CN113388631B - 一种高产IgG1的重组菌及构建方法 - Google Patents
一种高产IgG1的重组菌及构建方法 Download PDFInfo
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Abstract
本发明公开了一种高产IgG1的重组菌及构建方法，利用合成生物学技术对大肠杆菌氨基酸合成路径中的基因进行过表达，以获得高产IgG1的重组大肠杆菌。相较于以前的研究成果，本发明的重组菌可以提高IgG1的表达量，本发明的方法具有操作简便，方便，易于大肠杆菌规模化生产IgG1抗体的特点。
Description
技术领域
本发明属于生物工程领域，特别是涉及一种IgG1的重组菌及构建方法。
背景技术
单克隆抗体在自身免疫性疾病和肿瘤的治疗中起着重要作用，在医药市场上占据很大份额。2017年，全球十大最畅销药物中有五种是单克隆抗体。目前，美国FDA和欧洲EMEA批准的几十种单克隆抗体药物中，免疫球蛋白G(IgG)占据最重要的地位。因此，提高免疫球蛋白G产量、结构稳定性和功能可靠性的研究引起了人们的广泛关注。
西妥昔单抗(Cetuximab)是FDA批准的表皮生长因子受体(EGFR)抑制剂的嵌合IgG1。西妥昔单抗通过抑制癌细胞增殖、血管再生和转移，并通过促进癌细胞凋亡来增强化学疗法和放射疗法，从而达到抗肿瘤的作用。西妥昔单抗的抗原结合片段通过细胞外表皮生长因子的结构域III结合，从而阻止受体的构象变化并阻断了其信号传导。西妥昔单抗已被广泛证明，与其他药物和放疗具有协同作用。
目前在大肠杆菌中表达IgG1的研究很少。2009年，Hakim等人发表在MAbs期刊上的文献“Inclonals”:IgGs and IgG-enzyme fusion proteins produced in an E.coliexpression-refolding system.公开了提取嵌合IgG轻链和重链的包涵体，并将其重新折叠组装，从而获得全长的嵌合IgG1。2015年Robinson等人发表在Nature Communications期刊上的文献Efficient expression of full-length antibodies in the cytoplasm ofengineered bacteria.公开了大肠杆菌Shuffle菌株可以产生可溶性全长抗体，最终获得1.1mg/L抗PA-63嵌合IgG1。
但是目前缺少大肠杆菌氨基酸合成途径对全长IgG1抗体表达影响的相关研究。
发明内容
本发明的目的是克服现有技术的不足，提供一种高产IgG1的重组菌。
本发明的第二个目的是提供一种高产IgG1的重组菌的构建方法。
本发明的技术方案概述如下：
一种高产IgG1的重组菌的构建方法，包括如下步骤：
(1)基因的体外合成和扩增
从NCBI数据库获得来源于人源的西妥昔单抗轻链的氨基酸序列和西妥昔单抗重链的氨基酸序列，将所述西妥昔单抗轻链简称LC，将所述西妥昔单抗重链简称HC；
将所述LC的氨基酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的LC；在优化后的LC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的LC，在体外合成和扩增；
将所HC的氨基酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的HC，在优化后的HC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的HC，在体外合成和扩增；
所述LC的氨基酸序列如SEQ ID NO.1所示；
所述HC的氨基酸序列如SEQ ID NO.2所示；
所述加酶切位点的LC的核苷酸序列如SEQ ID NO.3所示；
所述加酶切位点的HC的核苷酸序列如SEQ ID NO.4所示；
从NCBI数据库获得来源于大肠杆菌MG1655(Escherichia coli MG1655)的ilvE的核苷酸序列；
将所述ilvE的核苷酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的ilvE；在优化后的ilvE的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的ilvE，在体外合成和扩增；
从NCBI数据库获得来源于大肠杆菌MG1655(Escherichia coli MG1655)的aspC的核苷酸序列；
将所述aspC的核苷酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的aspC；在优化后的aspC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的aspC，在体外合成和扩增；
所述加酶切位点的ilvE的核苷酸序列如SEQ ID NO.5所示；
所述加酶切位点的aspC的核苷酸序列如SEQ ID NO.6所示；
(2)设计引物alaA-F和alaA-R，通过PCR方法从大肠杆菌MG1655(Escherichiacoli MG1655)的基因组DNA扩增出alaA核苷酸序列；设计引物gdhA-F和gdhA-R，通过PCR方法从大肠杆菌MG1655(Escherichia coli MG1655)的基因组DNA扩增出gdhA核苷酸序列；设计引物serB-F和serB-R，通过PCR方法从大肠杆菌MG1655(Escherichia coli MG1655)的基因组DNA扩增出serB核苷酸序列；设计引物pheA-F和pheA-R，通过PCR方法从大肠杆菌MG1655(Escherichia coli MG1655)的基因组DNA扩增出pheA核苷酸序列；alaA-F的核苷酸序列如SEQ ID NO.7所示；alaA-R的核苷酸序列如SEQ ID NO.8所示；gdhA-F的核苷酸序列如SEQ ID NO.9所示；gdhA-R的核苷酸序列如SEQ ID NO.10所示；serB-F的核苷酸序列如SEQ ID NO.11所示；serB-R的核苷酸序列如SEQ ID NO.12所示；pheA-F的核苷酸序列如SEQID NO.13所示；pheA-R的核苷酸序列如SEQ ID NO.14所示；所述alaA的核苷酸序列如SEQID NO.15所示；所述gdhA的核苷酸序列如SEQ ID NO.16所示；所述serB的核苷酸序列如SEQID NO.17所示；所述pheA的核苷酸序列如SEQ ID NO.18所示；
(3)将步骤(1)获得的加酶切位点的LC和加酶切位点的HC分别连接到p2A4UV5载体中，得到p2A4UV5-LC和p2A4UV5-HC；将p2A4UV5-LC用SpeI和PstI限制酶进行酶切，将p2A4UV5-HC用XbaI和PstI限制酶进行酶切，将p2A4UV5-HC酶切的核苷酸片段连接到p2A4UV5-LC酶切的核苷酸片段的3'端，得到p2A4UV5-AB；
将步骤(1)获得的所述加酶切位点的ilvE的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-IlvE；
将步骤(1)获得的所述加酶切位点的aspC的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-AspC；
将步骤(2)获得的所述alaA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-AlaA；
将步骤(2)获得的所述gdhA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-GdhA；
将步骤(2)获得的所述serB的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-SerB；
将步骤(2)获得的所述pheA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-PheA；
所述p2A4UV5载体的核苷酸序列如SEQ ID NO.19所示；所述p3C5BAD载体的核苷酸序列如SEQ ID NO.20所示；
(4)将p2A4UV5-AB和p3C5BAD-IlvE导入大肠杆菌中，得到重组菌2；将p2A4UV5-AB和p3C5BAD-AspC导入大肠杆菌中，得到重组菌3；将p2A4UV5-AB和p3C5BAD-AlaA导入大肠杆菌中，得到重组菌4；将p2A4UV5-AB和p3C5BAD-GdhA导入大肠杆菌中，得到重组菌5；将p2A4UV5-AB和p3C5BAD-SerB导入大肠杆菌中，得到重组菌6；将p2A4UV5-AB和p3C5BAD-PheA导入大肠杆菌中，得到重组菌7。
上述方法构建的一种高产IgG1的重组菌。
本发明的优点：
本发明的重组菌可以提高IgG1的表达量；本发明的重组菌的构建方法可以快速筛选出高表达IgG1的重组菌。
附图说明
图1为重组菌的IgG1表达量。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1
一种高产IgG1的重组菌的构建方法，包括如下步骤：
(1)基因的体外合成和扩增
从NCBI数据库获得来源于人源的西妥昔单抗轻链的氨基酸序列和西妥昔单抗重链的氨基酸序列，将西妥昔单抗轻链简称LC，将西妥昔单抗重链简称HC；
将LC的氨基酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的LC；在优化后的LC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的LC，在体外合成和扩增；
将HC的氨基酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的HC，在优化后的HC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的HC，在体外合成和扩增；
LC的氨基酸序列如SEQ ID NO.1所示；
HC的氨基酸序列如SEQ ID NO.2所示；
加酶切位点的LC的核苷酸序列如SEQ ID NO.3所示；
加酶切位点的HC的核苷酸序列如SEQ ID NO.4所示；
从NCBI数据库获得来源于大肠杆菌MG1655(Escherichia coli MG1655)的ilvE的核苷酸序列；
将ilvE的核苷酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的ilvE；在优化后的ilvE的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的ilvE，在体外合成和扩增；
从NCBI数据库获得来源于大肠杆菌MG1655(Escherichia coli MG1655)的aspC的核苷酸序列；
将aspC的核苷酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的aspC；在优化后的aspC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的aspC，在体外合成和扩增；
加酶切位点的ilvE的核苷酸序列如SEQ ID NO.5所示；
加酶切位点的aspC的核苷酸序列如SEQ ID NO.6所示；
(2)设计引物alaA-F和alaA-R，通过PCR方法从大肠杆菌MG1655(Escherichiacoli MG1655)的基因组DNA扩增出alaA核苷酸序列；设计引物gdhA-F和gdhA-R，通过PCR方法从大肠杆菌MG1655(Escherichia coli MG1655)的基因组DNA扩增出gdhA核苷酸序列；设计引物serB-F和serB-R，通过PCR方法从大肠杆菌MG1655(Escherichia coli MG1655)的基因组DNA扩增出serB核苷酸序列；设计引物pheA-F和pheA-R，通过PCR方法从大肠杆菌MG1655(Escherichia coli MG1655)的基因组DNA扩增出pheA核苷酸序列；alaA-F的核苷酸序列如SEQ ID NO.7所示；alaA-R的核苷酸序列如SEQ ID NO.8所示；gdhA-F的核苷酸序列如SEQ ID NO.9所示；gdhA-R的核苷酸序列如SEQ ID NO.10所示；serB-F的核苷酸序列如SEQ ID NO.11所示；serB-R的核苷酸序列如SEQ ID NO.12所示；pheA-F的核苷酸序列如SEQID NO.13所示；pheA-R的核苷酸序列如SEQ ID NO.14所示；alaA的核苷酸序列如SEQ IDNO.15所示；gdhA的核苷酸序列如SEQ ID NO.16所示；serB的核苷酸序列如SEQ ID NO.17所示；pheA的核苷酸序列如SEQ ID NO.18所示；
(3)将步骤(1)获得的加酶切位点的LC和加酶切位点的HC分别连接到p2A4UV5载体中，得到p2A4UV5-LC和p2A4UV5-HC；将p2A4UV5-LC用SpeI和PstI限制酶进行酶切，将p2A4UV5-HC用XbaI和PstI限制酶进行酶切，将p2A4UV5-HC酶切的核苷酸片段连接到p2A4UV5-LC酶切的核苷酸片段的3'端，得到p2A4UV5-AB；
将步骤(1)获得的所述加酶切位点的ilvE的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-IlvE；
将步骤(1)获得的所述加酶切位点的aspC的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-AspC；
将步骤(2)获得的所述alaA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-AlaA；
将步骤(2)获得的所述gdhA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-GdhA；
将步骤(2)获得的所述serB的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-SerB；
将步骤(2)获得的所述pheA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-PheA；
所述p2A4UV5载体的核苷酸序列如SEQ ID NO.19所示；
所述p3C5BAD载体的核苷酸序列如SEQ ID NO.20所示；
(4)将p2A4UV5-AB和p3C5BAD导入大肠杆菌中，得到重组菌1(对照)；将p2A4UV5-AB和p3C5BAD-IlvE导入大肠杆菌中，得到重组菌2；将p2A4UV5-AB和p3C5BAD-AspC导入大肠杆菌中，得到重组菌3；将p2A4UV5-AB和p3C5BAD-AlaA导入大肠杆菌中，得到重组菌4；将p2A4UV5-AB和p3C5BAD-GdhA导入大肠杆菌中，得到重组菌5；将p2A4UV5-AB和p3C5BAD-SerB导入大肠杆菌中，得到重组菌6；将p2A4UV5-AB和p3C5BAD-PheA导入大肠杆菌中，得到重组菌7。
实施例2
实施例1制备的重组菌1、重组菌2、重组菌3、重组菌4、重组菌5、重组菌6、重组菌7发酵：
(1)将重组菌1(对照)、重组菌2、重组菌3、重组菌4、重组菌5、重组菌6、重组菌7分别发酵，得到发酵液WT、ilvE、aspC、alaA、gdhA、serB、pheA；
发酵过程：将重组菌1(对照)、重组菌2、重组菌3、重组菌4、重组菌5、重组菌6、重组菌7，分别在含有(100μg/ml氨苄青霉素和34μg/ml氯霉素)的改进的M9培养基中培养，37℃和220rpm下培养16小时；将培养物以1:100比例转接入含有50mL改进的M9培养基的250mL摇瓶中，并在37℃下生长。当600nm处的光密度(OD600)达到约0.6时，加入异丙基硫代半乳糖苷(IPTG)和阿拉伯糖(L-arabinose)，使其终浓度分别为1mM和0.2mM，并在25℃和200rpm下生长16小时；
改进的M9培养基：6.8g/LNa2HPO4，3g/LKH2PO4，0.5g/LNaCl，2g/LNH4Cl，0.25g/LMgSO4·7H2O，11.1mg/LCaCl2，10mg/L硫胺素，2g/L酵母提取，3％(v/v)甘油和1mL/L的金属痕量储备液；
金属痕量储备液：27g/LFeCl3·6H2O，2g/LZnCl2，2g/LNa2MoO4·2H2O，1.9g/LCuSO4·5H2O，0.5g/LH3BO3和100mL/L浓HCl；通过Tris将pH调节至7.25。
(2)粗蛋白液的提取；
将得到的WT、ilvE、aspC、alaA、gdhA、serB、pheA发酵液在4℃和6500rpm下离心收集沉淀。细胞用0.01M PBS(pH＝7.0)洗涤。WT、ilvE、aspC、alaA、gdhA、serB、pheA沉淀用0.01MPBS(pH＝7.0)重悬细胞并进行超声破碎处理，5s脉冲，10s间歇，总工作时间为20分钟。然后，将细胞裂解物在10,000rpm和4℃下离心10分钟，收集上清液。然后使用0.45μm滤膜进行过滤。得到粗蛋白液WT、ilvE、aspC、alaA、gdhA、serB、pheA。
(3)ELISA(酶联免疫吸附剂测定，采用Matriks Biotek Cetuximab ElisaSHIKARI Q-CET试剂盒)；
第一步：96孔板，每孔加入100μlAssayBuffer，分别将10μl步骤(2)获得的粗蛋白液放入孔中，3个平行；再将10μl浓度分别是0μg/ml、10μg/ml、30μg/ml、100μg/ml、300μg/ml西妥昔单抗标准品加入孔中，3个平行；轻轻摇板进行简单混合，用封板膜封住反应孔，室温下孵化30min。
第二步：揭掉封板膜，吸弃孔内液体。用300μl稀释的WashBuffer洗涤3次，在干净的纸上拍干。
第三步：每孔加入100μl准备好的HRPConjugate。
第四步：用新的封板膜密封。室温孵化30min。
第五步：重复第二步。
第六步：每孔加入100μlTMBSubstrateSolution。
第七步：黑暗条件下室温孵化10min(不封膜)。
第八步：加入100μlStopSolution停止反应。轻轻摇板进行简单混合，孔内颜色由蓝变黄。
第九步：以空白孔调零，30min内使用酶标仪在450nm测定每孔的吸光度，见图1。
序列表
<110> 天津大学
<120> 一种高产IgG1的重组菌及构建方法
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 237
<212> PRT
<213> 智人(Homo sapiens)
<400> 1
Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe Ser
1 5 10 15
Ile Ala Thr Asn Ala Tyr Ala Asp Ile Leu Leu Thr Gln Ser Pro Val
20 25 30
Ile Leu Ser Val Ser Pro Gly Glu Arg Val Ser Phe Ser Cys Arg Ala
35 40 45
Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Arg Thr Asn
50 55 60
Gly Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser Gly
65 70 75 80
Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
85 90 95
Ser Ile Asn Ser Val Glu Ser Glu Asp Ile Ala Asp Tyr Tyr Cys Gln
100 105 110
Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Ala Gly Thr Lys Leu Glu
115 120 125
Leu Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
130 135 140
Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
145 150 155 160
Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
165 170 175
Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
180 185 190
Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
195 200 205
Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
210 215 220
Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
225 230 235
<210> 2
<211> 471
<212> PRT
<213> 智人(Homo sapiens)
<400> 2
Met Lys Lys Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe Ser
1 5 10 15
Ile Ala Thr Asn Ala Tyr Ala Gln Val Gln Leu Lys Gln Ser Gly Pro
20 25 30
Gly Leu Val Gln Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr Val Ser
35 40 45
Gly Phe Ser Leu Thr Asn Tyr Gly Val His Trp Val Arg Gln Ser Pro
50 55 60
Gly Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr
65 70 75 80
Asp Tyr Asn Thr Pro Phe Thr Ser Arg Leu Ser Ile Asn Lys Asp Asn
85 90 95
Ser Lys Ser Gln Val Phe Phe Lys Met Asn Ser Leu Gln Ser Asn Asp
100 105 110
Thr Ala Ile Tyr Tyr Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu
115 120 125
Phe Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Ser
130 135 140
Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
145 150 155 160
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
165 170 175
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
195 200 205
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
210 215 220
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val
225 230 235 240
Glu Pro Lys Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
245 250 255
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
260 265 270
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
275 280 285
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
290 295 300
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
305 310 315 320
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
325 330 335
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
340 345 350
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
355 360 365
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
370 375 380
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
385 390 395 400
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
405 410 415
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
420 425 430
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
435 440 445
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
450 455 460
Leu Ser Leu Ser Pro Gly Lys
465 470
<210> 3
<211> 722
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
catatgaaaa agaacatagc gtttcttctt gcatctatgt tcgttttttc tattgctaca 60
aacgcgtatg cagacatcct gctgacccag tctccggtta tcctgtctgt ttctccgggt 120
gaacgtgttt ctttctcttg ccgtgcttct cagtctatcg gtaccaacat ccactggtac 180
cagcagcgta ccaacggttc tccgcgtctg ctgatcaaat acgcttctga atctatctct 240
ggtatcccgt ctcgtttctc tggttctggt tctggtaccg acttcaccct gtctatcaac 300
tctgttgaat ctgaagacat cgctgactac tactgccagc agaacaacaa ctggccgacc 360
accttcggtg ctggtaccaa actggaactg aaacgtaccg ttgctgctcc gtctgttttc 420
atcttcccgc cgtctgacga acagctgaaa tctggtaccg cttctgttgt ttgcctgctg 480
aacaacttct acccgcgtga agctaaagtt cagtggaaag ttgacaacgc tctgcagtct 540
ggtaactctc aggaatctgt taccgaacag gactctaaag actctaccta ctctctgtct 600
tctaccctga ccctgtctaa agctgactac gaaaaacaca aagtttacgc ttgcgaagtt 660
acccaccagg gtctgtcttc tccggttacc aaatctttca accgtggtga atgctaacta 720
gt 722
<210> 4
<211> 1424
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
catatgaaaa agaacatagc gtttcttctt gcatctatgt tcgttttttc tattgctaca 60
aacgcgtatg cacaggttca gctgaaacag tctggtccgg gtctggttca gccgtctcag 120
tctctgtcta tcacctgcac cgtttctggt ttctctctga ccaactacgg tgttcactgg 180
gttcgtcagt ctccgggtaa aggtctggaa tggctgggtg ttatctggtc tggtggtaac 240
accgactaca acaccccgtt cacctctcgt ctgtctatca acaaagacaa ctctaaatct 300
caggttttct tcaaaatgaa ctctctgcag tctaacgaca ccgctatcta ctactgcgct 360
cgtgctctga cctactacga ctacgaattt gcttactggg gtcagggtac tctcgttacc 420
gtaagcgctg cttctaccaa aggtccgtct gttttcccgc tggctccgtc ttctaaatct 480
acctctggtg gtaccgctgc tctgggttgc ctggttaaag actacttccc ggaaccggtt 540
accgtttctt ggaactctgg tgctctgacc tctggtgttc acaccttccc ggctgttctg 600
cagtcttctg gtctgtactc tctgtcttct gttgttaccg ttccgtcttc ttctctgggt 660
acccagacct acatctgcaa cgttaaccac aaaccgtcta acaccaaagt tgacaaacgt 720
gttgaaccga aatctgacaa aacccacacc tgcccgccgt gcccggctcc ggaactgctg 780
ggtggtccgt ctgttttcct gttcccgccg aaaccgaaag acaccctgat gatctctcgt 840
accccggaag ttacctgcgt tgttgttgac gtttctcacg aagacccgga agttaaattc 900
aactggtacg ttgacggtgt tgaagttcac aacgctaaaa ccaaaccgcg tgaagaacag 960
tacaactcta cctaccgtgt tgtttctgtt ctgaccgttc tgcaccagga ctggctgaac 1020
ggtaaagaat acaaatgcaa agtttctaac aaagctctgc cggctccgat cgaaaaaacc 1080
atctctaaag ctaaaggtca gccgagggag ccgcaggtat acaccctccc gccgtctcgt 1140
gacgaactga ccaaaaacca ggtttctctg acctgcctgg ttaaaggttt ctacccgtct 1200
gacatcgctg ttgaatggga atctaacggt cagccggaaa acaactacaa aaccaccccg 1260
ccggttctgg actctgacgg ttctttcttc ctgtactcta aactgaccgt tgacaaatct 1320
cgttggcagc agggtaacgt tttctcttgc tctgttatgc acgaagctct gcacaaccac 1380
tacacccaga aatctctgtc tctgtctccg ggtaaataac tagt 1424
<210> 5
<211> 941
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
catatgacca ccaaaaaagc tgactacatc tggttcaacg gtgaaatggt tcgttgggaa 60
gacgctaaag ttcacgttat gtctcacgct ctgcactacg gtacctctgt tttcgaaggt 120
atccgttgct acgactctca caaaggtccg gttgttttcc gtcaccgtga acacatgcag 180
cgtctgcacg actctgctaa aatctacaga tttccggtaa gccagtctat cgacgaactg 240
atggaagcgt gccgtgacgt tatccgtaaa aacaacctga cctctgctta catccgtccg 300
ctgatcttcg ttggtgacgt tggtatgggt gttaacccgc cggctggtta ctctaccgac 360
gttataatag ctgcgttccc gtggggtgcg tacctgggtg ctgaagctct ggaacagggt 420
atcgacgcta tggtttcttc ttggaaccgt gctgctccga acaccatccc gaccgctgct 480
aaagctggtg gtaactacct gtcttctctg ctggttggtt ctgaagctcg tcgtcacggt 540
taccaggaag gtatcgctct ggacgttaac ggttacatct ctgaaggtgc tggtgaaaac 600
ctgttcgaag ttaaagacgg tgttctgttc accccgccgt tcacctcttc tgctctgccg 660
ggtatcaccc gtgacgctat catcaaactg gctaaagaac tgggtatcga agttcgtgaa 720
caggttctgt ctcgtgaatc tctgtacctg gctgacgaag ttttcatgtc tggtaccgct 780
gctgaaatca ccccggttcg ttctgttgac ggtatccagg ttggtgaagg tcgttgcggt 840
ccggttacca aacgtatcca gcaggctttc ttcggtctgt tcaccggtga aaccgaagac 900
aaatggggtt ggctggacca ggttaaccag taataactag t 941
<210> 6
<211> 1202
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
catatgttcg aaaacatcac cgctgctccg gctgacccga tcctgggtct ggctgacctg 60
ttccgtgctg acgaacgtcc gggtaaaatc aacctgggta tcggtgttta caaagacgaa 120
accggtaaaa ccccggttct gacctctgtt aaaaaagctg aacagtacct gctggaaaac 180
gaaaccacca aaaactacct gggtatcgac ggtatcccgg aatttggtcg ttgcacccag 240
gaactgctgt tcggtaaagg ttctgctctg atcaacgaca aacgtgctcg taccgctcag 300
accccgggtg gtaccggtgc tctgcgtgtt gctgctgact tcctggctaa aaacacctct 360
gttaaacgtg tttgggtttc taacccgtct tggccgaacc acaaatctgt tttcaactct 420
gctggtctgg aagttcgtga atacgcttac tacgacgctg aaaaccacac cctggacttc 480
gacgctctga tcaactctct gaacgaagct caggctggtg acgttgttct gttccacggt 540
tgctgccaca acccgaccgg tatcgacccg accctggaac agtggcagac cctggctcag 600
ctgtctgttg aaaaaggttg gctgccgctg ttcgacttcg cttaccaggg tttcgctcgt 660
ggtctggaag aagacgctga aggtctccgt gcgttcgctg ctatgcataa agaactgatc 720
gttgcttctt cttactctaa aaacttcggt ctgtacaacg aacgtgttgg tgcttgcacc 780
ctggttgctg ctgactctga aaccgttgac cgtgctttct ctcagatgaa agctgctatc 840
cgtgctaact actctaaccc gccggctcac ggtgcttctg ttgttgctac catcctgtct 900
aacgacgctc tgcgtgctat ctgggaacag gaactgaccg acatgcgtca gcgtatccag 960
cgtatgcgtc agctgttcgt taacaccctg caagaaaaag gtgctaaccg tgacttctct 1020
ttcatcatca aacagaatgg catgttcagc ttctctggtc tgactaaaga acaggttctg 1080
cgtctgcgtg aagaatttgg tgtttacgct gttgcttctg gtcgtgttaa cgttgctggt 1140
atgaccccgg acaacatggc tccgctgtgc gaagctatcg ttgctgttct gtaataacta 1200
gt 1202
<210> 7
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggaattccat atgtccccca ttgaaa 26
<210> 8
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggactagtta cagctgatga taaccagaaa gg 32
<210> 9
<211> 31
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggaattccat atggatcaga catattctct g 31
<210> 10
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ggactagtta aatcacaccc tgcgccagca tc 32
<210> 11
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggaattccat atgcctaaca ttacctg 27
<210> 12
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggactagtta cttctgattc aggctgcc 28
<210> 13
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggaattccat atgacatcgg aaaacccg 28
<210> 14
<211> 32
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ggactagtca ggttggatca acaggcacta cg 32
<210> 15
<211> 1226
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 15
catatgtccc ccattgaaaa atccagcaaa ttagagaatg tctgttatga catccgtggt 60
ccggtgctga aagaagcaaa acgcctggaa gaagaaggta acaaggtact gaaactgaac 120
atcggcaacc cagccccgtt cggttttgac gcgccagatg aaatcctcgt tgacgtgata 180
cgcaacctgc ctaccgctca agggtattgc gattccaaag gtctttactc cgcgcgtaaa 240
gccatcatgc agcactacca ggctcgtggc atgcgtgatg ttaccgtgga agatatttac 300
atcggcaatg gtgtatcgga gcttatcgtt caggcaatgc aggcattgct gaacagcggg 360
gacgaaatgt tggttcctgc accagattac ccactctgga ccgcggcggt ttcgctttcc 420
agcggtaaag cggtgcatta tctttgcgat gaatcctctg actggttccc ggacctcgat 480
gatattcgcg ctaaaattac gcctcgtacg cgtgggatcg ttattatcaa cccaaataac 540
ccaaccggcg cggtatattc caaagagctt ttaatggaga ttgtggagat tgcacgtcag 600
cataatctca ttatcttcgc cgatgaaatt tatgacaaaa ttctctacga cgacgctgag 660
catcactcaa ttgcgccgct ggcacctgac ctgctgacca ttacctttaa cggactgtcg 720
aaaacgtacc gcgttgcagg cttccgtcag gggtggatgg tgttgaacgg gccgaaaaaa 780
cacgccaaag gctacatcga aggtctggaa atgctggctt caatgcgcct gtgtgctaac 840
gttcctgcgc aacacgccat tcagaccgcg ctaggtggtt atcagagcat cagtgaattt 900
attacccctg gcggtcgtct ttatgagcag cgtaaccgcg cgtgggaact gatcaacgat 960
attccgggcg tttcctgcgt gaaacctcgt ggtgcgctgt atatgttccc gaaaatcgac 1020
gccaaacgct ttaacattca cgacgatcag aaaatggtgt tggatttcct gttgcaggaa 1080
aaagttctgt tggtgcaagg gacggcattc aactggccgt ggccggatca cttccgcatt 1140
gtcacgctac cgcgtgtcga tgatatcgag ctgtctttga gcaagttcgc gcgtttcctt 1200
tctggttatc atcagctgta actagt 1226
<210> 16
<211> 1352
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 16
catatggatc agacatattc tctggagtca ttcctcaacc atgtccaaaa gcgcgacccg 60
aatcaaaccg agttcgcgca agccgttcgt gaagtaatga ccacactctg gccttttctt 120
gaacaaaatc caaaatatcg ccagatgtca ttactggagc gtctggttga accggagcgc 180
gtgatccagt ttcgcgtggt atgggttgat gatcgcaacc agatacaggt caaccgtgca 240
tggcgtgtgc agttcagctc tgccatcggc ccgtacaaag gcggtatgcg cttccatccg 300
tcagttaacc tttccattct caaattcctc ggctttgaac aaaccttcaa aaatgccctg 360
actactctgc cgatgggcgg tggtaaaggc ggcagcgatt tcgatccgaa aggaaaaagc 420
gaaggtgaag tgatgcgttt ttgccaggcg ctgatgactg aactgtatcg ccacctgggc 480
gcggataccg acgttccggc aggtgatatc ggggttggtg gtcgtgaagt cggctttatg 540
gcggggatga tgaaaaagct ctccaacaat accgcctgcg tcttcaccgg taagggcctt 600
tcatttggcg gcagtcttat tcgcccggaa gctaccggct acggtctggt ttatttcaca 660
gaagcaatgc taaaacgcca cggtatgggt tttgaaggga tgcgcgtttc cgtttctggc 720
tccggcaacg tcgcccagta cgctatcgaa aaagcgatgg aatttggtgc tcgtgtgatc 780
actgcgtcag actccagcgg cactgtagtt gatgaaagcg gattcacgaa agagaaactg 840
gcacgtctta tcgaaatcaa agccagccgc gatggtcgag tggcagatta cgccaaagaa 900
tttggtctgg tctatctcga aggccaacag ccgtggtctc taccggttga tatcgccctg 960
ccttgcgcca cccagaatga actggatgtt gacgccgcgc atcagcttat cgctaatggc 1020
gttaaagccg tcgccgaagg ggcaaatatg ccgaccacca tcgaagcgac tgaactgttc 1080
cagcaggcag gcgtactatt tgcaccgggt aaagcggcta atgctggtgg cgtcgctaca 1140
tcgggcctgg aaatggcaca aaacgctgcg cgcctgggct ggaaagccga gaaagttgac 1200
gcacgtttgc atcacatcat gctggatatc caccatgcct gtgttgagca tggtggtgaa 1260
ggtgagcaaa ccaactacgt gcagggcgcg aacattgccg gttttgtgaa ggttgccgat 1320
gcgatgctgg cgcagggtgt gatttaacta gt 1352
<210> 17
<211> 977
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 17
catatgccta acattacctg gtgcgacctg cctgaagatg tctctttatg gccgggtctg 60
cctctttcat taagtggtga tgaagtgatg ccactggatt accacgcagg tcgtagcggc 120
tggctgctgt atggtcgtgg gctggataaa caacgtctga cccaatacca gagcaaactg 180
ggtgcggcga tggtgattgt tgccgcctgg tgcgtggaag attatcaggt gattcgtctg 240
gcaggttcac tcaccgcacg ggctacacgc ctggcccacg aagcgcagct ggatgtcgcc 300
ccgctgggga aaatcccgca cctgcgcacg ccgggtttgc tggtgatgga tatggactcc 360
accgccatcc agattgaatg tattgatgaa attgccaaac tggccggaac gggcgagatg 420
gtggcggaag taaccgaacg ggcgatgcgc ggcgaactcg attttaccgc cagcctgcgc 480
agccgtgtgg cgacgctgaa aggcgctgac gccaatattc tgcaacaggt gcgtgaaaat 540
ctgccgctga tgccaggctt aacgcaactg gtgctcaagc tggaaacgct gggctggaaa 600
gtggcgattg cctccggcgg ctttactttc tttgctgaat acctgcgcga caagctgcgc 660
ctgaccgccg tggtagccaa tgaactggag atcatggacg gtaaatttac cggcaatgtg 720
atcggcgaca tcgtagacgc gcagtacaaa gcgaaaactc tgactcgcct cgcgcaggag 780
tatgaaatcc cgctggcgca gaccgtggcg attggcgatg gagccaatga cctgccgatg 840
atcaaagcgg cagggctggg gattgcctac catgccaagc caaaagtgaa tgaaaaggcg 900
gaagtcacca tccgtcacgc tgacctgatg ggggtattct gcatcctctc aggcagcctg 960
aatcagaagt aactagt 977
<210> 18
<211> 1169
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 18
catatgacat cggaaaaccc gttactggcg ctgcgagaga aaatcagcgc gctggatgaa 60
aaattattag cgttactggc agaacggcgc gaactggccg tcgaggtggg aaaagccaaa 120
ctgctctcgc atcgcccggt acgtgatatt gatcgtgaac gcgatttgct ggaaagatta 180
attacgctcg gtaaagcgca ccatctggac gcccattaca ttactcgcct gttccagctc 240
atcattgaag attccgtatt aactcagcag gctttgctcc aacaacatct caataaaatt 300
aatccgcact cagcacgcat cgcttttctc ggccccaaag gttcttattc ccatcttgcg 360
gcgcgccagt atgctgcccg tcactttgag caattcattg aaagtggctg cgccaaattt 420
gccgatattt ttaatcaggt ggaaaccggc caggccgact atgccgtcgt accgattgaa 480
aataccagct ccggtgccat aaacgacgtt tacgatctgc tgcaacatac cagcttgtcg 540
attgttggcg agatgacgtt aactatcgac cattgtttgt tggtctccgg cactactgat 600
ttatccacca tcaatacggt ctacagccat ccgcagccat tccagcaatg cagcaaattc 660
cttaatcgtt atccgcactg gaagattgaa tataccgaaa gtacgtctgc ggcaatggaa 720
aaggttgcac aggcaaaatc accgcatgtt gctgcgttgg gaagcgaagc tggcggcact 780
ttgtacggtt tgcaggtact ggagcgtatt gaagcaaatc agcgacaaaa cttcacccga 840
tttgtggtgt tggcgcgtaa agccattaac gtgtctgatc aggttccggc gaaaaccacg 900
ttgttaatgg cgaccgggca acaagccggt gcgctggttg aagcgttgct ggtactgcgc 960
aaccacaatc tgattatgac ccgtctggaa tcacgcccga ttcacggtaa tccatgggaa 1020
gagatgttct atctggatat tcaggccaat cttgaatcag cggaaatgca aaaagcattg 1080
aaagagttag gggaaatcac ccgttcaatg aaggtattgg gctgttaccc aagtgagaac 1140
gtagtgcctg ttgatccaac ctgactagt 1169
<210> 19
<211> 3544
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
ggatccttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 60
gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 120
tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca 180
ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 240
ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 300
ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 360
aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 420
cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 480
gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 540
ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaagct 600
tgaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 660
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 720
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 780
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 840
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 900
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 960
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 1020
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 1080
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 1140
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 1200
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 1260
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 1320
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 1380
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 1440
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 1500
ataacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 1560
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 1620
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 1680
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 1740
atttccccga aaagtgccac ctgacgtcgc gttgcgctca ctgcccgctt tccagtcggg 1800
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 1860
tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc tgattgccct 1920
tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc 1980
gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct tcggtatcgt 2040
cgtatcccac taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca 2100
ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat 2160
tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg 2220
ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg 2280
ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca 2340
gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg ttgatgggtg 2400
tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa 2460
tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa 2520
gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca 2580
cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt 2640
gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt 2700
gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg 2760
ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac 2820
cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc ctgaattgac 2880
tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggtgtccg 2940
ggatctattt cagataaaaa aaatccttag ctttcgctaa ggatgatttc tggaattcgc 3000
cgctcctagg ccgatggcgc gccgagaggc tttacacttt atgcttccgg ctcgtataat 3060
gtgtggaatt gtgagcggat aacaatttct agaaataatt ttgtttaact ttaagaagga 3120
gatatacata tgtaactagt gactcgagcc aggcatcaaa taaaacgaaa ggctcagtcg 3180
aaagactggg cctttcgttt tatctgtttt tgtcggtgaa cgctctctac tagagtcaca 3240
ctggctcacc ttcgggtggg cctttctgcg tttatagcta gcgcctctgc agtccggcaa 3300
aaaagggcaa ggtgtcacca ccctgccctt tttctttaaa accgaaaaga ttacttcgcg 3360
ttatgcaggc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 3420
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 3480
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 3540
cgtt 3544
<210> 20
<211> 4191
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tgccacctga cgtctaagaa aaggaatatt cagcaatttg cccgtgccga agaaaggccc 60
acccgtgaag gtgagccagt gagttgattg ctacgtaatt agttagttag cccttagtga 120
ctcgaattcc ctgtcaaatg gacgaagcag ggattctgca aaccctatgc tactccgtca 180
agccgtcaat tgtctgattc gttaccaatt atgacaactt gacggctaca tcattcactt 240
tttcttcaca accggcacgg aactcgctcg ggctggcccc ggtgcatttt ttaaataccc 300
gcgagaaata gagttgatcg tcaaaaccaa cattgcgacc gacggtggcg ataggcatcc 360
gggtggtgct caaaagcagc ttcgcctggc tgatacgttg gtcctcgcgc cagcttaaga 420
cgctaatccc taactgctgg cggaaaagat gtgacagacg cgacggcgac aagcaaacat 480
gctgtgcgac gctggcgata tcaaaattgc tgtctgccag gtgatcgctg atgtactgac 540
aagcctcgcg tacccgatta tccatcggtg gatggagcga ctcgttaatc gcttccatgc 600
gccgcagtaa caattgctca agcagattta tcgccagcag ctccgaatag cgcccttccc 660
cttgcccggc gttaatgatt tgcccaaaca ggtcgctgaa atgcggctgg tgcgcttcat 720
ccgggcgaaa gaaccccgta ttggcaaata ttgacggcca gttaagccat tcatgccagt 780
aggcgcgcgg acgaaagtaa acccactggt gataccattc gcgagcctcc ggatgacgac 840
cgtagtgatg aatctctcct ggcgggaaca gcaaaatatc acccggtcgg caaacaaatt 900
ctcgtccctg atttttcacc accccctgac cgcgaatggt gagattgaga atataacctt 960
tcattcccag cggtcggtcg ataaaaaaat cgagataacc gttggcctca atcggcgtta 1020
aacccgccac cagatgggca ttaaacgagt atcccggcag caggggatca ttttgcgctt 1080
cagccatact tttcatactc ccgccattca gagaagaaac caattgtcca tattgcatca 1140
gacattgccg tcactgcgtc ttttactggc tcttctcgct aaccaaaccg gtaaccccgc 1200
ttattaaaag cattctgtaa caaagcggga ccaaagccat gacaaaaacg cgtaacaaaa 1260
gtgtctataa tcacggcaga aaagtccaca ttgattattt gcacggcgtc acactttgct 1320
atgccatagc atttttatcc ataagattag cggatcctac ctgacgcttt ttatcgcaac 1380
tctctactgt ttctccatac ccgttttttt gggctctaga aataattttg tttaacttta 1440
agaaggagat atacatatgt aactagtgac tcgagccagg catcaaataa aacgaaaggc 1500
tcagtcgaaa gactgggcct ttcgttttat ctgtttttgt cggtgaacgc tctctactag 1560
agtcacactg gctcaccttc gggtgggcct ttctgcgttt atagctagcg cctctgcagg 1620
agtcactaag ggttagttag ttagattagc agaaagtcaa aagcctccga ccggaggctt 1680
ttgactaaaa cttcccttgg ggttatcatt ggggctcact caaaggcggt aatcagataa 1740
aaaaaatcct tagctttcgc taaggatgat ttctgctaga gatggaatag actggatgga 1800
ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc 1860
tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga 1920
tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga 1980
acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga 2040
ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat 2100
ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt 2160
ccactgagcg tcagacccct taataagatg atcttcttga gatcgttttg gtctgcgcgt 2220
aatctcttgc tctgaaaacg aaaaaaccgc cttgcagggc ggtttttcga aggttctctg 2280
agctaccaac tctttgaacc gaggtaactg gcttggagga gcgcagtcac caaaacttgt 2340
cctttcagtt tagccttaac cggcgcatga cttcaagact aactcctcta aatcaattac 2400
cagtggctgc tgccagtggt gcttttgcat gtctttccgg gttggactca agacgatagt 2460
taccggataa ggcgcagcgg tcggactgaa cggggggttc gtgcatacag tccagcttgg 2520
agcgaactgc ctacccggaa ctgagtgtca ggcgtggaat gagacaaacg cggccataac 2580
agcggaatga caccggtaaa ccgaaaggca ggaacaggag agcgcacgag ggagccgcca 2640
ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccaccactga tttgagcgtc 2700
agatttcgtg atgcttgtca ggggggcgga gcctatggaa aaacggcttt gccgcggccc 2760
tctcacttcc ctgttaagta tcttcctggc atcttccagg aaatctccgc cccgttcgta 2820
agccatttcc gctcgccgca gtcgaacgac cgagcgtagc gagtcagtga gcgaggaagc 2880
ggaatatatc ctgtatcaca tattctgctg acgcaccggt gcagcctttt ttctcctgcc 2940
acatgaagca cttcactgac accctcatca gtgccaacat agtaagccag tatacactcc 3000
gctagcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag gcctgaatcg 3060
ccccatcatc cagccagaaa gtgagggagc cacggttgat gagagctttg ttgtaggtgg 3120
accagttggt gattttgaac ttttgctttg ccacggaacg gtctgcgttg tcgggaagat 3180
gcgtgatctg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc cacgttgtgt 3240
ctcaaaatct ctgatgttac attgcacaag ataaaaatat atcatcatga acaataaaac 3300
tgtctgctta cataaacagt aatacaaggg gtgtttacta gaggttgatc gggcacgtaa 3360
gaggttccaa ctttcaccat aatgaaataa gatcactacc gggcgtattt tttgagttat 3420
cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcacggga tataccaccg 3480
ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat 3540
gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa 3600
ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aacgctcacc 3660
cggagtttcg tatggccatg aaagacggtg agctggtgat ctgggatagt gttcaccctt 3720
gttacaccgt tttccatgag caaactgaaa cgttttcgtc cctctggagt gaataccacg 3780
acgatttccg gcagtttctc cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc 3840
tggcctattt ccctaaaggg tttattgaga atatgttttt tgtctcagcc aatccctggg 3900
tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt 3960
tcacgatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgatccagg 4020
ttcatcatgc cgtttgtgat ggcttccatg tcggccgcat gcttaatgaa ttacaacagt 4080
actgtgatga gtggcagggc ggggcgtaat aatactagct ccggcaaaaa aacgggcaag 4140
gtgtcaccac cctgcccttt ttctttaaaa ccgaaaagat tacttcgcgt t 4191
Claims (2)
1.一种高产IgG1的重组菌的构建方法，其特征是包括如下步骤：
(1)基因的体外合成和扩增
从NCBI数据库获得来源于人源的西妥昔单抗轻链的氨基酸序列和西妥昔单抗重链的氨基酸序列，将所述西妥昔单抗轻链简称LC，将所述西妥昔单抗重链简称HC；
将所述LC的氨基酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的LC；在优化后的LC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的LC，在体外合成和扩增；
将所述HC的氨基酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的HC，在优化后的HC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的HC，在体外合成和扩增；
所述LC的氨基酸序列如SEQ ID NO.1所示；
所述HC的氨基酸序列如SEQ ID NO.2所示；
所述加酶切位点的LC的核苷酸序列如SEQ ID NO.3所示；
所述加酶切位点的HC的核苷酸序列如SEQ ID NO.4所示；
从NCBI数据库获得来源于大肠杆菌MG1655(Escherichia coli MG1655)的ilvE的核苷酸序列；
将所述ilvE的核苷酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的ilvE；在优化后的ilvE的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的ilvE，在体外合成和扩增；
从NCBI数据库获得来源于大肠杆菌MG1655(Escherichia coli MG1655)的aspC的核苷酸序列；
将所述aspC的核苷酸序列在JCAT网站，通过输入：避免XbaI，NdeI，SpeI和HindIII限制性酶切位点为条件，得到优化后的aspC；在优化后的aspC的核苷酸序列5'、3'端分别加入NdeI限制性酶切位点和SpeI限制性酶切位点，得到加酶切位点的aspC，在体外合成和扩增；
所述加酶切位点的ilvE的核苷酸序列如SEQ ID NO.5所示；
所述加酶切位点的aspC的核苷酸序列如SEQ ID NO.6所示；
(2)设计引物alaA-F和alaA-R，通过PCR方法从大肠杆菌MG1655的基因组DNA扩增出alaA核苷酸序列；
设计引物gdhA-F和gdhA-R，通过PCR方法从大肠杆菌MG1655的基因组DNA扩增出gdhA核苷酸序列；
设计引物serB-F和serB-R，通过PCR方法从大肠杆菌MG1655的基因组DNA扩增出serB核苷酸序列；
设计引物pheA-F和pheA-R，通过PCR方法从大肠杆菌MG1655的基因组DNA扩增出pheA核苷酸序列；
alaA-F的核苷酸序列如SEQ ID NO.7所示；alaA-R的核苷酸序列如SEQ ID NO.8所示；gdhA-F的核苷酸序列如SEQ ID NO.9所示；gdhA-R的核苷酸序列如SEQ ID NO.10所示；serB-F的核苷酸序列如SEQ ID NO.11所示；serB-R的核苷酸序列如SEQ ID NO.12所示；pheA-F的核苷酸序列如SEQ ID NO.13所示；pheA-R的核苷酸序列如SEQ ID NO.14所示；所述alaA的核苷酸序列如SEQ ID NO.15所示；所述gdhA的核苷酸序列如SEQ ID NO.16所示；所述serB的核苷酸序列如SEQ ID NO.17所示；所述pheA的核苷酸序列如SEQ ID NO.18所示；
(3)将步骤(1)获得的加酶切位点的LC和加酶切位点的HC分别连接到p2A4UV5载体中，得到p2A4UV5-LC和p2A4UV5-HC；将p2A4UV5-LC用SpeI和PstI限制酶进行酶切，将p2A4UV5-HC用XbaI和PstI限制酶进行酶切，将p2A4UV5-HC酶切的核苷酸片段连接到p2A4UV5-LC酶切的核苷酸片段的3'端，得到p2A4UV5-AB；
将步骤(1)获得的所述加酶切位点的ilvE的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-IlvE；
将步骤(1)获得的所述加酶切位点的aspC的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-AspC；
将步骤(2)获得的所述alaA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-AlaA；将步骤(2)获得的所述gdhA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-GdhA；
将步骤(2)获得的所述serB的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-SerB；
将步骤(2)获得的所述pheA的核苷酸序列连接到p3C5BAD载体中得到p3C5BAD-PheA；
所述p2A4UV5载体的核苷酸序列如SEQ ID NO.19所示；所述p3C5BAD载体的核苷酸序列如SEQ ID NO.20所示；
(4)将p2A4UV5-AB和p3C5BAD-IlvE导入大肠杆菌中，得到重组菌2；将p2A4UV5-AB和p3C5BAD-AspC导入大肠杆菌中，得到重组菌3；将p2A4UV5-AB和p3C5BAD-AlaA导入大肠杆菌中，得到重组菌4；将p2A4UV5-AB和p3C5BAD-GdhA导入大肠杆菌中，得到重组菌5；将p2A4UV5-AB和p3C5BAD-SerB导入大肠杆菌中，得到重组菌6；将p2A4UV5-AB和p3C5BAD-PheA导入大肠杆菌中，得到重组菌7。
2.权利要求1的方法构建的一种高产IgG1的重组菌。
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