CN113215185B - 一种用于重组表达棘白菌素b脱酰基酶的重组基因序列 - Google Patents
一种用于重组表达棘白菌素b脱酰基酶的重组基因序列 Download PDFInfo
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Abstract
本发明公开了一种用于重组表达棘白菌素B脱酰基酶的重组基因序列。本发明对实验室以获得的棘白菌素B脱酰基酶进行了克隆,并首次在大肠杆菌中成功的活性表达了重组棘白菌素B脱酰基酶。以往的多亚基单链结构蛋白在大肠杆菌中很难成熟,在我们的发明中采用将亚基拆分后共表达的方法,成功实现了在大肠杆菌中的活性表达。由于这是研究中首次将棘白菌素B脱酰基酶于大肠杆菌中表达,为其他的多亚基类似蛋白酶提供了一种新的异源表达思路,同时也为了后续的工业化生产奠定了一定基础。
Description
技术领域
本发明涉及生物工程技术领域,特别是涉及一种用于重组表达棘白菌素B脱酰基酶的重组基因序列。
背景技术
真菌感染是指由真菌微生物引起的感染。近年来,真菌感染造成的死亡人数不断增多,其原因与皮质激素、细胞毒性药物、光谱抗生素及免疫抑制剂等在临床中的广泛应用有关。棘白菌素类药物作为与细胞壁合成酶作用的抑制剂普遍存在抗菌活性强,与其他药物不存在拮抗作用等特点,其中尤以阿尼芬净最为突出,其具有较大的分布容积和光谱抗菌性,不经肝脏代谢,不存在交叉耐药性等优点,而生物法催化生产阿尼芬净前体已经成为国内外抗真菌药物研究的热点。其中棘白菌素B脱酰基酶(Echinocandin B decaylase,ECBD)属于N端亲核水解酶超家族。Echinocandin B(ECB)都可以被ECB脱酰基酶在酰胺键上裂解,生成环状六肽Echinocandin B核(ECBN),这是半合成抗真菌抗生素阿尼芬净的关键前体。
信号肽工程通过筛选不同信号肽,将目的蛋白定位于周质空间或穿过细菌外膜直接到达胞外培养基。大肠杆菌的周质空间内含有一系列的酶,并提供了一个氧化的环境,这些都有利于二硫键的正确形成,并增强硫基蛋白的正确折叠使活性蛋白质的产量得到提高,而且研究发现胞周质或胞外分泌表达能增强某些基因产物的可溶性。
亚基重构(Subunit reconsititution)是指针对多亚基蛋白的一种结构域再安排,与循环排列类似(Circular permutation),多见于抗体基因在大肠杆菌中的表达,由于在目的基因中含有内含子或连接肽等无功能活性的基因,将原本单链多亚基的蛋白利用基因工程的方法将其独立表达后再利用蛋白的自组装形成活性结构域,该过程本身不会导致对现有蛋白质序列的任何氨基酸取代,而只是重组了多肽链中残基的顺序。而且此方法规避了单链多亚基蛋白在大肠杆菌中复杂的成熟过程,更有利于拥有复杂基因结构的蛋白在大肠杆菌中的成功表达。
近几年来,棘白菌素B脱酰基酶的异源表达宿主主要为链霉菌,如变铅青链霉菌、天蓝色链霉菌等。但是链霉菌的基因分子操作很不便利,可供使用的载体与改造方法也有限(如更换质粒的拷贝数、更换强启动子与信号肽等方法)。而且其发酵周期与代谢调控与大肠杆菌相比复杂很多。
因此,构建一株能够活性表达、遗传背景清晰的棘白菌素B脱酰基酶重组菌具有重要意义。
发明内容
本发明针对现有技术中存在的上述问题,提供了一种用于重组表达棘白菌素B脱酰基酶的重组基因序列。
一种用于重组表达棘白菌素B脱酰基酶的重组基因序列,核苷酸序列如SEQ IDNo.20所示。该重组基因序列是用同一个启动子同时启动表达棘白菌素B脱酰基酶α亚基和β亚基,且两个亚基对应的基因序列前分别有RBS结合位点(核糖体结合位点),可以由同一启动子同时启动两个亚基的表达。在RBS结合位点与亚基之间还设有OmpA信号肽,用于将两个亚基进行分泌表达,表达到细胞周质中,且OmpA信号肽分泌效率最高。
本发明又提供了所述重组基因序列的重组表达载体。
本发明又提供了所述重组表达载体的基因工程菌,由宿主菌中转入所述重组表达载体得到。优选的,宿主菌为大肠杆菌,比如用于表达的大肠杆菌BL21(DE3)。
本发明还提供了所述的重组基因序列、所述的重组表达载体、所述的基因工程菌在重组表达棘白菌素B脱酰基酶中的应用。
本发明还提供了一种重组表达棘白菌素B脱酰基酶的方法,将所述基因工程菌在培养基中发酵表达棘白菌素B脱酰基酶。优选的,发酵培养基的组分为:蛋白胨12g/L,酵母粉24g/L,甘油5g/L,KH2PO4 2.31g/L,K2HPO4 12.54g/L。
本发明对实验室以获得的棘白菌素B脱酰基酶进行了克隆,并首次在大肠杆菌中成功的活性表达了重组棘白菌素B脱酰基酶。以往的多亚基单链结构蛋白在大肠杆菌中很难成熟,在我们的发明中采用将亚基拆分后共表达的方法,成功实现了在大肠杆菌中的活性表达。由于这是研究中首次将棘白菌素B脱酰基酶于大肠杆菌中表达,为其他的多亚基类似蛋白酶提供了一种新的异源表达思路,同时也为了后续的工业化生产奠定了一定基础。
附图说明
图1为棘白菌素B脱酰基酶来源质粒示意图。
图2为棘白菌素B脱酰基酶(ECBD)重组子的PCR验证(M:2000bp Marker)结果图。
图3为密码子优化前后的棘白菌素B脱酰基酶的SDS-PAGE分析结果图。
图4为不同信号肽的分泌效率SDS-PAGE分析结果图。
图5为重组棘白菌素B脱酰基酶的不同表达方式示意图。
图6为重组棘白菌素B脱酰基酶的SDS-PAGE分析结果图。
图7为不同双亚基表达方式的酶活比较结果图。
具体实施方式
所用培养基与溶液:
LB(g/L):胰蛋白胨10,酵母膏5,NaCl 10,pH 7.0;
发酵培养基:蛋白胨12g/L,酵母粉24g/L,甘油5g/L,KH2PO4 2.31g/L,K2HPO412.54g/L,pH 7.5。接种前添加卡那霉素至终浓度50μg·mL-1。
0.1M PBS缓冲液:称取适量的KH2PO4与K2HPO4,用ddH2O配制为pH为7的溶液。
C112一步克隆连接酶、PCR所用酶均购自于诺维赞生物科技(南京)有限公司。
棘白菌素B脱酰基酶酶活测定方法:
在35℃下,在总共1mL反应混合物中测定ECBD活性,该混合物由pH 7、0.1M磷酸钠缓冲液和0.2g/L ECB组成。反应在600rpm下进行30分钟。反应结束后,离心以终止反应。之后采用HPLC检测产物生成。一个单位ECBD活性(U)定义为在35℃,pH 7的标准条件下每分钟产生1μg ECBN所需的酶量。比活性(U/g)定义为每克干细胞所具有的活性。
大肠杆菌感受态细胞的制作:
(1)取-80℃甘油保藏的E.coli菌液划线LB固体平板,37℃恒温倒置培养24h;
(2)从平板上挑取单菌落接种于5mL LB液体培养基,37℃,180rpm过夜培养;
(3)以10%接种量转接到50mL LB液体培养基,同样条件继续培养2~3h至OD 600达到0.4左右;
(4)无菌环境中将菌液转入50mL灭菌预冷的离心管,冰浴20min;
(5)菌液分装于2根离心管,置于冷冻离心机4℃,5000rpm离心5min;
(6)弃尽上清,各用5mL冰水预冷过的0.1mol·L-1无菌CaCl2溶液吹打重悬菌体沉淀,冰浴20min;
(7)再次将离心管放到离心机4℃,5000rpm离心5min,于超净工作台弃尽上清废液,各加2mL预冷的CaCl2溶液再一次重悬菌体沉淀,冰浴20min。
(8)向感受态细胞中加入等量冰水预冷的15%甘油-CaCl2溶液,充分混匀;
(9)将混合液以100μL每管的量分装到1.5mL无菌EP管中,-80℃低温保藏备用。
取上述感受态细胞与重组质粒pET28a-ECBD混合,进行热激转化,将转化液涂布于LB(含终浓度50μg·mL-1的卡那霉素)平板,使具有卡那抗性的重组细胞生长形成菌落。
实施例1
表达棘白菌素B脱酰基酶的重组大肠杆菌的构建方法。
为实现棘白菌素B脱酰基酶基因在大肠杆菌中的表达,根据棘白菌素B脱酰基酶基因设计引物如下:
上游引物ECBD-F(小写字母表示同源臂):
5'-agaaggagagaattcGTGACGTCCTCGTACATGCGCCTGAA-3';
下游引物ECBD-R(小写字母表示同源臂):
5'-tgcggccgcaagcttTCAGCGTCCCCGCTGTGCCA-3'。
线性化扩增质粒引物如下:
上游引物28a-F:5'-AAGCTTGCGGCCGCACTC-3';
下游引物28a-R:5'-GAATTCTCTCCTTCTTAAAGTTAAACAA-3'。
以实验室保存的克隆有ECBD野生型酶编码基因的pSET-152-PermE*质粒(图1)DNA为模板,ECBD-F及ECBD-R为引物扩增序列如SEQ ID NO.1所示的目的基因,SEQ ID NO.1即是原始的棘白菌素B脱酰基酶(ECBD),其中信号肽编码序列1-96bp,α亚基编码序列97-645bp,连接肽编码序列646-690bp,β亚基编码序列691-2364bp。PCR体系为(100μL):PhantaMax Super-Fidelity酶2μL;质粒DNA模板1μL;ECBD-F 1μL;ECBD-R 1μL;dNTP 2μL,buffer50μL,ddH2O 43μL。PCR条件为:95℃预变性5min;95℃变性30s,55℃退火30s,72℃延伸2min,30个循环;72℃后延伸5min。
将扩增获得的带有同源臂的目的基因与质粒pET-28a(+)用一步克隆酶连接,体系如下:
一步克隆连接体系(20μL):目的基因与载体按摩尔质量比3∶1的比率混合,37℃连接30min并转化E.coli BL21(DE3)感受态细胞。涂布于LB(含终浓度50μg·mL-1的卡那霉素)平板上培养12-16h。随机挑取若干株菌进行菌落PCR验证如图2,验证正确的进行序列测定,测序正确的即为重组质粒pET28a-ECBD。如图2所示在未经密码子优化之前的ECBD基因在大肠杆菌中表达失败,但是经过密码子优化后获得适合在大肠杆菌中表达的ECBD序列SEQ IDNO.2,异源表达成功,如图3,ECBD的前体得到了大量表达,但是无催化活性。
实施例2
重组棘白菌素B脱酰基酶高效率分泌信号肽的筛选。
由于实施例1中经过密码子优化的ECBD基因构建的重组载体并没有获得活性表达,这可能是由于异源基因上的信号肽在大肠杆菌中不能正确识别,导致目标蛋白不能达到合适的亚细胞空间进行成熟,所以我们挑选了四种大肠杆菌中常用的信号肽,来使ECB脱酰基酶达到正确的亚细胞空间。
实验中将从以下四种信号肽筛选:FhuD、PelB、DsbA和OmpA。
氨基酸序列如下:
FhuD:MSGLPLISRRRLLTAMALSPLLWQMNTAHA;
PelB:MKYLLPTAAAGLLLLAAQPAMA;
DsbA:MKKIWLALAGLVLAFSASA;
OmpA:MKKTAIAIAVALAGFATVAQA。
并分别设计了所需信号肽***的引物序列,利用PCR Overlap来替换棘白菌素B脱酰基酶原有的信号肽序列。所用引物下:
FhuD-F:
5'-GACCGCGATGGCGCTGAGCCCGCTGCTGTGGCAGATGAACACCGCGCATGCCGGTGGCTACGCGGCGCTGATCCGTC-3',
FhuD-R:
5'-TCAGCGCCATCGCGGTCAGCAGGCGGCGGCGGCTAATCAGCGGCAGGCCGCTCATGAATTCTCTCCTTCTTAAAGTTAAA-3',
PelB-F:
5'-GCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGGTGGCTACGCGGCGCTGATCCGTC-3',
PelB-R:
5'-GAGCAGCAGACCAGCAGCAGCGGTCGGCAGCAGGTATTTCATGAATTCTCTCCTTCTTAAAGTTAAA-3',
DsbA-F:
5'-CTGGCGGGCCTGGTGCTGGCGTTTAGCGCTAGCGCCGGTGGCTACGCGGCGCTGATCCGTC-3',
DsbA-R:
5'-CACCAGGCCCGCCAGCGCCAGCCAAATTTTTTTCATGAATTCTCTCCTTCTTAAAGTTAAA-3',
OmpA-F:
5'-GCACTGGCTGGTTTCGCTACCGTAGCGCAGGCCGGTGGCTACGCGGCGCTGATCCGTC-3',
OmpA-R:
5'-ACCAGCCAGTGCCACTGCAATCGCGATAGCTGTCTTTTTCATGAATTCTCTCCTTCTTAAAGTTAAA-3'。
以pET-28a-ECBD为模板,利用上述引物进行全质粒扩增。PCR体系为(100μL):Phanta Max Super-Fidelity酶2μL;质粒DNA模板1μL;Sigs-F 1μL;Sigs-R 1μL;dNTP 2μL,buffer50μL,ddH2O 43μL。PCR条件为:95℃预变性5min;95℃变性30s,59℃退火1min,72℃延伸2min,20个循环;72℃后延伸5min。
取上述PCR产物感受态混合,进行热激转化,将转化液涂布于LB(含终浓度50μg·mL-1的卡那霉素)平板,使具有卡那抗性的重组细胞生长形成菌落。将测序验证成功后的含有FhuD(基因序列如SEQ ID No.3所示)、PelB(基因序列如SEQ ID No.4所示)、DsbA(基因序列如SEQ ID No.5所示)和OmpA(基因序列如SEQ ID No.6所示)四种不同信号肽的棘白菌素B脱酰基酶基因在大肠杆菌中的分泌效率进行筛选,如图4所示,其中OmpA信号肽分泌效率最高。
实施例3
重组棘白菌素B脱酰基酶在大肠杆菌中不同表达方式的筛选:
如实施例2中所示,虽然筛选到较高分泌效率的信号肽,ECBD的前体得到了大量表达(图4),但是无催化活性。这是由于ECBD前体无法在大肠杆菌中正确裂解,因此我们接下来尝试共表达α和β亚基。我们假设α和β亚基可以在大肠杆菌中形成异二聚体,而没有连接肽将ECBD前体中的这两个亚基分开。为了证明这一假设,在α亚基编码区的末端添加了终止密码子,并在β亚基编码区之前***了T7启动子和核糖体结合位点的序列(图5,pT7-α/T7-β,质粒骨架pET-28a,***部分的序列如SEQ ID No.19所示,在实施例2质粒基础上进行改造)。将质粒pT7-α/T7-β导入E.coli BL21(DE3)中获得基因工程菌T7-α/T7-β,检测到ECB水解活性(图7),并且通过SDS-PAGE分析观察到具有β亚基理论分子量大小的清晰条带(图6)。然而,可溶形式和包涵体中的α亚基的表达均较弱。
接下来,我们研究了增加α亚基表达水平对粗酶溶液活性的影响。考虑到α和β亚基编码区之前的两个T7启动子可能相互竞争与pT7-α/T7-β中的转录调节子结合,因此去除β亚基编码区之前的T7启动子以增加α亚基的表达从而构建质粒pT7-α/β(图5,***部分的序列如SEQ ID No.20所示,在上述pT7-α/T7-β质粒的基础上进行改造)将质粒pT7-α/β导入E.coli BL21(DE3)中获得基因工程菌T7-α/β。与菌株T7-α/T7-β相比,使用一个T7启动子(菌株T7-α/β)共表达α和β亚基明显增加了α亚基的水平并降低了β亚基。菌株T7-α/β的活性(菌体破碎后的粗酶液活性)是菌株T7-α/T7-β的两倍左右,证明α亚基的表达水平对ECB脱酰基酶的活性至关重要(图7)。
序列表
<110> 浙江工业大学
<120> 一种用于重组表达棘白菌素B脱酰基酶的重组基因序列
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2364
<212> DNA
<213> 犹他游动放线菌(Actinoplanes utahensis)
<400> 1
gtgacgtcct cgtacatgcg cctgaaagca gcagcgatcg ccttcggtgt gatcgtggcg 60
accgcagccg tgccgtcacc cgcttccggc agggaacatg acggcggcta tgcggccctg 120
atccgccggg cctcgtacgg cgtcccgcac atcaccgccg acgacttcgg gagcctcggt 180
ttcggcgtcg ggtacgtgca ggccgaggac aacatctgcg tcatcgccga gagcgtagtg 240
acggccaacg gtgagcggtc gcggtggttc ggtgcgaccg ggccggacga cgccgatgtg 300
cgcagcgacc tcttccaccg caaggcgatc gacgaccgcg tcgccgagcg gctcctcgaa 360
gggccccgcg acggcgtgcg ggcgccgtcg gacgacgtcc gggaccagat gcgcggcttc 420
gtcgccggct acaaccactt cctacgccgc accggcgtgc accgcctgac cgacccggcg 480
tgccgcggca aggcctgggt gcgcccgctc tccgagatcg atctctggcg tacgtcgtgg 540
gacagcatgg tccgggccgg ttccggggcg ctgctcgacg gcatcgtcgc cgcgacgcca 600
cctacagccg ccgggcccgc gtcagccccg gaggcacccg acgccgccgc gatcgccgcc 660
gccctcgacg ggacgagcgc gggcatcggc agcaacgcgt acggcctcgg cgcgcaggcc 720
accgtgaacg gcagcgggat ggtgctggcc aacccgcact tcccgtggca gggcgccgca 780
cgcttctacc ggatgcacct caaggtgccc ggccgctacg acgtcgaggg cgcggcgctg 840
atcggcgacc cgatcatcgg gatcgggcac aaccgcacgg tcgcctggag ccacaccgtc 900
tccaccgccc gccggttcgt gtggcaccgc ctgagcctcg tgcccggcga ccccacctcc 960
tattacgtcg acggccggcc cgagcggatg cgcgcccgca cggtcacggt ccagaccggc 1020
agcggcccgg tcagccgcac cttccacgac acccgctacg gcccggtggc cgtgatgccg 1080
ggcaccttcg actggacgcc ggccaccgcg tacgccatca ccgacgtcaa cgcgggcaac 1140
aaccgcgcct tcgacgggtg gctgcggatg ggccaggcca aggacgtccg ggcgctcaag 1200
gcggtcctcg accggcacca gttcctgccc tgggtcaacg tgatcgccgc cgacgcgcgg 1260
ggcgaggccc tctacggcga tcattcggtc gtcccccggg tgaccggcgc gctcgctgcc 1320
gcctgcatcc cggcgccgtt ccagccgctc tacgcctcca gcggccaggc ggtcctggac 1380
ggttcccggt cggactgcgc gctcggcgcc gaccccgacg ccgcggtccc gggcattctc 1440
ggcccggcga gcctgccggt gcggttccgc gacgactacg tcaccaactc caacgacagt 1500
cactggctgg ccagcccggc cgccccgctg gaaggcttcc cgcggatcct cggcaacgaa 1560
cgcaccccgc gcagcctgcg cacccggctc gggctggacc agatccagca gcgcctcgcc 1620
ggcacggacg gtctgcccgg caagggcttc accaccgccc ggctctggca ggtcatgttc 1680
ggcaaccgga tgcacggcgc cgaactcgcc cgcgacgacc tggtcgcgct ctgccgccgc 1740
cagccgaccg cgaccgcctc gaacggcgcg atcgtcgacc tcaccgcggc ctgcacggcg 1800
ctgtcccgct tcgatgagcg tgccgacctg gacagccggg gcgcgcacct gttcaccgag 1860
ttcgccctcg cgggcggaat caggttcgcc gacaccttcg aggtgaccga tccggtacgc 1920
accccgcgcc gtctgaacac cacggatccg cgggtacgga cggcgctcgc cgacgccgtg 1980
caacggctcg ccggcatccc cctcgacgcg aagctgggag acatccacac cgacagccgc 2040
ggcgaacggc gcatccccat ccacggtggc cgcggggaag caggcacctt caacgtgatc 2100
accaacccgc tcgtgccggg cgtgggatac ccgcaggtcg tccacggaac atcgttcgtg 2160
atggccgtcg aactcggccc gcacggcccg tcgggacggc agatcctcac ctatgcgcag 2220
tcgacgaacc cgaactcacc ctggtacgcc gaccagaccg tgctctactc gcggaagggc 2280
tgggacacca tcaagtacac cgaggcgcag atcgcggccg acccgaacct gcgcgtctac 2340
cgggtggcac agcggggacg ctga 2364
<210> 2
<211> 2364
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaccagca gctacatgcg tctgaaagcg gcggcgattg cgtttggtgt gattgttgcg 60
accgcggcgg ttccgagccc ggcgagcggt cgtgaacatg atggtggcta cgcggcgctg 120
atccgtcgtg cgagctatgg cgtgccgcac attaccgcgg acgatttcgg tagcctgggt 180
tttggcgtgg gttatgttca ggcggaggat aacatctgcg ttattgcgga aagcgtggtt 240
accgcgaacg gtgaacgtag ccgttggttt ggtgcgaccg gtccggatga tgcggatgtg 300
cgtagcgacc tgttccaccg taaggcgatc gacgatcgtg ttgcggagcg tctgctggaa 360
ggtccgcgtg atggtgtgcg tgcgccgagc gatgatgttc gtgatcaaat gcgtggtttc 420
gtggcgggct acaaccactt tctgcgtcgt accggcgttc atcgtctgac cgatccggcg 480
tgccgtggta aagcgtgggt gcgtccgctg agcgagatcg acctgtggcg taccagctgg 540
gatagcatgg tgcgtgcggg tagcggtgcg ctgctggatg gcattgttgc ggcgaccccg 600
ccgaccgcgg cgggtccggc gagcgcgccg gaagcgccgg acgcggcggc gatcgcggcg 660
gcgctggatg gtaccagcgc gggcattggt agcaacgcgt atggtctggg tgcgcaggcg 720
accgtgaacg gtagcggtat ggttctggcg aacccgcact tcccgtggca gggtgcggcg 780
cgtttttacc gtatgcacct gaaagtgccg ggtcgttatg acgttgaggg tgcggcgctg 840
atcggcgatc cgatcattgg cattggtcac aaccgtaccg ttgcgtggag ccacaccgtt 900
agcaccgcgc gtcgtttcgt gtggcatcgt ctgagcctgg ttccgggtga cccgaccagc 960
tactatgttg atggtcgtcc ggaacgtatg cgtgcgcgta ccgtgaccgt tcaaaccggt 1020
agcggtccgg ttagccgtac cttccacgac acccgttacg gtccggtggc ggttatgccg 1080
ggcacctttg attggacccc ggcgaccgcg tatgcgatca ccgacgttaa cgcgggtaac 1140
aaccgtgcgt tcgatggttg gctgcgtatg ggccaggcga aggacgtgcg tgcgctgaaa 1200
gcggttctgg atcgtcacca atttctgccg tgggtgaacg ttattgcggc ggatgcgcgt 1260
ggtgaggcgc tgtacggcga tcacagcgtg gttccgcgtg tgaccggtgc gctggcggcg 1320
gcgtgcattc cggcgccgtt tcagccgctg tatgcgagca gcggtcaagc ggttctggat 1380
ggtagccgta gcgattgcgc gctgggtgcg gacccggatg cggcggtgcc gggcatcctg 1440
ggtccggcga gcctgccggt gcgtttccgt gacgattacg ttaccaacag caacgacagc 1500
cattggctgg cgagcccggc ggcgccgctg gaaggttttc cgcgtatcct gggtaacgag 1560
cgtaccccgc gtagcctgcg tacccgtctg ggtctggacc agattcagca acgtctggcg 1620
ggtaccgatg gtctgccggg caagggtttc accaccgcgc gtctgtggca agtgatgttt 1680
ggtaaccgta tgcacggcgc ggaactggcg cgtgacgatc tggttgcgct gtgccgtcgt 1740
caaccgaccg cgaccgcgag caacggtgcg atcgtggatc tgaccgcggc gtgcaccgcg 1800
ctgagccgtt tcgatgaacg tgcggacctg gatagccgtg gtgcgcacct gttcaccgag 1860
tttgcgctgg cgggtggcat tcgtttcgcg gacacctttg aagtgaccga tccggttcgt 1920
accccgcgtc gtctgaacac caccgacccg cgtgtgcgta ccgcgctggc ggatgcggtt 1980
caacgtctgg cgggtatccc gctggacgcg aaactgggcg acattcacac cgatagccgt 2040
ggtgaacgtc gtattccgat tcatggtggc cgtggcgagg cgggtacctt caacgttatc 2100
accaacccgc tggtgccggg cgttggttac ccgcaggtgg ttcacggtac cagctttgtg 2160
atggcggtgg agctgggtcc gcatggtccg agcggtcgtc agattctgac ctatgcgcaa 2220
agcaccaacc cgaacagccc gtggtacgcg gaccaaaccg tgctgtatag ccgtaagggc 2280
tgggatacca tcaaatacac cgaagcgcag attgcggcgg acccgaacct gcgtgtgtat 2340
cgtgttgcgc aacgtggtcg ttaa 2364
<210> 3
<211> 90
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgagcggcc tgccgctgat tagccgccgc cgcctgctga ccgcgatggc gctgagcccg 60
ctgctgtggc agatgaacac cgcgcatgcc 90
<210> 4
<211> 66
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60
atggcc 66
<210> 5
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgaaaaaaa tttggctggc gctggcgggc ctggtgctgg cgtttagcgc tagcgcc 57
<210> 6
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaaaaaga cagctatcgc gattgcagtg gcactggctg gtttcgctac cgtagcgcag 60
gcc 63
<210> 7
<211> 30
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ser Gly Leu Pro Leu Ile Ser Arg Arg Arg Leu Leu Thr Ala Met
1 5 10 15
Ala Leu Ser Pro Leu Leu Trp Gln Met Asn Thr Ala His Ala
20 25 30
<210> 8
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala
1 5 10 15
Ala Gln Pro Ala Met Ala
20
<210> 9
<211> 19
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser
1 5 10 15
Ala Ser Ala
<210> 10
<211> 21
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Lys Lys Thr Ala Ile Ala Ile Ala Val Ala Leu Ala Gly Phe Ala
1 5 10 15
Thr Val Ala Gln Ala
20
<210> 11
<211> 77
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gaccgcgatg gcgctgagcc cgctgctgtg gcagatgaac accgcgcatg ccggtggcta 60
cgcggcgctg atccgtc 77
<210> 12
<211> 80
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tcagcgccat cgcggtcagc aggcggcggc ggctaatcag cggcaggccg ctcatgaatt 60
ctctccttct taaagttaaa 80
<210> 13
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gcactggctg gtttcgctac cgtagcgcag gccggtggct acgcggcgct gatccgtc 58
<210> 14
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gagcagcaga ccagcagcag cggtcggcag caggtatttc atgaattctc tccttcttaa 60
agttaaa 67
<210> 15
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ctggcgggcc tggtgctggc gtttagcgct agcgccggtg gctacgcggc gctgatccgt 60
c 61
<210> 16
<211> 61
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
caccaggccc gccagcgcca gccaaatttt tttcatgaat tctctccttc ttaaagttaa 60
a 61
<210> 17
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
gcactggctg gtttcgctac cgtagcgcag gccggtggct acgcggcgct gatccgtc 58
<210> 18
<211> 67
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
accagccagt gccactgcaa tcgcgatagc tgtctttttc atgaattctc tccttcttaa 60
agttaaa 67
<210> 19
<211> 2529
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 60
tgtttaactt taagaaggag agaattcatg aaaaagacag ctatcgcgat tgcagtggca 120
ctggctggtt tcgctaccgt agcgcaggcc catgatggtg gctacgcggc gctgatccgt 180
cgtgcgagct atggcgtgcc gcacattacc gcggacgatt tcggtagcct gggttttggc 240
gtgggttatg ttcaggcgga ggataacatc tgcgttattg cggaaagcgt ggttaccgcg 300
aacggtgaac gtagccgttg gtttggtgcg accggtccgg atgatgcgga tgtgcgtagc 360
gacctgttcc accgtaaggc gatcgacgat cgtgttgcgg agcgtctgct ggaaggtccg 420
cgtgatggtg tgcgtgcgcc gagcgatgat gttcgtgatc aaatgcgtgg tttcgtggcg 480
ggctacaacc actttctgcg tcgtaccggc gttcatcgtc tgaccgatcc ggcgtgccgt 540
ggtaaagcgt gggtgcgtcc gctgagcgag atcgacctgt ggcgtaccag ctgggatagc 600
atggtgcgtg cgggtagcgg tgcgctgctg gatggcattg ttgcggcgac cccgccgacc 660
gcggcgggtc cggcgagcgc gccggaagcg ccggacgcgt aaggatccta atacgactca 720
ctatagggga attgtgagcg gataacaatt ccccatctta gtatttgttt aactttaaga 780
aggagaccta ggatgaaaaa gacagctatc gcgattgcag tggcactggc tggtttcgct 840
accgtagcgc aggccagcaa cgcgtatggt ctgggtgcgc aggcgaccgt gaacggtagc 900
ggtatggttc tggcgaaccc gcacttcccg tggcagggtg cggcgcgttt ttaccgtatg 960
cacctgaaag tgccgggtcg ttatgacgtt gagggtgcgg cgctgatcgg cgatccgatc 1020
attggcattg gtcacaaccg taccgttgcg tggagccaca ccgttagcac cgcgcgtcgt 1080
ttcgtgtggc atcgtctgag cctggttccg ggtgacccga ccagctacta tgttgatggt 1140
cgtccggaac gtatgcgtgc gcgtaccgtg accgttcaaa ccggtagcgg tccggttagc 1200
cgtaccttcc acgacacccg ttacggtccg gtggcggtta tgccgggcac ctttgattgg 1260
accccggcga ccgcgtatgc gatcaccgac gttaacgcgg gtaacaaccg tgcgttcgat 1320
ggttggctgc gtatgggcca ggcgaaggac gtgcgtgcgc tgaaagcggt tctggatcgt 1380
caccaatttc tgccgtgggt gaacgttatt gcggcggatg cgcgtggtga ggcgctgtac 1440
ggcgatcaca gcgtggttcc gcgtgtgacc ggtgcgctgg cggcggcgtg cattccggcg 1500
ccgtttcagc cgctgtatgc gagcagcggt caagcggttc tggatggtag ccgtagcgat 1560
tgcgcgctgg gtgcggaccc ggatgcggcg gtgccgggca tcctgggtcc ggcgagcctg 1620
ccggtgcgtt tccgtgacga ttacgttacc aacagcaacg acagccattg gctggcgagc 1680
ccggcggcgc cgctggaagg ttttccgcgt atcctgggta acgagcgtac cccgcgtagc 1740
ctgcgtaccc gtctgggtct ggaccagatt cagcaacgtc tggcgggtac cgatggtctg 1800
ccgggcaagg gtttcaccac cgcgcgtctg tggcaagtga tgtttggtaa ccgtatgcac 1860
ggcgcggaac tggcgcgtga cgatctggtt gcgctgtgcc gtcgtcaacc gaccgcgacc 1920
gcgagcaacg gtgcgatcgt ggatctgacc gcggcgtgca ccgcgctgag ccgtttcgat 1980
gaacgtgcgg acctggatag ccgtggtgcg cacctgttca ccgagtttgc gctggcgggt 2040
ggcattcgtt tcgcggacac ctttgaagtg accgatccgg ttcgtacccc gcgtcgtctg 2100
aacaccaccg acccgcgtgt gcgtaccgcg ctggcggatg cggttcaacg tctggcgggt 2160
atcccgctgg acgcgaaact gggcgacatt cacaccgata gccgtggtga acgtcgtatt 2220
ccgattcatg gtggccgtgg cgaggcgggt accttcaacg ttatcaccaa cccgctggtg 2280
ccgggcgttg gttacccgca ggtggttcac ggtaccagct ttgtgatggc ggtggagctg 2340
ggtccgcatg gtccgagcgg tcgtcagatt ctgacctatg cgcaaagcac caacccgaac 2400
agcccgtggt acgcggacca aaccgtgctg tatagccgta agggctggga taccatcaaa 2460
tacaccgaag cgcagattgc ggcggacccg aacctgcgtg tgtatcgtgt tgcgcaacgt 2520
ggtcgttaa 2529
<210> 20
<211> 2479
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt 60
tgtttaactt taagaaggag agaattcatg aaaaagacag ctatcgcgat tgcagtggca 120
ctggctggtt tcgctaccgt agcgcaggcc catgatggtg gctacgcggc gctgatccgt 180
cgtgcgagct atggcgtgcc gcacattacc gcggacgatt tcggtagcct gggttttggc 240
gtgggttatg ttcaggcgga ggataacatc tgcgttattg cggaaagcgt ggttaccgcg 300
aacggtgaac gtagccgttg gtttggtgcg accggtccgg atgatgcgga tgtgcgtagc 360
gacctgttcc accgtaaggc gatcgacgat cgtgttgcgg agcgtctgct ggaaggtccg 420
cgtgatggtg tgcgtgcgcc gagcgatgat gttcgtgatc aaatgcgtgg tttcgtggcg 480
ggctacaacc actttctgcg tcgtaccggc gttcatcgtc tgaccgatcc ggcgtgccgt 540
ggtaaagcgt gggtgcgtcc gctgagcgag atcgacctgt ggcgtaccag ctgggatagc 600
atggtgcgtg cgggtagcgg tgcgctgctg gatggcattg ttgcggcgac cccgccgacc 660
gcggcgggtc cggcgagcgc gccggaagcg ccggacgcgt aaccatctta gtatttgttt 720
aactttaaga aggagaccta ggatgaaaaa gacagctatc gcgattgcag tggcactggc 780
tggtttcgct accgtagcgc aggccagcaa cgcgtatggt ctgggtgcgc aggcgaccgt 840
gaacggtagc ggtatggttc tggcgaaccc gcacttcccg tggcagggtg cggcgcgttt 900
ttaccgtatg cacctgaaag tgccgggtcg ttatgacgtt gagggtgcgg cgctgatcgg 960
cgatccgatc attggcattg gtcacaaccg taccgttgcg tggagccaca ccgttagcac 1020
cgcgcgtcgt ttcgtgtggc atcgtctgag cctggttccg ggtgacccga ccagctacta 1080
tgttgatggt cgtccggaac gtatgcgtgc gcgtaccgtg accgttcaaa ccggtagcgg 1140
tccggttagc cgtaccttcc acgacacccg ttacggtccg gtggcggtta tgccgggcac 1200
ctttgattgg accccggcga ccgcgtatgc gatcaccgac gttaacgcgg gtaacaaccg 1260
tgcgttcgat ggttggctgc gtatgggcca ggcgaaggac gtgcgtgcgc tgaaagcggt 1320
tctggatcgt caccaatttc tgccgtgggt gaacgttatt gcggcggatg cgcgtggtga 1380
ggcgctgtac ggcgatcaca gcgtggttcc gcgtgtgacc ggtgcgctgg cggcggcgtg 1440
cattccggcg ccgtttcagc cgctgtatgc gagcagcggt caagcggttc tggatggtag 1500
ccgtagcgat tgcgcgctgg gtgcggaccc ggatgcggcg gtgccgggca tcctgggtcc 1560
ggcgagcctg ccggtgcgtt tccgtgacga ttacgttacc aacagcaacg acagccattg 1620
gctggcgagc ccggcggcgc cgctggaagg ttttccgcgt atcctgggta acgagcgtac 1680
cccgcgtagc ctgcgtaccc gtctgggtct ggaccagatt cagcaacgtc tggcgggtac 1740
cgatggtctg ccgggcaagg gtttcaccac cgcgcgtctg tggcaagtga tgtttggtaa 1800
ccgtatgcac ggcgcggaac tggcgcgtga cgatctggtt gcgctgtgcc gtcgtcaacc 1860
gaccgcgacc gcgagcaacg gtgcgatcgt ggatctgacc gcggcgtgca ccgcgctgag 1920
ccgtttcgat gaacgtgcgg acctggatag ccgtggtgcg cacctgttca ccgagtttgc 1980
gctggcgggt ggcattcgtt tcgcggacac ctttgaagtg accgatccgg ttcgtacccc 2040
gcgtcgtctg aacaccaccg acccgcgtgt gcgtaccgcg ctggcggatg cggttcaacg 2100
tctggcgggt atcccgctgg acgcgaaact gggcgacatt cacaccgata gccgtggtga 2160
acgtcgtatt ccgattcatg gtggccgtgg cgaggcgggt accttcaacg ttatcaccaa 2220
cccgctggtg ccgggcgttg gttacccgca ggtggttcac ggtaccagct ttgtgatggc 2280
ggtggagctg ggtccgcatg gtccgagcgg tcgtcagatt ctgacctatg cgcaaagcac 2340
caacccgaac agcccgtggt acgcggacca aaccgtgctg tatagccgta agggctggga 2400
taccatcaaa tacaccgaag cgcagattgc ggcggacccg aacctgcgtg tgtatcgtgt 2460
tgcgcaacgt ggtcgttaa 2479
Claims (7)
1.一种用于重组表达棘白菌素B脱酰基酶的重组基因序列,其特征在于,核苷酸序列如SEQ ID No.20所示。
2.包含如权利要求1所述重组基因序列的重组表达载体。
3.包含如权利要求2所述重组表达载体的基因工程菌,其特征在于,由宿主菌中转入所述重组表达载体得到。
4.如权利要求3所述基因工程菌,其特征在于,宿主菌为大肠杆菌。
5.如权利要求1所述的重组基因序列、如权利要求2所述的重组表达载体、如权利要求3或4所述的基因工程菌在重组表达棘白菌素B脱酰基酶中的应用。
6.一种重组表达棘白菌素B脱酰基酶的方法,其特征在于,将权利要求4所述基因工程菌在培养基中发酵表达棘白菌素B脱酰基酶。
7.如权利要求6所述的方法,其特征在于,发酵培养基的组分为:蛋白胨12g/L,酵母粉24g/L,甘油5g/L,KH2PO4 2.31g/L,K2HPO4 12.54g/L。
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