct gaatagagtt cataaacaat cctgcatgat
  181  aaccatcaca aacagaatga tgtacctgta aagatagcgg taaatatatt gaattacctt
  241  tattaatgaa ttttcctgct gtaataatgg gtagaaggta attactatta ttattgatat
  301  ttaagttaaa cccagtaaat gaagtccatg gaataataga aagagaaaaa gcattttcag
  361  gtataggtgt tttgggaaac aatttccccg aaccattata tttctctaca tcagaaaggt
  421  ataaatcata aaactctttg aagtcattct ttacaggagt ccaaatacca gagaatgttt
  481  tagatacacc atcaaaaatt gtataaagtg gctctaactt atcccaataa cctaactctc
  541  cgtcgctatt gtaaccagtt ctaaaagctg tatttgagtt tatcaccctt gtcactaaga
  601  aaataaatgc agggtaaaat ttatatcctt cttgttttat gtttcggtat aaaacactaa
  661  tatcaatttc tgtggttata ctaaaagtcg tttgttggtt caaataatga ttaaatatct
  721  cttttctctt ccaattgtct aaatcaattt tattaaagtt catttgatat gcctcctaaa
  781  tttttatcta aagtgaattt aggaggctta cttgtctgct ttcttcatta gaatcaatcc
  841  ttttttaaaa gtcaatatta ctgtaacata aatatatatt ttaaaaatat cccactttat
  901  ccaattttcg tttgttgaac taatgggtgc tttagttgaa gaataaaaga ccacattaaa
  961  aaatgtggtc ttttgtgttt ttttaaagga tttgagcgta gcgaaaaatc cttttctttc
 1021  ttatcttgat aataagggta actattgccg atcgtccatt ccgacagcat cgccagtcac
 1081  tatggcgtgc tgctagcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
 1141  ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
 1201  aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt
 1261  cgagctcagg ccttaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
 1321  aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
 1381  tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct
 1441  tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc
 1501  gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt
 1561  cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca
 1621  ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat
 1681  tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg
 1741  ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg
 1801  ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca
 1861  gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg
 1921  tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa
 1981  tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa
 2041  gattgtgcac cgccgtttta caggcttcga cgccgcttcg ttctaccatc gacaccacca
 2101  cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt
 2161  gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt
 2221  gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg
 2281  ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac
 2341  cggcatactc tgcgacatcg tataacgtta ctggtttcat caaaatcgtc tccctccgtt
 2401  tgaatatttg attgatcgta accagatgaa gcactctttc cactatccct acagtgttat
 2461  ggcttgaaca atcacgaaac aataattggt acgtacgatc tttcagccga ctcaaacatc
 2521  aaatcttaca aatgtagtct ttgaaagtat tacatatgta agatttaaat gcaaccgttt
 2581  tttcggaagg aaatgatgac ctcgtttcca ccggaattag cttggtacca gctattgtaa
 2641  cataatcggt acgggggtga aaaagctaac ggaaaaggga gcggaaaaga atgatgtaag
 2701  cgtgaaaaat tttttatctt atcacttgaa attggaaggg agattcttta ttataagaat
 2761  tgtggaattg tgagcggata acaattccca attaaaggag gaaggatcct ctagagtcga
 2821  cgtccccggg gcagcccgcc taatgagcgg gcttttttca cgtcacgcgt ccatggagat
 2881  ctttgtctgc aactgaaaag tttatacctt acctggaaca aatggttgaa acatacgagg
 2941  ctaatatcgg cttattagga atagtccctg tactaataaa atcaggtgga tcagttgatc
 3001  agtatatttt ggacgaagct cggaaagaat ttggagatga cttgcttaat tccacaatta
 3061  aattaaggga aagaataaag cgatttgatg ttcaaggaat cacggaagaa gatactcatg
 3121  ataaagaagc tctaaaacta ttcaataacc ttacaatgga attgatcgaa agggtggaag
 3181  gttaatggta cgaaaattag gggatctacc tagaaagcca caaggcgata ggtcaagctt
 3241  aaagaaccct tacatggatc ttacagattc tgaaagtaaa gaaacaacag aggttaaaca
 3301  aacagaacca aaaagaaaaa aagcattgtt gaaaacaatg aaagttgatg tttcaatcca
 3361  taataagatt aaatcgctgc acgaaattct ggcagcatcc gaagggaatt catattactt
 3421  agaggatact attgagagag ctattgataa gatggttgag acattacctg agagccaaaa
 3481  aactttttat gaatatgaat taaaaaaaag aaccaacaaa ggctgagaca gactccaaac
 3541  gagtctgttt ttttaaaaaa aatattagga gcattgaata tatattagag aattaagaaa
 3601  gacatgggaa taaaaatatt ttaaatccag taaaaatatg ataagattat ttcagaatat
 3661  gaagaactct gtttgttttt gatgaaaaaa caaacaaaaa aaatccacct aacggaatct
 3721  caatttaact aacagcggcc aaactgagaa gttaaatttg agaaggggaa aaggcggatt
 3781  tatacttgta tttaactatc tccattttaa cattttatta aaccccatac aagtgaaaat
 3841  cctcttttac actgttcctt taggtgatcg cggagggaca ttatgagtga agtaaaccta
 3901  aaaggaaata cagatgaatt agtgtattat cgacagcaaa ccactggaaa taaaatcgcc
 3961  aggaagagaa tcaaaaaagg gaaagaagaa gtttattatg ttgctgaaac ggaagagaag
 4021  atatggacag aagagcaaat aaaaaacttt tctttagaca aatttggtac gcatatacct
 4081  tacatagaag gtcattatac aatcttaaat aattacttct ttgatttttg gggctatttt
 4141  ttaggtgctg aaggaattgc gctctatgct cacctaactc gttatgcata cggcagcaaa
 4201  gacttttgct ttcctagtct acaaacaatc gctaaaaaaa tggacaagac tcctgttaca
 4261  gttagaggct acttgaaact gcttgaaagg tacggtttta tttggaaggt aaacgtccgt
 4321  aataaaacca aggataacac agaggaatcc ccgattttta agattagacg taaggttcct
 4381  ttgctttcag aagaactttt aaatggaaac cctaatattg aaattccaga tgacgaggaa
 4441  gcacatgtaa agaaggcttt aaaaaaggaa aaagagggtc ttccaaaggt tttgaaaaaa
 4501  gagcacgatg aatttgttaa aaaaatgatg gatgagtcag aaacaattaa tattccagag
 4561  gccttacaat atgacacaat gtatgaagat atactcagta aaggagaaat tcgaaaagaa
 4621  atcaaaaaac aaatacctaa tcctacaaca tcttttgaga gtatatcaat gacaactgaa
 4681  gaggaaaaag tcgacagtac tttaaaaagc gaaatgcaaa atcgtgtctc taagccttct
 4741  tttgatacct ggtttaaaaa cactaagatc aaaattgaaa ataaaaattg tttattactt
 4801  gtaccgagtg aatttgcatt tgaatggatt aagaaaagat atttagaaac aattaaaaca
 4861  gtccttgaag aagctggata tgttttcgaa aaaatcgaac taagaaaagt gcaataaact
 4921  gctgaagtat ttcagcagtt ttttttattt agaaatagtg aaaaaaatat aatcagggag
 4981  gtatcaatat ttaatgagta ctgatttaaa tttatttaga ctggaattaa taattaacac
 5041  gtagactaat taaaatttaa tgagggataa agaggataca aaaatattaa tttcaatccc
 5101  tattaaattt taacaagggg gggattaaaa tttaattaga ggtttatcca caagaaaaga
 5161  ccctaataaa atttttacta gggttataac actgattaat ttcttaatgg gggagggatt
 5221  aaaatttaat gacaaagaaa acaatctttt aagaaaagct tttaaaagat aataataaaa
 5281  agagctttgc gattaagcaa aactctttac tttttcattg acattatcaa attcatcgat
 5341  ttcaaattgt tgttgtatca taaagttaat tctgttttgc acaacctttt caggaatata
 5401  aaacacatct gaggcttgtt ttataaactc agggtcgcta aagtcaatgt aacgtagcat
 5461  atgatatggt atagcttcca cccaagttag cctttctgct tcttctgaat gtttttcata
 5521  tacttccatg ggtatctcta aatgattttc ctcatgtagc aaggtatgag caaaaagttt
 5581  atggaattga tagttcctct ctttttcttc aactttttta tctaaaacaa acactttaac
 5641  atctgagtca atgtaagcat aagatgtttt tccagtcata atttcaatcc caaatctttt
 5701  agacagaaat tctggacgta aatcttttgg tgaaagaatt tttttatgta gcaatatatc
 5761  cgatacagca ccttctaaaa gcgttggtga atagggcatt ttacctatct cctctcattt
 5821  tgtggaataa aaatagtcat attcgtccat ctacctatcc tattatcgaa cagttgaact
 5881  ttttaatcaa ggatcagtcc tttttttcat tattcttaaa ctgtgctctt aactttaaca
 5941  actcgatttg tttttccaga tctcgagggt aactagcctc gccgatcccg caagaggccc
 6001  ggcagtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
 6061  aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
 6121  attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
 6181  cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
 6241  aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
 6301  ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
 6361  gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
 6421  attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
 6481  tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
 6541  tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
 6601  atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
 6661  agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
 6721  aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
 6781  caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
 6841  ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
 6901  gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
 6961  tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
 7021  atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
 7081  tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
 7141  accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
 7201  gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
 7261  caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
 7321  tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
 7381  ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
 7441  tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
 7501  gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
 7561  tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
 7621  gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
 7681  gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
 7741  ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
 7801  ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
 7861  ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
 7921  tgagcgagga agcggaagag cgcccaatac gcatgc
    A+

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