1.简介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生产等方面的贡献而广为人知。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,且一般认为安全的有机体;(二)无明显的密码子偏好性;(三)可直接将功能性胞外蛋白分泌到培养基中(目前,大约60%的市售酶由芽孢杆菌生产);(四)具备包含转录,翻译,蛋白质折叠、分泌机制,遗传操作和大规模发酵的大信息量机体。但是下述两个障碍减少了枯草芽孢杆菌的使用:(一)产生一定数目的识别并降解外源蛋白的胞外蛋白酶;(二)载体质粒稳定性。个障碍已因蛋白酶缺失株的构建而基本解决 。第二个因引入使用θ-复制模式质粒被完全克服,如由天然质粒pAMβ1和pBS72衍生的一些质粒(Jannière等,1990;Titok等,2003)。最近,基于大肠杆菌 - 枯草杆菌穿梭质粒pMTLBS72的四种不同表达载体的构建和使用展示出全面的结构稳定性,业已出版(Nguyen等,2005)。两个新的载体pHT01和pHT43允许在细胞质中高水平表达重组蛋白,其中pHT43载体引导重组蛋白到培养基。这两个载体基于强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。pHT01衍生载体可与8×His标签(pHT08),链球菌标签(pHT9)或C - Myc的标签(pHT10)相结合。2. pHT 载体所有在枯草芽孢杆菌的groESL操纵子之前使强启动子与lac操纵子融合的载体都可通过加入IPTG进行诱导。尽管当未添加诱导物时表达组件的背景表达水平很低,还是成功从约1300种诱导因子中筛选出一种来使用bgaB报道基因(Phan等,2005)。当分别将htpG和pbpE基因融合到groE启动子时,加入IPTG后,表达的重组蛋白可能分别占细胞总蛋白的10%和13%(Phan等,2005)。热纤梭菌的amyQα-淀粉酶和纤维素酶A、B的高水平表达实验证实。该载体还插入了一个高效SD序列以及一个多克隆位点(BamH I, Xba I, Aat II, Sma I)。编码α-淀粉酶的amyQ基因的信号肽编码区域与pHT01的SD序列融合,构成了pHT43,以此获得分泌的重组蛋白。
LOCUS pHT43 8057 bp DNA circular 12-JAN-2014SOURCE ORGANISM COMMENT This file is created by Vector NTI http://www.biofeng.com/COMMENT VNTAUTHORNAME|biofeng.com|BASE COUNT 2503 a 1584 c 1703 g 2267 t ORIGIN 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 gaataaagac cacattaaaa 961 aatgtggtct tttgtgtttt tttaaaggat ttgagcgtag cgaaaaatcc ttttctttct 1021 tatcttgata ataagggtaa ctattgccga tcgtccattc cgacagcatc gccagtcact 1081 atggcgtgct gctagcgcca ttcgccattc aggctgcgca actgttggga agggcgatcg 1141 gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc aaggcgatta 1201 agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc cagtgaattc 1261 gagctcaggc cttaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 1321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 1381 attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt 1441 caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg 1501 aaaatcctgt ttgatggtgg ttgacggcgg gatataacat gagctgtctt cggtatcgtc 1561 gtatcccact accgagatat ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat 1621 tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt 1681 cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc 1741 tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc 1801 cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag 1861 atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt 1921 ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat 1981 ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag 2041 attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac 2101 gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg 2161 cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg 2221 tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt 2281 tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc 2341 ggcatactct gcgacatcgt ataacgttac tggtttcatc aaaatcgtct ccctccgttt 2401 gaatatttga ttgatcgtaa ccagatgaag cactctttcc actatcccta cagtgttatg 2461 gcttgaacaa tcacgaaaca ataattggta cgtacgatct ttcagccgac tcaaacatca 2521 aatcttacaa atgtagtctt tgaaagtatt acatatgtaa gatttaaatg caaccgtttt 2581 ttcggaagga aatgatgacc tcgtttccac cggaattagc ttggtaccag ctattgtaac 2641 ataatcggta cgggggtgaa aaagctaacg gaaaagggag cggaaaagaa tgatgtaagc 2701 gtgaaaaatt ttttatctta tcacttgaaa ttggaaggga gattctttat tataagaatt 2761 gtggaattgt gagcggataa caattcccaa ttaaaggagg aaggatcaat gattcaaaaa 2821 cgaaagcgga cagtttcgtt cagacttgtg cttatgtgca cgctgttatt tgtcagtttg 2881 ccgattacaa aaacatcagc cgtaggatcc tctagagtcg acgtccccgg ggcagcccgc 2941 ctaatgagcg ggcttttttc acgtcacgcg tccatggaga tctttgtctg caactgaaaa 3001 gtttatacct tacctggaac aaatggttga aacatacgag gctaatatcg gcttattagg 3061 aatagtccct gtactaataa aatcaggtgg atcagttgat cagtatattt tggacgaagc 3121 tcggaaagaa tttggagatg acttgcttaa ttccacaatt aaattaaggg aaagaataaa 3181 gcgatttgat gttcaaggaa tcacggaaga agatactcat gataaagaag ctctaaaact 3241 attcaataac cttacaatgg aattgatcga aagggtggaa ggttaatggt acgaaaatta 3301 ggggatctac ctagaaagcc acaaggcgat aggtcaagct taaagaaccc ttacatggat 3361 cttacagatt ctgaaagtaa agaaacaaca gaggttaaac aaacagaacc aaaaagaaaa 3421 aaagcattgt tgaaaacaat gaaagttgat gtttcaatcc ataataagat taaatcgctg 3481 cacgaaattc tggcagcatc cgaagggaat tcatattact tagaggatac tattgagaga 3541 gctattgata agatggttga gacattacct gagagccaaa aaacttttta tgaatatgaa 3601 ttaaaaaaaa gaaccaacaa aggctgagac agactccaaa cgagtctgtt tttttaaaaa 3661 aaatattagg agcattgaat atatattaga gaattaagaa agacatggga ataaaaatat 3721 tttaaatcca gtaaaaatat gataagatta tttcagaata tgaagaactc tgtttgtttt 3781 tgatgaaaaa acaaacaaaa aaaatccacc taacggaatc tcaatttaac taacagcggc 3841 caaactgaga agttaaattt gagaagggga aaaggcggat ttatacttgt atttaactat 3901 ctccatttta acattttatt aaaccccata caagtgaaaa tcctctttta cactgttcct 3961 ttaggtgatc gcggagggac attatgagtg aagtaaacct aaaaggaaat acagatgaat 4021 tagtgtatta tcgacagcaa accactggaa ataaaatcgc caggaagaga atcaaaaaag 4081 ggaaagaaga agtttattat gttgctgaaa cggaagagaa gatatggaca gaagagcaaa 4141 taaaaaactt ttctttagac aaatttggta cgcatatacc ttacatagaa ggtcattata 4201 caatcttaaa taattacttc tttgattttt ggggctattt tttaggtgct gaaggaattg 4261 cgctctatgc tcacctaact cgttatgcat acggcagcaa agacttttgc tttcctagtc 4321 tacaaacaat cgctaaaaaa atggacaaga ctcctgttac agttagaggc tacttgaaac 4381 tgcttgaaag gtacggtttt atttggaagg taaacgtccg taataaaacc aaggataaca 4441 cagaggaatc cccgattttt aagattagac gtaaggttcc tttgctttca gaagaacttt 4501 taaatggaaa ccctaatatt gaaattccag atgacgagga agcacatgta aagaaggctt 4561 taaaaaagga aaaagagggt cttccaaagg ttttgaaaaa agagcacgat gaatttgtta 4621 aaaaaatgat ggatgagtca gaaacaatta atattccaga ggccttacaa tatgacacaa 4681 tgtatgaaga tatactcagt aaaggagaaa ttcgaaaaga aatcaaaaaa caaataccta 4741 atcctacaac atcttttgag agtatatcaa tgacaactga agaggaaaaa gtcgacagta 4801 ctttaaaaag cgaaatgcaa aatcgtgtct ctaagccttc ttttgatacc tggtttaaaa 4861 acactaagat caaaattgaa aataaaaatt gtttattact tgtaccgagt gaatttgcat 4921 ttgaatggat taagaaaaga tatttagaaa caattaaaac agtccttgaa gaagctggat 4981 atgttttcga aaaaatcgaa ctaagaaaag tgcaataaac tgctgaagta tttcagcagt 5041 tttttttatt tagaaatagt gaaaaaaata taatcaggga ggtatcaata tttaatgagt 5101 actgatttaa atttatttag actggaatta ataattaaca cgtagactaa ttaaaattta 5161 atgagggata aagaggatac aaaaatatta atttcaatcc ctattaaatt ttaacaaggg 5221 ggggattaaa atttaattag aggtttatcc acaagaaaag accctaataa aatttttact 5281 agggttataa cactgattaa tttcttaatg ggggagggat taaaatttaa tgacaaagaa 5341 aacaatcttt taagaaaagc ttttaaaaga taataataaa aagagctttg cgattaagca 5401 aaactcttta ctttttcatt gacattatca aattcatcga tttcaaattg ttgttgtatc 5461 ataaagttaa ttctgttttg cacaaccttt tcaggaatat aaaacacatc tgaggcttgt 5521 tttataaact cagggtcgct aaagtcaatg taacgtagca tatgatatgg tatagcttcc 5581 acccaagtta gcctttctgc ttcttctgaa tgtttttcat atacttccat gggtatctct 5641 aaatgatttt cctcatgtag caaggtatga gcaaaaagtt tatggaattg atagttcctc 5701 tctttttctt caactttttt atctaaaaca aacactttaa catctgagtc aatgtaagca 5761 taagatgttt ttccagtcat aatttcaatc ccaaatcttt tagacagaaa ttctggacgt 5821 aaatcttttg gtgaaagaat ttttttatgt agcaatatat ccgatacagc accttctaaa 5881 agcgttggtg aatagggcat tttacctatc tcctctcatt ttgtggaata aaaatagtca 5941 tattcgtcca tctacctatc ctattatcga acagttgaac tttttaatca aggatcagtc 6001 ctttttttca ttattcttaa actgtgctct taactttaac aactcgattt gtttttccag 6061 atctcgaggg taactagcct cgccgatccc gcaagaggcc cggcagtcag gtggcacttt 6121 tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 6181 tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 6241 gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 6301 ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 6361 agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 6421 agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 6481 tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 6541 tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 6601 cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 6661 aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 6721 tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 6781 tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 6841 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 6901 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 6961 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 7021 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 7081 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 7141 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 7201 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 7261 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 7321 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 7381 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 7441 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 7501 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 7561 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 7621 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 7681 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 7741 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 7801 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 7861 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 7921 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 7981 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 8041 gcgcccaata cgcatgc
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