pHT43

载体基本信息

载体质粒图谱和多克隆位点信息

载体简介

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。

载体序列

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