pHT43

价格:2000元

联系方式:I47-825O-882O

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载体基本信息

出品公司: MoBiTec GmbH, Germany
载体名称: pHT43
质粒类型: 枯草芽孢杆菌表达载体
高拷贝/低拷贝: 高拷贝
启动子: Pgrac
克隆方法: 多克隆位点,限制性内切酶
载体大小: 8057bp
5' 测序引物及序列: LacI-R:GGCATACTCTGCGACATCGT
3' 测序引物及序列: --
载体标签: N-amyQ
载体抗性: 氨苄和氯霉素
筛选标记: --
备注: pHT43载体通过IPTG诱导,能够高水平分泌表达目的蛋白。
产品目录号: pBS002
稳定性: 瞬时表达
组成型: 诱导型
病毒/非病毒: 非病毒

载体质粒图谱和多克隆位点信息

pHT43载体图谱



pHT43多克隆位点

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。第一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-2014
SOURCE      
  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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