載體基本信息

載體質(zhì)粒圖譜和多克隆位點(diǎn)信息

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載體簡(jiǎn)介

1.簡(jiǎn)介
革蘭氏陽(yáng)性菌因其在農(nóng)業(yè)，醫(yī)療和食品生物技術(shù)和重組蛋白生產(chǎn)等方面的貢獻(xiàn)而廣為人知。在所有革蘭氏陽(yáng)性菌中，枯草芽孢桿菌載體因下列原因尤為引人矚目。（一）無(wú)致病性，且一般認(rèn)為安全的有機(jī)體；（二）無(wú)明顯的密碼子偏好性；（三）可直接將功能性胞外蛋白分泌到培養(yǎng)基中（目前，大約60％的市售酶由芽孢桿菌生產(chǎn)）；（四）具備包含轉(zhuǎn)錄，翻譯，蛋白質(zhì)折疊、分泌機(jī)制，遺傳操作和大規(guī)模發(fā)酵的大信息量機(jī)體。

但是下述兩個(gè)障礙減少了枯草芽孢桿菌的使用：（一）產(chǎn)生一定數(shù)目的識(shí)別并降解外源蛋白的胞外蛋白酶；（二）載體質(zhì)粒穩(wěn)定性。個(gè)障礙已因蛋白酶缺失株的構(gòu)建而基本解決 。第二個(gè)因引入使用θ-復(fù)制模式質(zhì)粒被完全克服，如由天然質(zhì)粒pAMβ1和pBS72衍生的一些質(zhì)粒（Jannière等，1990；Titok等，2003）。

最近，基于大腸桿菌 - 枯草桿菌穿梭質(zhì)粒pMTLBS72的四種不同表達(dá)載體的構(gòu)建和使用展示出全面的結(jié)構(gòu)穩(wěn)定性，業(yè)已出版（Nguyen等，2005）。

兩個(gè)新的載體pHT01和pHT43允許在細(xì)胞質(zhì)中高水平表達(dá)重組蛋白，其中pHT43載體引導(dǎo)重組蛋白到培養(yǎng)基。這兩個(gè)載體基于強(qiáng)σA-依賴性啟動(dòng)子的枯草桿菌groE操縱子通過(guò)添加lac操縱子改造成為一種高效可控的（IPTG誘導(dǎo)的）啟動(dòng)子。pHT01衍生載體可與8×His標(biāo)簽（pHT08），鏈球菌標(biāo)簽（pHT9）或C - Myc的標(biāo)簽（pHT10）相結(jié)合。

2. pHT 載體
所有在枯草芽孢桿菌的groESL操縱子之前使強(qiáng)啟動(dòng)子與lac操縱子融合的載體都可通過(guò)加入IPTG進(jìn)行誘導(dǎo)。盡管當(dāng)未添加誘導(dǎo)物時(shí)表達(dá)組件的背景表達(dá)水平很低，還是成功從約1300種誘導(dǎo)因子中篩選出一種來(lái)使用bgaB報(bào)道基因（Phan等，2005）。當(dāng)分別將htpG和pbpE基因融合到groE啟動(dòng)子時(shí)，加入IPTG后，表達(dá)的重組蛋白可能分別占細(xì)胞總蛋白的10%和13%（Phan等，2005）。熱纖梭菌的amyQα-淀粉酶和纖維素酶A、B的高水平表達(dá)實(shí)驗(yàn)證實(shí)。該載體還插入了一個(gè)高效SD序列以及一個(gè)多克隆位點(diǎn)（BamH I, Xba I, Aat II, Sma I）。編碼α-淀粉酶的amyQ基因的信號(hào)肽編碼區(qū)域與pHT01的SD序列融合，構(gòu)成了pHT43，以此獲得分泌的重組蛋白。

載體序列

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