CN112680434B - 一种提高蛋白质谷氨酰胺酶分泌表达的方法 - Google Patents

一种提高蛋白质谷氨酰胺酶分泌表达的方法 Download PDF

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CN112680434B
CN112680434B CN202110132935.5A CN202110132935A CN112680434B CN 112680434 B CN112680434 B CN 112680434B CN 202110132935 A CN202110132935 A CN 202110132935A CN 112680434 B CN112680434 B CN 112680434B
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张国强
堵国成
殷鑫鑫
周景文
陈坚
李江华
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Wuhan Sunhy Biological Co ltd
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Abstract

本发明公开了一种提高蛋白质谷氨酰胺酶分泌表达的方法,属于基因工程技术领域。在本发明中通过改造信号肽,实现了蛋白质谷氨酰胺酶在枯草芽孢杆菌中高效的胞外表达,解决了蛋白质谷氨酰胺酶胞外分泌不足的问题。本发明通过改造信号肽,使蛋白质谷氨酰胺酶的分泌酶活由0.44U/mL提高到3.2U/mL,酶活提高7.27倍,应用本发明中的信号肽明显提高了蛋白质谷氨酰胺酶的胞外分泌,降低了工业上生产蛋白质谷氨酰胺酶的成本,同时也为枯草芽孢杆菌外源分泌蛋白表达量的提高奠定了基础。

Description

一种提高蛋白质谷氨酰胺酶分泌表达的方法
技术领域
本发明涉及一种提高蛋白质谷氨酰胺酶分泌表达的方法,属于基因工程技术领域。
背景技术
蛋白质谷氨酰胺酶(protein-glutaminase,EC3.5.1.44,简称PG)是一类特异性谷氨酰胺脱酰胺水解酶。蛋白质谷氨酰胺酶主要应用于食品工业,大量研究验证该酶能显著改善小麦谷蛋白、米谷蛋白等植物蛋白的溶解度、乳化性和起泡性等功能,蛋白质谷氨酰胺酶的脱酰胺作用不需要提前对蛋白质进行预处理且特异性强,只作用于蛋白质的侧链的谷氨酰胺,对天冬酰胺无脱酰胺作用,且不会对蛋白质的结构造成不良变化。但是目前蛋白质谷氨酰胺酶的应用受到制约,其中蛋白分泌表达量低是其中一个重要因素。
与产蛋白质谷氨酰胺酶的野生解朊金黄杆菌相比,枯草芽孢杆菌作为传统的工业生产菌,具有极强的蛋白质分泌能力,是蛋白质谷氨酰胺酶表达和分泌的良好系统。目前研究表明枯草芽孢杆菌的启动子与信号肽等表达元件的选择与优化对外源蛋白表达有重要影响。其中,信号肽最主要功能就是引导分泌蛋白或膜蛋白,在细胞内不同区域或不同细胞器进行正确定位,可提高外源蛋白质的可溶性,避免因包涵体复性带来的产物纯化等困难。然而信号肽对蛋白的表达分泌往往具有特异性,同一种蛋白质在不同信号肽的作用下,表达分泌效率往往相差甚远,缺乏普适应的规律。
因此,获得高效、特异性的信号肽序列,改善蛋白质谷氨酰胺酶蛋白分泌途径,提高蛋白质谷氨酰胺酶表达效率,是很有难度的事情,但具有重要的应用价值。
发明内容
本发明提供一种提高蛋白质谷氨酰胺酶分泌表达的方法,所述方法包括:以含有SEQ ID NO.1~8任一所示氨基酸序列的信号肽引导蛋白质谷氨酰胺酶的分泌表达。
在本发明的一种实施方式中,所述蛋白质谷氨酰胺酶的氨基酸序列如GenBank登录号:AB046594.1所示,编码所述蛋白质谷氨酰胺酶的核苷酸序列如SEQ ID NO.9所示。
在本发明的一种实施方式中,编码SEQ ID NO.1所示信号肽的核苷酸序列如SEQID NO.22所示。编码SEQ ID NO.2所示信号肽的核苷酸序列如SEQ ID NO.23所示。编码SEQID NO.3所示信号肽的核苷酸序列如SEQ ID NO.24所示。编码SEQ ID NO.4所示信号肽的核苷酸序列如SEQ ID NO.25所示。编码SEQ ID NO.5所示信号肽的核苷酸序列如SEQ IDNO.26所示。编码SEQ ID NO.6所示信号肽的核苷酸序列如SEQ ID NO.27所示。编码SEQ IDNO.7所示信号肽的核苷酸序列如SEQ ID NO.28所示。编码SEQ ID NO.8所示信号肽的核苷酸序列如SEQ ID NO.29所示。
在本发明的一种实施方式中,所述分泌表达是通过构建重组菌实现。
在本发明的一种实施方式中,所述重组菌是以细菌为宿主。
在本发明的一种实施方式中,所述重组菌是重组枯草芽孢杆菌。
在本发明的一种实施方式中,所述重组枯草芽孢杆菌的出发菌株为枯草芽孢杆菌WB600。
在本发明的一种实施方式中,所述重组枯草芽孢杆菌,以枯草芽孢杆菌WB600为出发菌株,将SEQ ID NO.9所示基因编码的蛋白质谷氨酰胺酶基因N端融合核苷酸序列为SEQID NO.22~29所示的信号肽形成的重组基因进行表达。
本发明的第二个目的是提供一种重组菌,所述重组菌以含有SEQ ID NO.1~8任一所示氨基酸序列的信号肽引导蛋白质谷氨酰胺酶的分泌表达。
在本发明的一种实施方式中,所述重组菌是重组枯草芽孢杆菌。
在本发明的一种实施方式中,所述重组枯草芽孢杆菌的构建方法,在编码蛋白质谷氨酰胺酶基因的N端融合SEQ ID NO.1~8任一所示的信号肽,得到重组基因,并将该重组基因与表达质粒pP43NMK连接所构成的重组质粒转化到大肠杆菌JM109中,抽提质粒转化到枯草芽孢杆菌WB600中。
在本发明的一种实施方式中,所述蛋白质谷氨酰胺酶的氨基酸序列如GenBank登录号:AB046594.1所示;编码蛋白质谷氨酰胺酶的基因序列如SEQ ID NO.9所示。
在本发明的一种实施方式中,所述重组基因是采用基因合成和一步克隆法得到的含有改造信号肽和蛋白质谷氨酰胺酶基因序列的重组基因。
在本发明的一种实施方式中,所述重组枯草芽孢杆菌的构建方法如下:合成SEQID NO.22~29任一所示的信号肽,分别通过一步克隆的方法将每条信号肽的DNA片段与插入到目的载体的RBS之后,RBS序列如SEQ ID NO.21所示,得到重组质粒,转入E.coli JM109中,经质粒的抽提后导入宿主菌细胞中,得到重组枯草芽孢杆菌。
本发明另外提供了一种生产蛋白质谷氨酰胺酶的方法,是使用上述的重组菌进行发酵生产蛋白质谷氨酰胺酶。可选地,所述重组菌为重组枯草芽孢杆菌。
在本发明的一种实施方式中,所述方法是将所述重组枯草芽孢杆菌于37℃培养36h。
在本发明的一种实施方式中,所述方法是将所述重组枯草芽孢杆菌接种至发酵培养基发酵。
在本发明的一种实施方式中,所述接种是按体积接种4%的种子液。
在本发明的一种实施方式中,所述种子液是将所述重组菌接种于LB培养基中,于37℃,220rpm培养8~12h,获得种子液。
在本发明的一种实施方式中,所述方法是先进行种子培养,再进行发酵;所述种子培养是将所构建好的重组质粒转化到枯草芽孢杆菌WB600中,现用现转。从平板上挑取枯草芽孢杆菌单菌落,接种至装液量为25mL LB培养基的250mL三角瓶中,37℃,220r/min培养8~12h,所述摇瓶发酵是将培养10h的种子液以4%(V/V)的接种量接入含50mg/L卡那霉素的发酵培养基中,装液量为25mL/250mL,37℃,220r/min培养36h。
本发明还要求保护所述重组菌在食品、生物领域的应用。
本发明还要求保护SEQ ID NO.8所述的信号肽(YdeJ)在提高蛋白质谷氨酰胺酶分泌表达效果或在蛋白质谷氨酰胺酶生产方面的应用。
有益效果:
在本发明中通过改造信号肽,实现了蛋白质谷氨酰胺酶在枯草芽孢杆菌中高效的胞外表达,解决了蛋白质谷氨酰胺酶胞外分泌不足的问题。本发明通过改造信号肽,使蛋白质谷氨酰胺酶的分泌酶活由0.44U/mL提高到3.26U/mL,酶活提高7.4倍,应用本发明中的信号肽明显提高了蛋白质谷氨酰胺酶的胞外分泌,降低了工业上生产蛋白质谷氨酰胺酶的成本,同时也为枯草芽孢杆菌外源分泌蛋白表达量的提高奠定了基础。
附图说明
图1为构建重组质粒的流程图;
图2为电泳图;其中GlmU、LipA、YmzC、NprE、YfkN、SpoIIIJ、NprB、YdeJ为含有SecA启动子且分别含有GlmU、LipA、YmzC、NprE、YfkN、SpoIIIJ、NprB、YdeJ信号肽的重组质粒转化到枯草芽孢杆菌WB 600感受态得到的重组菌株;
图3为重组菌株发酵酶活。
具体实施方式
信号肽GlmU、LipA、YmzC、NprE、YfkN、SpoIIIJ、NprB、YdeJ的氨基酸序列依次如SEQID NO.1~8所示,编码上述信号肽的基因的核苷酸序列依次如SEQ ID NO.22~29所示。
编码蛋白质谷氨酰胺酶的基因PG的核苷酸序列如SEQ ID NO.9所示。
LB/种子培养基(g/L):胰蛋白胨10、酵母粉5、NaCl 10。发酵培养基(g/L):酵母膏24、胰蛋白胨12、甘油10、K2HPO4 16.43、KH2PO4 2.32。
蛋白质谷氨酰胺酶的酶活测定方法:蛋白质谷氨酰胺酶能特异性地水解蛋白质中谷氨酰胺残基上的氨酰基,释放出游离的氨,氨在硝普钠的催化作用下,以次氯酸钠为氧化剂,与苯酚生成蓝绿色的靛酚蓝染料,对波长为630nm的光谱具有特异的吸收作用,通过测定OD630即可反映PG酶的相对活力。
酶活定义:100μL酶液每分钟水解二肽Cbz-Gln-Gly释放1μmol氨为1个酶活力单位(U/ml)。方法:取1mL二肽Cbz-Cln-Gly溶液(称取0.337g二肽Cbz-Gln-Gly,溶于0.176mol/LPBS缓冲液(pH6.5)中,并定容至100mL),滴加100μL待测样品(100μL无活性的酶液作为空白对照),混匀后置于37℃恒温箱中孵育30min,滴加1mL 0.4mol/L三氯乙酸溶液,混匀并终止反应。取60μL反应液与240μL蒸馏水、300μL显色试剂A(称取2.023g苯酚和7.5mg亚硝基铁氰化钠,溶于双蒸水并定容至50mL)、150μL显色试剂B(称取2.5g KOH溶于双蒸水并定容至50mL)和300μL显色试剂C(称取10.2g无水K2CO3溶于双蒸水,滴加417μL NaClO溶液,加水定容至50mL)充分混匀,置于37℃恒温箱中孵育20min,冷却,测量体系在630nm处的光吸收值。
实施例1:出发菌株重组质粒的构建
将序列如SEQ ID NO.31所示的6×His组氨酸标签加入蛋白质谷氨酰胺酶(GenBank:AB046594.1)的C端,合成全长共981bp的核苷酸序列(由生工完成),设计两对引物SEQ ID NO.11和SEQ ID NO.12、SEQ ID NO.13和SEQ ID NO.14经过一步PCR分别得到序列为SEQ ID NO.9的目的片段和SEQ ID NO.10的具有粘性末端的pP43NMK载体片段,用一步克隆法37℃连接30min,将连接产物通过化学法转化至大肠杆菌JM109感受态细胞,转化液涂在含有氨苄青霉素(100mg/L)的LB平板上,转化子的测序由上海生工完成,测序无误后得到出发菌株的重组质粒。
实施例2:枯草芽孢杆菌感受态的制备
将出发菌株的重组质粒,柱回收后转化待用,转化枯草芽孢杆菌WB600,具体操作如下:
(1)从甘油管中挑取一环枯草芽孢杆菌WB600菌液,在LB平板上划线,37℃恒温过夜培养;
(2)从平板上挑取枯草芽孢杆菌WB600单菌落,接种至含有2mL SPI的14mL摇菌管中,37℃,220r/min培养过夜;
(3)取200μL步骤2过夜培养物接种至含有2mL SPII的14mL摇菌管中,37℃、220r/min培养1.5~2h;
(4)步骤3分装前10min加入20μL EGTA。
(5)将步骤3培养物分装500μL于1.5mL EP管中,即得到枯草芽孢杆菌WB600感受态。
实施例3:含有启动子SecA重组质粒的构建
设计引物,序列如SEQ ID NO.15和SEQ ID NO.16所示,以实施例1构建的重组质粒为模板进行一步PCR得到具有粘性末端的pP43NMK片段,同时设计引物SEQ ID NO.17和SEQID NO.18,从枯草芽孢杆菌基因组上一步PCR得到启动子SecA片段,将上述PCR得到的两个片段进行一步克隆,将连接产物通过化学法转化至大肠杆菌JM109感受态细胞,转化液涂在含有氨苄青霉素(100mg/L)的LB平板上,转化子的测序由上海生工完成,测序无误最终将启动子P43更换成启动子SecA。
实施例4:含有不同信号肽重组枯草芽孢杆菌的构建
设计上下游引物,上游引物序列如SEQ ID NO.19所示,下游序列如SEQ ID NO.20所示,以实施例3的重组质粒为模板进行PCR扩增,得到的片段序列如SEQ ID NO.30所示,作为后续一步克隆的载体片段;合成编码SEQ ID NO.1~8所示信号肽的基因并在每条信号肽上游加入如SEQ ID NO.32所示的同源片段gtaagagaggaatgtacac,下游加入如SEQ IDNO.33所示的同源片段GATTCAAATGGCAATCAAGAAATTAATG,分别命名为GlmU、LipA、YmzC、NprE、YfkN、SpoIIIJ、NprB、YdeJ(核苷酸序列分别为SEQ ID NO.22~29所示)作为一步克隆的插入片段插入到目的载体RBS之后,RBS序列如SEQ ID NO.21所示,用一步克隆法连接后转化、测序得到信号肽改造后的重组质粒。将5~10μL重组质粒转入到实施例2制备的枯草芽孢杆菌WB600感受态中,37℃、220r/min培养2h后,8000rpm离心2min,弃去400μL上清后混匀,涂布到含有卡那霉素(50mg/L)的LB平板上,37℃恒温过夜培养得到转化子;挑取LB平板上转化子进行菌落PCR,通过核酸电泳得到大小正确的条带(图2),说明得到重组枯草芽孢杆菌。
实施例5:构建重组枯草芽孢杆菌的摇瓶发酵培养
将实施例4的信号肽GlmU、LipA、YmzC、NprE、YfkN、SpoIIIJ、NprB、YdeJ用到重组枯草芽孢杆菌中为生产菌株,同时实施例4构建的GlmU重组枯草芽孢杆菌为对照,挑取实施例4中的8种重组枯草芽孢杆菌单菌落,接种至装液量为25mL LB培养基的250mL三角瓶中,37℃,220r/min培养8~12h,然后按照4%的接种量再次转接至装液量为25mL发酵培养基的250mL三角瓶中,37℃,220r/min培养36h后测发酵液上清酶活。
结果如图3所示,重组枯草芽孢杆菌GlmU、LipA、YmzC、NprE、YfkN、SpoIIIJ、NprB和YdeJ的发酵酶活分别为0.44U/mL、0.48U/mL、0.02U/mL、0.57U/mL、0.91U/mL、0.54U/mL、2.02U/mL和3.26U/mL。其中菌株YdeJ酶活最高,达3.26U/mL,与菌株GlmU(对照)相比,酶活提高7.4倍。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQ ID NO.1
MDRRDNGGQYMDKRFAVVLAAGQGTRMKSKLYKVLHP
SEQ ID NO.2
MKFVKRRIIALVTILMLSVTSLFALQPSAKA
SEQ ID NO.3
MFESEAELRRIRIALVWIAVFLLFGACG
SEQ ID NO.4
MGLGKKLSVAVAASFMSLSISLPGVQA
SEQ ID NO.5
MRIQKRRTHVENILRILLPPIMILSLILPTPPIHA
SEQ ID NO.6
MLLKRRIGLLLSMVGVFMLLAGCS
SEQ ID NO.7
MRNLTKTSLLLAGLCTAAQMVFVTHASA
SEQ ID NO.8
MKKRRKICYCNTALLLMILLAGCT
SEQ ID NO.9
ATGAAAAACCTTTTTCTGAGCATGATGGCGTTTGTTACAGTTCTGACCTTTAATAGCTGCGCTGATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTTTCCGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACGGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTCTCTTTTATGCTGACAGCACAATTTTATGAGATTAAACCTACAAAAGAAAATGAACAATATATTGGTATGCTTAGACAAGCAGTTAAAAATGAATCACCGGTCCATATTTTTCTTAAACCGAATAGCAATGAAATTGGCAAAGTTGAATCAGCGAGCCCTGAAGATGTTAGATACTTTAAAACAATTCTCACGAAGGAAGTGAAAGGCCAGACGAATAAACTGGCCAGCGTCATTCCGGACGTGGCGACGCTGAATAGCCTTTTTAACCAAATCAAGAATCAAAGTTGTGGAACATCAACAGCATCAAGCCCGTGCATTACATTTAGATATCCGGTTGATGGCTGCTATGCGAGAGCACATAAAATGAGACAGATTCTTATGAATAATGGCTATGATTGCGAAAAACAATTTGTCTATGGCAATCTGAAAGCAAGCACAGGCACATGCTGCGTCGCATGGAGCTATCATGTGGCAATTCTGGTTTCATATAAAAATGCGTCAGGCGTAACAGAAAAACGCATTATTGATCCGTCATTATTTTCATCAGGCCCGGTGACAGATACGGCATGGAGAAATGCGTGCGTGAATACAAGCTGCGGCTCAGCAAGCGTTAGCAGCTATGCAAATACAGCGGGAAATGTTTATTATCGTTCACCGTCAAATAGCTATCTTTATGATAATAATCTGATTAATACAAATTGCGTTCTGACAAAATTTAGCCTTCTGAGCGGCTGTAGCCCGAGCCCGGCACCGGATGTTTCATCATGCGGCTTTcatcatcatcatcatcatTAA
SEQ ID NO.10
catcatcatTAAtgatgaaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattccttaaggaacgtacagacggcttaaaagcctttaaaaacgtttttaaggggtttgtagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgggataaagtaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgcctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgcgcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagctggactaaaaggcatgcaatttcataatcaaagagagcgaaaaagtagaacgaatgatgatattgaccatgagcgaacacgtgaaaattatgatttgaaaaatgataaaaatattgattacaacgaacgtgtcaaagaaattattgaatcacaaaaaacaggtacaagaaaaacgaggaaagatgctgttcttgtaaatgagttgctagtaacatctgaccgagatttttttgagcaactggatcctgataggtggtatgttttcgcttgaacttttaaatacagccattgaacatacggttgatttaataactgacaaacatcaccctcttgctaaagcggccaaggacgctgccgccggggctgtttgcgtttttgccgtgatttcgtgtatcattggtttacttatttttttgccaaagctgtaatggctgaaaattcttacatttattttacatttttagaaatgggcgtgaaaaaaagcgcgcgattatgtaaaatataaagtgatagcggtaccattataggtaagagaggaatgtacacATGAAAAACCTTTTTC
SEQ ID NO.11
ATGAAAAACCTTTTTCTGAGCATGATGGCGTTTG
SEQ ID NO.12
TTAatgatgatgatgatgatgAAAGCCGCATGATG
SEQ ID NO.13
GAAAAAGGTTTTTCATgtgtacattcctctcttacctataatg
SEQ ID NO.14
catcatcatTAAtgatgaaagcttggcgtaatc
SEQ ID NO.15
GAGGTATACATGGACTAGgtaagagaggaatg
SEQ ID NO.16
CAAGCTCTCCTCggatccagttgctcaaaaaaatctcg
SEQ ID NO.17
GAGGAGAGCTTGGACATCGTC
SEQ ID NO.18
CTAGTCCATGTATACCTCTCATTATC
SEQ ID NO.19
GATTCAAATGGCAATCAAGAAATTAATG
SEQ ID NO.20
Gtgtacattcctctcttac
SEQ ID NO.21
Gtaagagaggaatgtacac
SEQ ID NO.22
ATGGATAggAGGGATAATGGAGGCCAATACATGGATAAGCGGTTTGCAGTTGTGTTAGCAGCTGGTCAAGGAACAAGAATGAAATCAAAGCTATATAAAGTTCTTCATCCT
SEQ ID NO.23
atgaaatttgtaaaaagaaggatcattgcacttgtaacaattttgatgctgtctgttacatcgctgtttgcgttgcagccgtcagcaaaagcc
SEQ ID NO.24
Atgtttgaaagtgaagcagaactgagacgaatcaggattgcacttgtatggatagctgtctttttactgttcggggcgtgcggg
SEQ ID NO.25
Atgggtttaggtaagaaattgtctgttgctgtcgctgcttcgtttatgagtttatcaatcagcctgccaggtgttcaggct
SEQ ID NO.26
Atgagaatacagaaaagacgaacacacgtcgaaaacattctccgtattcttttgcccccaattatgatacttagcctaatcctcccaacaccacccattcatgca
SEQ ID NO.27
atgttgttgaaaaggagaatagggttgctattaagtatggttggcgtattcatgcttttggctggatgctcg
SEQ ID NO.28
Atgcgcaacttgaccaagacatctctattactggccggcttatgcacagcggcccaaatggtttttgtaacacatgcctcagct
SEQ ID NO.29
Atgaagaaacgcagaaagatatgttattgcaatactgccctgctgcttatgattttgcttgctggatgtacg
SEQ ID NO.30
GATTCAAATGGCAATCAAGAAATTAATGGCAAAGAAAAACTTTCCGTTAATGATTCAAAACTGAAAGATTTTGGCAAAACGGTTCCGGTTGGCATTGATGAAGAAAATGGCATGATTAAAGTCTCTTTTATGCTGACAGCACAATTTTATGAGATTAAACCTACAAAAGAAAATGAACAATATATTGGTATGCTTAGACAAGCAGTTAAAAATGAATCACCGGTCCATATTTTTCTTAAACCGAATAGCAATGAAATTGGCAAAGTTGAATCAGCGAGCCCTGAAGATGTTAGATACTTTAAAACAATTCTCACGAAGGAAGTGAAAGGCCAGACGAATAAACTGGCCAGCGTCATTCCGGACGTGGCGACGCTGAATAGCCTTTTTAACCAAATCAAGAATCAAAGTTGTGGAACATCAACAGCATCAAGCCCGTGCATTACATTTAGATATCCGGTTGATGGCTGCTATGCGAGAGCACATAAAATGAGACAGATTCTTATGAATAATGGCTATGATTGCGAAAAACAATTTGTCTATGGCAATCTGAAAGCAAGCACAGGCACATGCTGCGTCGCATGGAGCTATCATGTGGCAATTCTGGTTTCATATAAAAATGCGTCAGGCGTAACAGAAAAACGCATTATTGATCCGTCATTATTTTCATCAGGCCCGGTGACAGATACGGCATGGAGAAATGCGTGCGTGAATACAAGCTGCGGCTCAGCAAGCGTTAGCAGCTATGCAAATACAGCGGGAAATGTTTATTATCGTTCACCGTCAAATAGCTATCTTTATGATAATAATCTGATTAATACAAATTGCGTTCTGACAAAATTTAGCCTTCTGAGCGGCTGTAGCCCGAGCCCGGCACCGGATGTTTCATCATGCGGCTTTcatcatcatcatcatcatTAAtgatgaaagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattccttaaggaacgtacagacggcttaaaagcctttaaaaacgtttttaaggggtttgtagacaaggtaaaggataaaacagcacaattccaagaaaaacacgatttagaacctaaaaagaacgaatttgaactaactcataaccgagaggtaaaaaaagaacgaagtcgagatcagggaatgagtttataaaataaaaaaagcacctgaaaaggtgtctttttttgatggttttgaacttgttctttcttatcttgatacatatagaaataacgtcatttttattttagttgctgaaaggtgcgttgaagtgttggtatgtatgtgttttaaagtattgaaaacccttaaaattggttgcacagaaaaaccccatctgttaaagttataagtgactaaacaaataactaaatagatgggggtttcttttaatattatgtgtcctaatagtagcatttattcagatgaaaaatcaagggttttagtggacaagacaaaaagtggaaaagtgagaccatggagagaaaagaaaatcgctaatgttgattactttgaacttctgcatattcttgaatttaaaaaggctgaaagagtaaaagattgtgctgaaatattagagtataaacaaaatcgtgaaacaggcgaaagaaagttgtatcgagtgtggttttgtaaatccaggctttgtccaatgtgcaactggaggagagcaatgaaacatggcattcagtcacaaaaggttgttgctgaagttattaaacaaaagccaacagttcgttggttgtttctcacattaacagttaaaaatgtttatgatggcgaagaattaaataagagtttgtcagatatggctcaaggatttcgccgaatgatgcaatataaaaaaattaataaaaatcttgttggttttatgcgtgcaacggaagtgacaataaataataaagataattcttataatcagcacatgcatgtattggtatgtgtggaaccaacttattttaagaatacagaaaactacgtgaatcaaaaacaatggattcaattttggaaaaaggcaatgaaattagactatgatccaaatgtaaaagttcaaatgattcgaccgaaaaataaatataaatcggatatacaatcggcaattgacgaaactgcaaaatatcctgtaaaggatacggattttatgaccgatgatgaagaaaagaatttgaaacgtttgtctgatttggaggaaggtttacaccgtaaaaggttaatctcctatggtggtttgttaaaagaaatacataaaaaattaaaccttgatgacacagaagaaggcgatttgattcatacagatgatgacgaaaaagccgatgaagatggattttctattattgcaatgtggaattgggaacggaaaaattattttattaaagagtagttcaacaaacgggccagtttgttgaagattagatgctataattgttattaaaaggattgaaggatgcttaggaagacgagttattaatagctgaataagaacggtgctctccaaatattcttatttagaaaagcaaatctaaaattatctgaaaagggaatgagaatagtgaatggaccaataataatgactagagaagaaagaatgaagattgttcatgaaattaaggaacgaatattggataaatatggggatgatgttaaggctattggtgtttatggctctcttggtcgtcagactgatgggccctattcggatattgagatgatgtgtgtcatgtcaacagaggaagcagagttcagccatgaatggacaaccggtgagtggaaggtggaagtgaattttgatagcgaagagattctactagattatgcatctcaggtggaatcagattggccgcttacacatggtcaatttttctctattttgccgatttatgattcaggtggatacttagagaaagtgtatcaaactgctaaatcggtagaagcccaaacgttccacgatgcgatttgtgcccttatcgtagaagagctgtttgaatatgcaggcaaatggcgtaatattcgtgtgcaaggaccgacaacatttctaccatccttgactgtacaggtagcaatggcaggtgccatgttgattggtctgcatcatcgcatctgttatacgacgagcgcttcggtcttaactgaagcagttaagcaatcagatcttccttcaggttatgaccatctgtgccagttcgtaatgtctggtcaactttccgactctgagaaacttctggaatcgctagagaatttctggaatgggattcaggagtggacagaacgacacggatatatagtggatgtgtcaaaacgcataccattttgaacgatgacctctaataattgttaatcatgttggttacgtatttattaacttctcctagtattagtaattatcatggctgtcatggcgcattaacggaataaagggtgtgcttaaatcgggccattttgcgtaataagaaaaaggattaattatgagcgaattgaattaataataaggtaatagatttacattagaaaatgaaaggggattttatgcgtgagaatgttacagtctatcccggcattgccagtcggggatattaaaaagagtataggtttttattgggataaagtaggtttcactttggttcaccatgaagatggattcgcagttctaatgtgtaatgaggttcggattcatctatgggaggcaagtgatgaaggctggcgcctcgtagtaatgattcaccggtttgtacaggtgcggagtcgtttattgctggtactgctagttgccgcattgaagtagagggaattgatgaattatatcaacatattaagcctttgggcattttgcaccccaatacatcattaaaagatcagtggtgggatgaacgagactttgcagtaattgatcccgacaacaatttgattagcttttttcaacaaataaaaagctaaaatctattattaatctgttcagcaatcgggcgcgattgctgaataaaagatacgagagacctctcttgtatcttttttattttgagtggttttgtccgttacactagaaaaccgaaagacaataaaaattttattcttgctgagtctggctttcggtaagctagacaaaacggacaaaataaaaattggcaagggtttaaaggtggagattttttgagtgatcttctcaaaaaatactacctgtcccttgctgatttttaaacgagcacgagagcaaaacccccctttgctgaggtggcagagggcaggtttttttgtttcttttttctcgtaaaaaaaagaaaggtcttaaaggttttatggttttggtcggcactgccgcgcctcgcagagcacacactttatgaatataaagtatagtgtgttatactttacttggaagtggttgccggaaagagcgaaaatgcctcacatttgtgccacctaaaaaggagcgatttacatatgagttatgcagtttgtagaatgcaaaaagtgaaatcagctggactaaaaggcatgcaatttcataatcaaagagagcgaaaaagtagaacgaatgatgatattgaccatgagcgaacacgtgaaaattatgatttgaaaaatgataaaaatattgattacaacgaacgtgtcaaagaaattattgaatcacaaaaaacaggtacaagaaaaacgaggaaagatgctgttcttgtaaatgagttgctagtaacatctgaccgagatttttttgagcaactggatccGAGGAGAGCTTGGACATCGTCCGTCAGAAACGCTTTAATTTAAAGCCGATGGATAGTGAAGAAGCGATCTTGCAAATGAATATGCTCGGCCATAATTTCTTTGTTTTCACAAATGCGGAAACAAACCTTACAAATGTCGTGTACCGCAGAAATGACGGGAAATATGGCTTAATTGAACCGACTGAATAATGAAGAGAAGCCTTCCGTGATGTCCGCGGAAGGTTTTTGTTTTTCTTATTTGCAAATTCTTTGGAAATAACAAAAGGTATGATATGATAATGAGAGGTATACATGGACTAGgtaagagaggaatgtacac
SEQ ID NO.31
catcatcatcatcatcat
SEQ ID NO.32
Gtaagagaggaatgtacac
SEQ ID NO.33
GATTCAAATGGCAATCAAGAAATTAATG
SEQUENCE LISTING
<110> 江南大学
<120> 一种提高蛋白质谷氨酰胺酶分泌表达的方法
<160> 33
<170> PatentIn version 3.3
<210> 1
<211> 37
<212> PRT
<213> 人工合成
<400> 1
Met Asp Arg Arg Asp Asn Gly Gly Gln Tyr Met Asp Lys Arg Phe Ala
1 5 10 15
Val Val Leu Ala Ala Gly Gln Gly Thr Arg Met Lys Ser Lys Leu Tyr
20 25 30
Lys Val Leu His Pro
35
<210> 2
<211> 31
<212> PRT
<213> 人工合成
<400> 2
Met Lys Phe Val Lys Arg Arg Ile Ile Ala Leu Val Thr Ile Leu Met
1 5 10 15
Leu Ser Val Thr Ser Leu Phe Ala Leu Gln Pro Ser Ala Lys Ala
20 25 30
<210> 3
<211> 28
<212> PRT
<213> 人工合成
<400> 3
Met Phe Glu Ser Glu Ala Glu Leu Arg Arg Ile Arg Ile Ala Leu Val
1 5 10 15
Trp Ile Ala Val Phe Leu Leu Phe Gly Ala Cys Gly
20 25
<210> 4
<211> 27
<212> PRT
<213> 人工合成
<400> 4
Met Gly Leu Gly Lys Lys Leu Ser Val Ala Val Ala Ala Ser Phe Met
1 5 10 15
Ser Leu Ser Ile Ser Leu Pro Gly Val Gln Ala
20 25
<210> 5
<211> 35
<212> PRT
<213> 人工合成
<400> 5
Met Arg Ile Gln Lys Arg Arg Thr His Val Glu Asn Ile Leu Arg Ile
1 5 10 15
Leu Leu Pro Pro Ile Met Ile Leu Ser Leu Ile Leu Pro Thr Pro Pro
20 25 30
Ile His Ala
35
<210> 6
<211> 24
<212> PRT
<213> 人工合成
<400> 6
Met Leu Leu Lys Arg Arg Ile Gly Leu Leu Leu Ser Met Val Gly Val
1 5 10 15
Phe Met Leu Leu Ala Gly Cys Ser
20
<210> 7
<211> 28
<212> PRT
<213> 人工合成
<400> 7
Met Arg Asn Leu Thr Lys Thr Ser Leu Leu Leu Ala Gly Leu Cys Thr
1 5 10 15
Ala Ala Gln Met Val Phe Val Thr His Ala Ser Ala
20 25
<210> 8
<211> 24
<212> PRT
<213> 人工合成
<400> 8
Met Lys Lys Arg Arg Lys Ile Cys Tyr Cys Asn Thr Ala Leu Leu Leu
1 5 10 15
Met Ile Leu Leu Ala Gly Cys Thr
20
<210> 9
<211> 981
<212> DNA
<213> 人工合成
<400> 9
atgaaaaacc tttttctgag catgatggcg tttgttacag ttctgacctt taatagctgc 60
gctgattcaa atggcaatca agaaattaat ggcaaagaaa aactttccgt taatgattca 120
aaactgaaag attttggcaa aacggttccg gttggcattg atgaagaaaa tggcatgatt 180
aaagtctctt ttatgctgac agcacaattt tatgagatta aacctacaaa agaaaatgaa 240
caatatattg gtatgcttag acaagcagtt aaaaatgaat caccggtcca tatttttctt 300
aaaccgaata gcaatgaaat tggcaaagtt gaatcagcga gccctgaaga tgttagatac 360
tttaaaacaa ttctcacgaa ggaagtgaaa ggccagacga ataaactggc cagcgtcatt 420
ccggacgtgg cgacgctgaa tagccttttt aaccaaatca agaatcaaag ttgtggaaca 480
tcaacagcat caagcccgtg cattacattt agatatccgg ttgatggctg ctatgcgaga 540
gcacataaaa tgagacagat tcttatgaat aatggctatg attgcgaaaa acaatttgtc 600
tatggcaatc tgaaagcaag cacaggcaca tgctgcgtcg catggagcta tcatgtggca 660
attctggttt catataaaaa tgcgtcaggc gtaacagaaa aacgcattat tgatccgtca 720
ttattttcat caggcccggt gacagatacg gcatggagaa atgcgtgcgt gaatacaagc 780
tgcggctcag caagcgttag cagctatgca aatacagcgg gaaatgttta ttatcgttca 840
ccgtcaaata gctatcttta tgataataat ctgattaata caaattgcgt tctgacaaaa 900
tttagccttc tgagcggctg tagcccgagc ccggcaccgg atgtttcatc atgcggcttt 960
catcatcatc atcatcatta a 981
<210> 10
<211> 6733
<212> DNA
<213> 人工合成
<400> 10
catcatcatt aatgatgaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa 60
ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg 120
gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca 180
gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg 240
tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg 300
gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg 360
ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa 420
ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg 480
acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc 540
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc 600
ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc 660
ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg 720
ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc 780
actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga 840
gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc 900
tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac 960
caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg 1020
atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc 1080
acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa 1140
ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta 1200
ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt 1260
tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag 1320
tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca 1380
gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc 1440
tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt 1500
tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag 1560
ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt 1620
tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat 1680
ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt 1740
gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc 1800
ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat 1860
cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag 1920
ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt 1980
ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg 2040
gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta 2100
ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc 2160
gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt 2220
aacctataaa aataggcgta tcacgaggcc ctttcgtctc gcgcgtttcg gtgatgacgg 2280
tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc 2340
cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggctggct 2400
taactatgcg gcatcagagc agattgtact gagagtgcac catatgcggt gtgaaatacc 2460
gcacagatgc gtaaggagaa aataccgcat caggcgccat tcgccattca ggctgcgcaa 2520
ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg 2580
atgtgctgca aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa 2640
aacgacggcc agtgaattcc ttaaggaacg tacagacggc ttaaaagcct ttaaaaacgt 2700
ttttaagggg tttgtagaca aggtaaagga taaaacagca caattccaag aaaaacacga 2760
tttagaacct aaaaagaacg aatttgaact aactcataac cgagaggtaa aaaaagaacg 2820
aagtcgagat cagggaatga gtttataaaa taaaaaaagc acctgaaaag gtgtcttttt 2880
ttgatggttt tgaacttgtt ctttcttatc ttgatacata tagaaataac gtcattttta 2940
ttttagttgc tgaaaggtgc gttgaagtgt tggtatgtat gtgttttaaa gtattgaaaa 3000
cccttaaaat tggttgcaca gaaaaacccc atctgttaaa gttataagtg actaaacaaa 3060
taactaaata gatgggggtt tcttttaata ttatgtgtcc taatagtagc atttattcag 3120
atgaaaaatc aagggtttta gtggacaaga caaaaagtgg aaaagtgaga ccatggagag 3180
aaaagaaaat cgctaatgtt gattactttg aacttctgca tattcttgaa tttaaaaagg 3240
ctgaaagagt aaaagattgt gctgaaatat tagagtataa acaaaatcgt gaaacaggcg 3300
aaagaaagtt gtatcgagtg tggttttgta aatccaggct ttgtccaatg tgcaactgga 3360
ggagagcaat gaaacatggc attcagtcac aaaaggttgt tgctgaagtt attaaacaaa 3420
agccaacagt tcgttggttg tttctcacat taacagttaa aaatgtttat gatggcgaag 3480
aattaaataa gagtttgtca gatatggctc aaggatttcg ccgaatgatg caatataaaa 3540
aaattaataa aaatcttgtt ggttttatgc gtgcaacgga agtgacaata aataataaag 3600
ataattctta taatcagcac atgcatgtat tggtatgtgt ggaaccaact tattttaaga 3660
atacagaaaa ctacgtgaat caaaaacaat ggattcaatt ttggaaaaag gcaatgaaat 3720
tagactatga tccaaatgta aaagttcaaa tgattcgacc gaaaaataaa tataaatcgg 3780
atatacaatc ggcaattgac gaaactgcaa aatatcctgt aaaggatacg gattttatga 3840
ccgatgatga agaaaagaat ttgaaacgtt tgtctgattt ggaggaaggt ttacaccgta 3900
aaaggttaat ctcctatggt ggtttgttaa aagaaataca taaaaaatta aaccttgatg 3960
acacagaaga aggcgatttg attcatacag atgatgacga aaaagccgat gaagatggat 4020
tttctattat tgcaatgtgg aattgggaac ggaaaaatta ttttattaaa gagtagttca 4080
acaaacgggc cagtttgttg aagattagat gctataattg ttattaaaag gattgaagga 4140
tgcttaggaa gacgagttat taatagctga ataagaacgg tgctctccaa atattcttat 4200
ttagaaaagc aaatctaaaa ttatctgaaa agggaatgag aatagtgaat ggaccaataa 4260
taatgactag agaagaaaga atgaagattg ttcatgaaat taaggaacga atattggata 4320
aatatgggga tgatgttaag gctattggtg tttatggctc tcttggtcgt cagactgatg 4380
ggccctattc ggatattgag atgatgtgtg tcatgtcaac agaggaagca gagttcagcc 4440
atgaatggac aaccggtgag tggaaggtgg aagtgaattt tgatagcgaa gagattctac 4500
tagattatgc atctcaggtg gaatcagatt ggccgcttac acatggtcaa tttttctcta 4560
ttttgccgat ttatgattca ggtggatact tagagaaagt gtatcaaact gctaaatcgg 4620
tagaagccca aacgttccac gatgcgattt gtgcccttat cgtagaagag ctgtttgaat 4680
atgcaggcaa atggcgtaat attcgtgtgc aaggaccgac aacatttcta ccatccttga 4740
ctgtacaggt agcaatggca ggtgccatgt tgattggtct gcatcatcgc atctgttata 4800
cgacgagcgc ttcggtctta actgaagcag ttaagcaatc agatcttcct tcaggttatg 4860
accatctgtg ccagttcgta atgtctggtc aactttccga ctctgagaaa cttctggaat 4920
cgctagagaa tttctggaat gggattcagg agtggacaga acgacacgga tatatagtgg 4980
atgtgtcaaa acgcatacca ttttgaacga tgacctctaa taattgttaa tcatgttggt 5040
tacgtattta ttaacttctc ctagtattag taattatcat ggctgtcatg gcgcattaac 5100
ggaataaagg gtgtgcttaa atcgggccat tttgcgtaat aagaaaaagg attaattatg 5160
agcgaattga attaataata aggtaataga tttacattag aaaatgaaag gggattttat 5220
gcgtgagaat gttacagtct atcccggcat tgccagtcgg ggatattaaa aagagtatag 5280
gtttttattg ggataaagta ggtttcactt tggttcacca tgaagatgga ttcgcagttc 5340
taatgtgtaa tgaggttcgg attcatctat gggaggcaag tgatgaaggc tggcgcctcg 5400
tagtaatgat tcaccggttt gtacaggtgc ggagtcgttt attgctggta ctgctagttg 5460
ccgcattgaa gtagagggaa ttgatgaatt atatcaacat attaagcctt tgggcatttt 5520
gcaccccaat acatcattaa aagatcagtg gtgggatgaa cgagactttg cagtaattga 5580
tcccgacaac aatttgatta gcttttttca acaaataaaa agctaaaatc tattattaat 5640
ctgttcagca atcgggcgcg attgctgaat aaaagatacg agagacctct cttgtatctt 5700
ttttattttg agtggttttg tccgttacac tagaaaaccg aaagacaata aaaattttat 5760
tcttgctgag tctggctttc ggtaagctag acaaaacgga caaaataaaa attggcaagg 5820
gtttaaaggt ggagattttt tgagtgatct tctcaaaaaa tactacctgt cccttgctga 5880
tttttaaacg agcacgagag caaaaccccc ctttgctgag gtggcagagg gcaggttttt 5940
ttgtttcttt tttctcgtaa aaaaaagaaa ggtcttaaag gttttatggt tttggtcggc 6000
actgccgcgc ctcgcagagc acacacttta tgaatataaa gtatagtgtg ttatacttta 6060
cttggaagtg gttgccggaa agagcgaaaa tgcctcacat ttgtgccacc taaaaaggag 6120
cgatttacat atgagttatg cagtttgtag aatgcaaaaa gtgaaatcag ctggactaaa 6180
aggcatgcaa tttcataatc aaagagagcg aaaaagtaga acgaatgatg atattgacca 6240
tgagcgaaca cgtgaaaatt atgatttgaa aaatgataaa aatattgatt acaacgaacg 6300
tgtcaaagaa attattgaat cacaaaaaac aggtacaaga aaaacgagga aagatgctgt 6360
tcttgtaaat gagttgctag taacatctga ccgagatttt tttgagcaac tggatcctga 6420
taggtggtat gttttcgctt gaacttttaa atacagccat tgaacatacg gttgatttaa 6480
taactgacaa acatcaccct cttgctaaag cggccaagga cgctgccgcc ggggctgttt 6540
gcgtttttgc cgtgatttcg tgtatcattg gtttacttat ttttttgcca aagctgtaat 6600
ggctgaaaat tcttacattt attttacatt tttagaaatg ggcgtgaaaa aaagcgcgcg 6660
attatgtaaa atataaagtg atagcggtac cattataggt aagagaggaa tgtacacatg 6720
aaaaaccttt ttc 6733
<210> 11
<211> 34
<212> DNA
<213> 人工合成
<400> 11
atgaaaaacc tttttctgag catgatggcg tttg 34
<210> 12
<211> 35
<212> DNA
<213> 人工合成
<400> 12
ttaatgatga tgatgatgat gaaagccgca tgatg 35
<210> 13
<211> 43
<212> DNA
<213> 人工合成
<400> 13
gaaaaaggtt tttcatgtgt acattcctct cttacctata atg 43
<210> 14
<211> 33
<212> DNA
<213> 人工合成
<400> 14
catcatcatt aatgatgaaa gcttggcgta atc 33
<210> 15
<211> 32
<212> DNA
<213> 人工合成
<400> 15
gaggtataca tggactaggt aagagaggaa tg 32
<210> 16
<211> 38
<212> DNA
<213> 人工合成
<400> 16
caagctctcc tcggatccag ttgctcaaaa aaatctcg 38
<210> 17
<211> 21
<212> DNA
<213> 人工合成
<400> 17
gaggagagct tggacatcgt c 21
<210> 18
<211> 26
<212> DNA
<213> 人工合成
<400> 18
ctagtccatg tatacctctc attatc 26
<210> 19
<211> 28
<212> DNA
<213> 人工合成
<400> 19
gattcaaatg gcaatcaaga aattaatg 28
<210> 20
<211> 19
<212> DNA
<213> 人工合成
<400> 20
gtgtacattc ctctcttac 19
<210> 21
<211> 19
<212> DNA
<213> 人工合成
<400> 21
gtaagagagg aatgtacac 19
<210> 22
<211> 111
<212> DNA
<213> 人工合成
<400> 22
atggatagga gggataatgg aggccaatac atggataagc ggtttgcagt tgtgttagca 60
gctggtcaag gaacaagaat gaaatcaaag ctatataaag ttcttcatcc t 111
<210> 23
<211> 93
<212> DNA
<213> 人工合成
<400> 23
atgaaatttg taaaaagaag gatcattgca cttgtaacaa ttttgatgct gtctgttaca 60
tcgctgtttg cgttgcagcc gtcagcaaaa gcc 93
<210> 24
<211> 84
<212> DNA
<213> 人工合成
<400> 24
atgtttgaaa gtgaagcaga actgagacga atcaggattg cacttgtatg gatagctgtc 60
tttttactgt tcggggcgtg cggg 84
<210> 25
<211> 81
<212> DNA
<213> 人工合成
<400> 25
atgggtttag gtaagaaatt gtctgttgct gtcgctgctt cgtttatgag tttatcaatc 60
agcctgccag gtgttcaggc t 81
<210> 26
<211> 105
<212> DNA
<213> 人工合成
<400> 26
atgagaatac agaaaagacg aacacacgtc gaaaacattc tccgtattct tttgccccca 60
attatgatac ttagcctaat cctcccaaca ccacccattc atgca 105
<210> 27
<211> 72
<212> DNA
<213> 人工合成
<400> 27
atgttgttga aaaggagaat agggttgcta ttaagtatgg ttggcgtatt catgcttttg 60
gctggatgct cg 72
<210> 28
<211> 84
<212> DNA
<213> 人工合成
<400> 28
atgcgcaact tgaccaagac atctctatta ctggccggct tatgcacagc ggcccaaatg 60
gtttttgtaa cacatgcctc agct 84
<210> 29
<211> 72
<212> DNA
<213> 人工合成
<400> 29
atgaagaaac gcagaaagat atgttattgc aatactgccc tgctgcttat gattttgctt 60
gctggatgta cg 72
<210> 30
<211> 7642
<212> DNA
<213> 人工合成
<400> 30
gattcaaatg gcaatcaaga aattaatggc aaagaaaaac tttccgttaa tgattcaaaa 60
ctgaaagatt ttggcaaaac ggttccggtt ggcattgatg aagaaaatgg catgattaaa 120
gtctctttta tgctgacagc acaattttat gagattaaac ctacaaaaga aaatgaacaa 180
tatattggta tgcttagaca agcagttaaa aatgaatcac cggtccatat ttttcttaaa 240
ccgaatagca atgaaattgg caaagttgaa tcagcgagcc ctgaagatgt tagatacttt 300
aaaacaattc tcacgaagga agtgaaaggc cagacgaata aactggccag cgtcattccg 360
gacgtggcga cgctgaatag cctttttaac caaatcaaga atcaaagttg tggaacatca 420
acagcatcaa gcccgtgcat tacatttaga tatccggttg atggctgcta tgcgagagca 480
cataaaatga gacagattct tatgaataat ggctatgatt gcgaaaaaca atttgtctat 540
ggcaatctga aagcaagcac aggcacatgc tgcgtcgcat ggagctatca tgtggcaatt 600
ctggtttcat ataaaaatgc gtcaggcgta acagaaaaac gcattattga tccgtcatta 660
ttttcatcag gcccggtgac agatacggca tggagaaatg cgtgcgtgaa tacaagctgc 720
ggctcagcaa gcgttagcag ctatgcaaat acagcgggaa atgtttatta tcgttcaccg 780
tcaaatagct atctttatga taataatctg attaatacaa attgcgttct gacaaaattt 840
agccttctga gcggctgtag cccgagcccg gcaccggatg tttcatcatg cggctttcat 900
catcatcatc atcattaatg atgaaagctt ggcgtaatca tggtcatagc tgtttcctgt 960
gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa 1020
agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc 1080
tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag 1140
aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt 1200
cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga 1260
atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg 1320
taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa 1380
aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt 1440
tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct 1500
gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct 1560
cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc 1620
cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt 1680
atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc 1740
tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat 1800
ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa 1860
acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa 1920
aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga 1980
aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct 2040
tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga 2100
cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc 2160
catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg 2220
ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat 2280
aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat 2340
ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg 2400
caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc 2460
attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa 2520
agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc 2580
actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt 2640
ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag 2700
ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt 2760
gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag 2820
atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac 2880
cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc 2940
gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca 3000
gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg 3060
ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat 3120
gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga 3180
tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc 3240
ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg 3300
ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga 3360
aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct 3420
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 3480
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 3540
ttgtaaaacg acggccagtg aattccttaa ggaacgtaca gacggcttaa aagcctttaa 3600
aaacgttttt aaggggtttg tagacaaggt aaaggataaa acagcacaat tccaagaaaa 3660
acacgattta gaacctaaaa agaacgaatt tgaactaact cataaccgag aggtaaaaaa 3720
agaacgaagt cgagatcagg gaatgagttt ataaaataaa aaaagcacct gaaaaggtgt 3780
ctttttttga tggttttgaa cttgttcttt cttatcttga tacatataga aataacgtca 3840
tttttatttt agttgctgaa aggtgcgttg aagtgttggt atgtatgtgt tttaaagtat 3900
tgaaaaccct taaaattggt tgcacagaaa aaccccatct gttaaagtta taagtgacta 3960
aacaaataac taaatagatg ggggtttctt ttaatattat gtgtcctaat agtagcattt 4020
attcagatga aaaatcaagg gttttagtgg acaagacaaa aagtggaaaa gtgagaccat 4080
ggagagaaaa gaaaatcgct aatgttgatt actttgaact tctgcatatt cttgaattta 4140
aaaaggctga aagagtaaaa gattgtgctg aaatattaga gtataaacaa aatcgtgaaa 4200
caggcgaaag aaagttgtat cgagtgtggt tttgtaaatc caggctttgt ccaatgtgca 4260
actggaggag agcaatgaaa catggcattc agtcacaaaa ggttgttgct gaagttatta 4320
aacaaaagcc aacagttcgt tggttgtttc tcacattaac agttaaaaat gtttatgatg 4380
gcgaagaatt aaataagagt ttgtcagata tggctcaagg atttcgccga atgatgcaat 4440
ataaaaaaat taataaaaat cttgttggtt ttatgcgtgc aacggaagtg acaataaata 4500
ataaagataa ttcttataat cagcacatgc atgtattggt atgtgtggaa ccaacttatt 4560
ttaagaatac agaaaactac gtgaatcaaa aacaatggat tcaattttgg aaaaaggcaa 4620
tgaaattaga ctatgatcca aatgtaaaag ttcaaatgat tcgaccgaaa aataaatata 4680
aatcggatat acaatcggca attgacgaaa ctgcaaaata tcctgtaaag gatacggatt 4740
ttatgaccga tgatgaagaa aagaatttga aacgtttgtc tgatttggag gaaggtttac 4800
accgtaaaag gttaatctcc tatggtggtt tgttaaaaga aatacataaa aaattaaacc 4860
ttgatgacac agaagaaggc gatttgattc atacagatga tgacgaaaaa gccgatgaag 4920
atggattttc tattattgca atgtggaatt gggaacggaa aaattatttt attaaagagt 4980
agttcaacaa acgggccagt ttgttgaaga ttagatgcta taattgttat taaaaggatt 5040
gaaggatgct taggaagacg agttattaat agctgaataa gaacggtgct ctccaaatat 5100
tcttatttag aaaagcaaat ctaaaattat ctgaaaaggg aatgagaata gtgaatggac 5160
caataataat gactagagaa gaaagaatga agattgttca tgaaattaag gaacgaatat 5220
tggataaata tggggatgat gttaaggcta ttggtgttta tggctctctt ggtcgtcaga 5280
ctgatgggcc ctattcggat attgagatga tgtgtgtcat gtcaacagag gaagcagagt 5340
tcagccatga atggacaacc ggtgagtgga aggtggaagt gaattttgat agcgaagaga 5400
ttctactaga ttatgcatct caggtggaat cagattggcc gcttacacat ggtcaatttt 5460
tctctatttt gccgatttat gattcaggtg gatacttaga gaaagtgtat caaactgcta 5520
aatcggtaga agcccaaacg ttccacgatg cgatttgtgc ccttatcgta gaagagctgt 5580
ttgaatatgc aggcaaatgg cgtaatattc gtgtgcaagg accgacaaca tttctaccat 5640
ccttgactgt acaggtagca atggcaggtg ccatgttgat tggtctgcat catcgcatct 5700
gttatacgac gagcgcttcg gtcttaactg aagcagttaa gcaatcagat cttccttcag 5760
gttatgacca tctgtgccag ttcgtaatgt ctggtcaact ttccgactct gagaaacttc 5820
tggaatcgct agagaatttc tggaatggga ttcaggagtg gacagaacga cacggatata 5880
tagtggatgt gtcaaaacgc ataccatttt gaacgatgac ctctaataat tgttaatcat 5940
gttggttacg tatttattaa cttctcctag tattagtaat tatcatggct gtcatggcgc 6000
attaacggaa taaagggtgt gcttaaatcg ggccattttg cgtaataaga aaaaggatta 6060
attatgagcg aattgaatta ataataaggt aatagattta cattagaaaa tgaaagggga 6120
ttttatgcgt gagaatgtta cagtctatcc cggcattgcc agtcggggat attaaaaaga 6180
gtataggttt ttattgggat aaagtaggtt tcactttggt tcaccatgaa gatggattcg 6240
cagttctaat gtgtaatgag gttcggattc atctatggga ggcaagtgat gaaggctggc 6300
gcctcgtagt aatgattcac cggtttgtac aggtgcggag tcgtttattg ctggtactgc 6360
tagttgccgc attgaagtag agggaattga tgaattatat caacatatta agcctttggg 6420
cattttgcac cccaatacat cattaaaaga tcagtggtgg gatgaacgag actttgcagt 6480
aattgatccc gacaacaatt tgattagctt ttttcaacaa ataaaaagct aaaatctatt 6540
attaatctgt tcagcaatcg ggcgcgattg ctgaataaaa gatacgagag acctctcttg 6600
tatctttttt attttgagtg gttttgtccg ttacactaga aaaccgaaag acaataaaaa 6660
ttttattctt gctgagtctg gctttcggta agctagacaa aacggacaaa ataaaaattg 6720
gcaagggttt aaaggtggag attttttgag tgatcttctc aaaaaatact acctgtccct 6780
tgctgatttt taaacgagca cgagagcaaa accccccttt gctgaggtgg cagagggcag 6840
gtttttttgt ttcttttttc tcgtaaaaaa aagaaaggtc ttaaaggttt tatggttttg 6900
gtcggcactg ccgcgcctcg cagagcacac actttatgaa tataaagtat agtgtgttat 6960
actttacttg gaagtggttg ccggaaagag cgaaaatgcc tcacatttgt gccacctaaa 7020
aaggagcgat ttacatatga gttatgcagt ttgtagaatg caaaaagtga aatcagctgg 7080
actaaaaggc atgcaatttc ataatcaaag agagcgaaaa agtagaacga atgatgatat 7140
tgaccatgag cgaacacgtg aaaattatga tttgaaaaat gataaaaata ttgattacaa 7200
cgaacgtgtc aaagaaatta ttgaatcaca aaaaacaggt acaagaaaaa cgaggaaaga 7260
tgctgttctt gtaaatgagt tgctagtaac atctgaccga gatttttttg agcaactgga 7320
tccgaggaga gcttggacat cgtccgtcag aaacgcttta atttaaagcc gatggatagt 7380
gaagaagcga tcttgcaaat gaatatgctc ggccataatt tctttgtttt cacaaatgcg 7440
gaaacaaacc ttacaaatgt cgtgtaccgc agaaatgacg ggaaatatgg cttaattgaa 7500
ccgactgaat aatgaagaga agccttccgt gatgtccgcg gaaggttttt gtttttctta 7560
tttgcaaatt ctttggaaat aacaaaaggt atgatatgat aatgagaggt atacatggac 7620
taggtaagag aggaatgtac ac 7642
<210> 31
<211> 18
<212> DNA
<213> 人工合成
<400> 31
catcatcatc atcatcat 18
<210> 32
<211> 19
<212> DNA
<213> 人工合成
<400> 32
gtaagagagg aatgtacac 19
<210> 33
<211> 28
<212> DNA
<213> 人工合成
<400> 33
gattcaaatg gcaatcaaga aattaatg 28

Claims (10)

1.一种提高蛋白质谷氨酰胺酶分泌表达的方法,其特征在于,所述方法包括:以含有SEQ ID NO.8所示氨基酸序列的信号肽引导蛋白质谷氨酰胺酶的分泌表达。
2.根据权利要求1所述的方法,其特征在于,所述蛋白质谷氨酰胺酶的氨基酸序列如GenBank登录号:AB046594.1所示。
3.根据权利要求1所述的方法,其特征在于,所述分泌表达是通过构建重组菌实现。
4.根据权利要求3所述的方法,其特征在于,所述重组菌是以细菌为宿主。
5.一种重组菌,其特征在于,所述重组菌以含有SEQ ID NO.8所示氨基酸序列的信号肽引导蛋白质谷氨酰胺酶的分泌表达。
6.根据权利要求5所述的重组菌,其特征在于,所述重组菌是重组枯草芽孢杆菌;所述重组枯草芽孢杆菌的构建方法是:在编码蛋白质谷氨酰胺酶基因的N端融合SEQ ID NO.8所示的信号肽,得到重组基因,并将该重组基因与表达质粒pP43NMK连接所构成的重组质粒转化到大肠杆菌JM109中,抽提质粒转化到枯草芽孢杆菌WB600中。
7.根据权利要求5所述的重组菌,其特征在于,所述重组菌以枯草芽孢杆菌WB600为出发菌株,将SEQ ID NO.9所示基因编码的蛋白质谷氨酰胺酶基因N端融合核苷酸序列为SEQID NO.29所示的信号肽形成的重组基因进行表达。
8.一种生产蛋白质谷氨酰胺酶的方法,其特征在于,是使用权利要求5-7任一所述的重组菌进行发酵生产蛋白质谷氨酰胺酶。
9.权利要求5-7任一所述的重组菌在食品、生物领域的应用。
10.SEQ ID NO.8所示的信号肽在提高蛋白质谷氨酰胺酶分泌表达效果或在蛋白质谷氨酰胺酶生产方面的应用。
CN202110132935.5A 2021-02-01 2021-02-01 一种提高蛋白质谷氨酰胺酶分泌表达的方法 Active CN112680434B (zh)

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