CN108315288B - 一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌及其构建方法与应用 - Google Patents

一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌及其构建方法与应用 Download PDF

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CN108315288B
CN108315288B CN201711176672.8A CN201711176672A CN108315288B CN 108315288 B CN108315288 B CN 108315288B CN 201711176672 A CN201711176672 A CN 201711176672A CN 108315288 B CN108315288 B CN 108315288B
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娄文勇
区晓阳
宗敏华
高嘉心
彭飞
徐培
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Abstract

本发明公开了一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌及其构建方法与应用。本发明以大肠杆菌DH5α工程菌为宿主，将克隆得到的甲酰胺酶基因和亚磷酸脱氢酶基因扩增成为融合基因，再连接在载体的多克隆位点上，并将得到的重组质粒转化入DH5α，提取质粒并转入表达菌株中，经诱导培养得到重组大肠杆菌。该重组大肠杆菌表达产生甲酰胺酶与氧化亚磷酸脱氢酶融合蛋白，该融合蛋白能同时分解甲酰胺生成NH4 +和氧化亚磷酸盐磷酸盐成为磷酸盐，为重组大肠杆菌工程菌提供生长繁殖所需要的氮源和磷源，而其他微生物由于缺乏拥有这两条途径，无法在含有甲酰胺和亚磷酸盐的MOPS培养基中生长繁殖，解决了大肠杆菌在工业发酵中染菌问题。

Description

一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌 及其构建方法与应用
技术领域
本发明涉及基因工程领域，具体涉及一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌及其构建方法与应用。
背景技术
大肠杆菌是目前使用最广泛的宿主之一，这是因为大肠杆菌基因组被研究的透彻，并且繁殖快、发酵周期短。所以，大肠杆菌在工业发酵行业中受到企业家密切关注和重视。但是，在大肠杆菌发酵过程中，染菌问题依然企业最关注的问题。一旦染菌，不但造成经济损失、浪费原理和时间，还会给废料处理增加难度。所以，要是能找到一种方法，能解决大肠杆菌在发酵过程中被杂菌污染的问题，在促进企业的利润增长，还是对于社会的发展，将是非常有意义的。
目前关于发酵染菌的研究多集中在发酵染菌的来源(途径)及相关的菌形、不同发酵阶段染菌对发酵的影响及防治措施、从设备角度的分析和防治等方面。为了降低染菌，企业一般是从提高设备要求和操作员的技术水平入手。但是，复杂的周边环境，和发酵仪器隐秘部位，都使得在发酵工程时常染菌。
针对以上的大肠杆菌发酵过程染菌现象，研究从营养物质底物谱的摄取进行研究，对大肠杆菌的营养代谢途径进行改造，使改造后的大肠杆菌工程菌能表达甲酰胺酶与亚磷酸脱氢酶融合蛋白，该融合蛋白能分解甲酰胺产生氨作为氮源和分解氧化亚磷酸盐成为磷酸盐作为磷源，改变其氮源和磷源的来源途径，使其在特定的MOPS培养基中能正常生长，而不同时具有这两条代谢途径的杂菌微生物就会由于缺乏氮源或磷源营养物质的来源而被“饿死”。一方面，重组菌能表达融合蛋白利用甲酰胺和亚磷酸盐；另一方面，重组菌还能表达外源基因，包括抗体和其他有价值的酶制剂。同时，为验证该构建的工程菌合成外源基因的能力，还把绿色荧光蛋白基因转化入该工程菌进行共表达。
发明内容
本发明的目的在于针对现有技术的不足，提供了一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌，具体为一种能高效表达甲酰胺酶与氧化亚磷酸脱氢酶融合蛋白的重组大肠杆菌。该重组大肠杆菌表达甲酰胺酶与氧化亚磷酸脱氢酶融合蛋白基因for-Linker-ptx，产生甲酰胺酶与氧化亚磷酸脱氢酶融合蛋白，该融合蛋白能同时分解甲酰胺生成NH4 +和氧化亚磷酸盐磷酸盐成为磷酸盐，为重组大肠杆菌工程菌提供生长繁殖所需要的氮源和磷源，而其他不同时具有这两条代谢途径的微生物则会由于缺乏氮源和磷源的代谢途径而被“饿死”。
本发明的目的还在于提供所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌的构建方法。该构建方法以大肠杆菌DH5α工程菌为宿主，利用重叠PCR技术，将克隆自类芽孢杆菌Paenibacillus pasadenensis.CS0611 的含Linker序列的甲酰胺酶基因for-Linker和肺炎克雷伯菌Klebsiella pneumonia.OU7的亚磷酸脱氢酶基因ptx扩增成为融合基因for-Linker-ptx，再连接在载体pGEX-2T的多克隆位点上，并将得到的重组质粒pGEX-2T-for- Linker-ptx转化入DH5α；验证成功后，提取pGEX-2T-for-Linker-ptx质粒，转化入大肠杆菌表达菌株E.coliBL21(DE3)中，得到重组表达菌株BL21(DE3) (pGEX-2T-for-Linker-ptx)，并继续在含有甲酰胺和亚磷酸盐的MOPS培养基中诱导培养重组大肠杆菌。
利用Linker把扩增得到的甲酰胺酶基因for和亚磷酸脱氢酶基因ptx连接在一起，使两个酶基因融合表达，发挥其酶催化功能，实现重组大肠杆菌在含有甲酰胺和亚磷酸盐的MOPS培养基中正常生长繁殖并使其功能得到表达。
本发明的目的还在于提供所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌的应用。
本发明的目的通过如下技术方案实现。
一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，包括如下步骤：
(1)设计引物，PCR扩增含Linker序列的甲酰胺酶基因(3'端含Linker 序列)
上游引物
Figure BDA0001478360100000031
下划线斜体为BamH I酶切点；下游引物A2(5'- CGACCCACCACCGCCCGAGCCACCGCCACCTCGCGCCGCGCCTCCCTTCG C-3')，下划线为Linker序列；
以类芽孢杆菌Paenibacillus pasadenensis.CS0611的基因组为模板，克隆出1041bp的含Linker序列的甲酰胺酶基因序列for-Linker；将克隆得到的基因序列for-Linker连接到载体pMD-19T Simple上，再将重组载体转化到大肠杆菌 DH5α，得到含有重组载体pMD-19T Simple-for-Linker的重组大肠杆菌 DH5α(pMD-19T Simple-for-Linker)；然后，以A1和B1(5'- TATAACGAGTTTCGGCAGCATCGACCCACCACCGCCCGAGCCA-3')扩增 for-Linker片段；
(2)设计引物，PCR扩增亚磷酸脱氢酶基因
上游引物B2(5'- TGGCTCGGGCGGTGGTGGGTCGATGCTGCCGAAACTCGTTATA-3')，下划线为Linker的部分DNA；下游引物B3
Figure BDA0001478360100000041
Figure BDA0001478360100000042
下划线斜体为EcoRⅠ酶切点；
以肺炎克雷伯菌Klebsiella pneumonia.OU7的基因组为模板，克隆出 1008bp的亚磷酸脱氢酶基因序列ptx，得到ptx片段；
(3)重叠PCR扩增得到融合基因for-Linker-ptx
以步骤(1)、步骤(2)扩增得到的for-Linker片段和ptx片段为模板，以 A1和引物B3分别为上游、下游引物，扩增得到甲酰胺酶与亚磷酸脱氢酶融合基因for-Linker-ptx；利用BamH I和EcoR Ⅰ对融合基因for-Linker-ptx进行双酶切，连接入同样酶切后的pGEX-2T表达质粒，转化入DH5α，得到阳性克隆子 DH5α(pGEX-for-Linker-ptx)；
(4)转化表达重组菌株
从重组大肠杆菌DH5α(pGEX-for-Linker-ptx)中提取重组质粒pGEX-for-Linker-ptx，并将重组质粒pGEX-for-Linker-ptx转化入大肠杆菌表达菌株 BL21(DE3)中，得到重组表达菌株BL21(DE3)(pGEX-for-Linker-ptx)；
(5)诱导培养重组大肠杆菌
将得到的重组表达菌株BL21(DE3)(pGEX-for-Linker-ptx)接种到LB培养基中，进行诱导培养后，收集菌体并加入到含有甲酰胺和亚磷酸盐的MOPS培养基中，继续培养得到重组大肠杆菌。
进一步地，步骤(1)中，所述类芽孢杆菌Paenibacillus pasadenensis. CS0611的含Linker序列的甲酰胺酶基因序列如序列表SEQ1所示，片段长度为 1041bp，含Linker序列(GGTGGCGGTGGCTCGGGCGGTGGTGGGTCG)，编码347个氨基酸。
进一步地，步骤(1)中，所述类芽孢杆菌Paenibacillus pasadenensis. CS0611于2014年10月8日保藏于中国典型培养物保藏中心，保藏单位地址为：中国武汉武汉大学，保藏号为 CCTCC NO：M2014458。
进一步地，步骤(1)中，所述PCR扩增的条件为：94℃反应5min；于98℃反应10s、55℃反应5s、72℃反应70s，并循环30次；再于72℃反应7min；最后降温至16℃。
进一步地，步骤(2)中，所述肺炎克雷伯菌Klebsiella pneumonia.OU7的亚磷酸脱氢酶基因序列如序列表SEQ2所示，片段长度为1008bp，编码336个氨基酸。
进一步地，步骤(2)中，所述肺炎克雷伯菌Klebsiella pneumonia.OU7于 2017年8月24日保藏于中国典型培养物保藏中心，保藏单位地址为：中国武汉武汉大学，保藏号为CCTCC NO：M 2017449。
进一步地，步骤(2)中，所述PCR扩增的条件为：94℃反应5min；于98℃反应10s、55℃反应5s、72℃反应70s，并循环30次；再于72℃反应7min；最后降温至16℃。
进一步地，步骤(3)中，所述融合基因for-Linker-ptx的基因序列如序列表SEQ3所示，片段长度为2049bp，位于5’端的是甲酰胺酶基因，位于3’端的是亚磷酸脱氢酶基因，甲酰胺酶基因片段与亚磷酸脱氢酶基因片段之间有片段长度30bp的Linker序列(GGTGGCGGTGGCTCGGGCGGTGGTGGGTCG) 连接。
Linker序列位于甲酰胺酶基因的3’末端，使表达的甲酰胺酶与亚磷酸脱氢酶之间有10个疏水性氨基酸作为链桥作用，使两个蛋白酶在形成有活性的空间三维结构时，不会互相影响。
进一步地，步骤(3)中，所述PCR扩增的条件为：94℃反应5min；于98℃反应10s、55℃反应5s、72℃反应70s，并循环30次；再于72℃反应7min；最后降温至16℃。
进一步地，步骤(5)中，所述诱导培养是在LB培养基中，37℃、180 rpm培养12～16小时后，再接种于新鲜的LB培养基中，37℃、180rpm诱导培养至重组菌浓度至OD600＝0.6，降温至20℃后，加入终浓度为0.2mM的异丙基 -β-D-硫代半乳糖苷(IPTG)进行诱导16h。
进一步地，步骤(5)中，所述收集菌体是将诱导培养结束后的菌液在4℃、8000rpm、离心5min后，再用预冷至4℃的生理盐水悬浮，再次4℃、8000 rpm、离心5min，重复生理盐水悬浮并离心的步骤两次，以洗除残留的LB培养基，收集菌体。
进一步地，步骤(5)中，所述含有甲酰胺和亚磷酸盐的MOPS培养基中，甲酰胺的终浓度为200mM，亚磷酸盐的终浓度为1.32mM。
进一步地，步骤(5)中，收集的菌体加入到含有甲酰胺和亚磷酸盐的 MOPS培养基中后，菌体浓度OD600＝0.1～0.15。
进一步地，步骤(5)中，所述继续诱导培养是在含有甲酰胺和亚磷酸盐的MOPS培养基中加入终浓度为0.2mM的IPTG，并于30℃、180rpm继续培养84～96小时。
MOPS培养基中缺乏基础营养成分NH4 +和HPO4 2-而添加有甲酰胺和亚磷酸盐，同时杂菌微生物不具备分解甲酰胺和氧化亚磷酸盐获取营养成分的功能，使杂菌微生物缺乏氮源和磷源，使重组大肠杆菌由于具有高效的分解甲酰胺和氧化亚磷酸的功能而获得充足的营养成分而成为优势菌。
由上述任一项所述方法构建得到的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌，在MOPS培养基中能利用甲酰胺和亚磷酸盐进行生长繁殖，并且能共表达外源绿色荧光蛋白基因，合成绿色荧光蛋白(GFP)，具有表达外源基因的功能。
所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌能表达融合蛋白来高效分解甲酰胺和氧化亚磷酸盐，应用于大肠杆菌工业发酵酶促合成包括抗体或有价值的蛋白酶制剂，具有非常深远的意义。
与现有技术相比，本发明具有如下优点和有益效果：
(1)本发明的重组大肠杆菌能表达甲酰胺酶和亚磷酸脱氢酶融合蛋白，该融合蛋白能同时分解甲酰胺生成NH4 +和氧化亚磷酸盐磷酸盐成为磷酸盐，为重组大肠杆菌的正常生长繁殖提供氮源和磷源，而其他微生物由于缺乏拥有这两条途径，无法在含有甲酰胺和亚磷酸盐的MOPS培养基中生长繁殖，解决了大肠杆菌在工业发酵中染菌问题，降低了发酵设备需求，并能发挥其本来的工程菌表达外源基因的功能；
(2)本发明通过基因工程的生物技术解决了大肠杆菌发酵过程中的染菌问题，并且不会丧失作为工程菌表达外源蛋白的特性；
(3)本发明的重组大肠杆菌应用于大肠杆菌工业发酵酶促合成包括抗体或有价值的酶制剂，使酶促合成包括抗体或有价值的酶制剂的过程高效，合成的物质纯度高，并且可以在生产发酵过程中防止杂菌微生物的入侵污染问题。
附图说明
图1为重组融合表达质粒pGEX-for-Linker-ptx的构建过程图；
图2为重组大肠杆菌BL21(DE3)(pGEX-for-Linker-ptx)在MOPS培养基中的生长曲线图；
图3为重组大肠杆菌BL21(DE3)(pGEX-for-Linker-ptx)在MOPS培养基中表达绿色荧光蛋白(GFP)的荧光图。
具体实施方式
为更好地理解本发明，以下结合实施例及附图对本发明技术方案作进一步阐述，但所描述的内容仅用于阐述本发明，而不应当也不限制本发明。
本发明具体实施例中采用的类芽孢杆菌Paenibacillus pasadenensis.CS0611于2014年10月8日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：M2014458，并已完成全基因组测试，扩增得到的含Linker序列的甲酰胺酶基因的片段如序列表SEQ1所示，长度为1041bp，含Linker序列，编码347个氨基酸。
本发明具体实施例中的肺炎克雷伯菌Klebsiella pneumonia.OU7于2017年 8月24日保藏于中国典型培养物保藏中心(湖北省武汉市武昌路珞珈山武汉大学，邮编430072)，保藏号为CCTCC NO：M 2017449，且肺炎克雷伯菌 Klebsiella pneumonia.OU7为通过如下方法培养自主筛选得到，通过全基因组测试，其中的亚磷酸脱氢酶基因的片段如序列表SEQ2所示，长度为1008bp，编码336个氨基酸。
实施例1
甲酰胺酶基因for(含Linker序列)的获取
将类芽孢杆菌Paenibacillus pasadenensis.CS0611利用LB培养基在37℃、180rpm培养一天；将培养好的菌体于4℃、8000rpm、离心5min收集菌体，用生理盐水洗涤2次，以去掉残留的培养基，然后按照OMEGA细菌基因组 DNA提取试剂盒的具体方法对类芽孢杆菌Paenibacillus pasadenensis.CS0611 基因组进行提取；
以提取的Paenibacillus pasadenensis.CS0611基因组为模板，以
Figure BDA0001478360100000111
Figure BDA0001478360100000112
和A2(5'- CGACCCACC ACCGCCCGAGCCACCGCCACCTCGCGCCGCGCCTCCCTTCG C-3')分别为上、下游引物，采用PCR扩增甲酰胺酶基因for-Linker；
PCR所用的酶试剂为TaKaRa公司的
Figure BDA0001478360100000113
HS DNA Polymerase with GCbuffer；PCR反应体系及条件如下：
PCR反应液组成(25μL)
Figure BDA0001478360100000114
PCR反应条件：94℃反应5min；再依次于98℃反应10s、55℃反应5s、 72℃反应80s，并循环30次；再于72℃反应7min；最后降温至16℃。
将PCR扩增得到的DNA产物利用1wt％琼脂糖胶电泳，并使用OMEGA公司胶回收试剂盒，按照说明书步骤进行切胶回收，然后送检测序，结果显示已得到一段长度为1041bp的含Linker序列的甲酰胺酶基因序列，命名为for-Linker。
实施例2
亚磷酸脱氢酶基因ptx的获得
亚磷酸脱氢酶基因来源于自主筛选的肺炎克雷伯菌Klebsiella pneumonia.OU7，通过自主筛选得到；
按照OMEGA细菌基因组DNA提取试剂盒的具体方法对自主筛选的肺炎克雷伯菌Klebsiella pneumonia.OU7基因组进行提取，以自主筛选的肺炎克雷伯菌 Klebsiellapneumonia.OU7的基因组为模板，以B2(5'- TGGCTCGGGCGGTGGTGGGTCGATGCTGCCGAAACTCGTTATA-3')和
Figure BDA0001478360100000121
Figure BDA0001478360100000122
分别为上、下游引物，采用PCR扩增亚磷酸脱氢酶基因ptx；
PCR所用的酶试剂为TaKaRa公司的
Figure BDA0001478360100000123
HS DNA Polymerase with GCbuffer；PCR反应体系及条件如下：
PCR反应液组成(25μL)
Figure BDA0001478360100000124
Figure BDA0001478360100000131
PCR反应条件：94℃反应5min；再依次于98℃反应10s、55℃反应5s、72℃反应70s，并循环30次；再于72℃反应7min；最后降温至16℃。
将PCR扩增得到的DNA产物利用1wt％琼脂糖胶电泳，并使用OMEGA公司胶回收试剂盒，按照说明书步骤进行切胶回收，然后送检测序，结果显示已得到一段长度为1008bp的亚磷酸脱氢酶基因序列，命名为ptx。
实施例3
重叠PCR扩增得到融合基因for-Linker-ptx
以扩增得到for-Linker和ptx片段为模板，以引物
Figure BDA0001478360100000132
Figure BDA0001478360100000133
和引物
Figure BDA0001478360100000135
Figure BDA0001478360100000134
分别为上游、下游引物，扩增得到融合基因for-Linker-ptx。
扩增条件如下：94℃反应5min；再依次于98℃反应10s、55℃反应5s、72℃反应150s，并循环30次；最后于72℃反应7min；最后降温至16℃。
实施例4
重组菌BL21(DE3)(pGEX-2T-for-Linker-ptx)的构建
将实施例3中得到的融合基因片段for-Linker-ptx和质粒pGEX-2T分别利用BamHI和EcoR I进行双酶切，上游引物下划线斜体为BamH I酶切点，下游引物下划线斜体为EcoRI酶切点，酶切条件为：37℃，酶切120min；
酶切体系：
Figure BDA0001478360100000141
将酶切后的产物分别进行切胶回收，回收方法及步骤参照OMEGA公司的胶回收试剂盒；回收后利用1wt％琼脂糖胶电泳，检测回收率；然后将回收的目的片段和质粒进行连接，连接试剂盒采用Thermo Fisher SCIENTIFIC公司的 T4DNA Ligase，连接体系如下：
Figure BDA0001478360100000142
融合基因与pGEX-2T质粒摩尔比为5：1。
将连接产物转化入大肠杆菌DH5α，转化步骤如下：将10μL连接产物与 100μL大肠杆菌DH5α感受态细胞混合，冰浴30min，42℃热激90s，再冰浴 2min，加入890μL LB培养基，37℃、180rpm摇床培养1h后，4000rpm离心5分钟收集菌体，取上清培养基890μL，将管底的菌体重悬，均匀地涂布在含氨苄抗性素LB固体平板(含100μg/mL氨苄青霉素钠)，37℃培养16小时，待平板长出转化子后，挑取转化子进行PCR验证并送检测序，并利用 DNAssist软件对测序结果进行ORF查找。
结果显示，得到融合基因序列(for-Linker-ptx)已正确地插入pGEX-2T的多克隆位点上，成功获得了pGEX-2T-for-Linker-ptx质粒，并转化入BL21(DE3)，得到重组菌BL21(DE3)(pGEX-2T-for-Linker-ptx)。
重组大肠杆菌BL21(DE3)(pGEX-2T-for-Linker-ptx)同化代谢利用甲酰胺和亚磷酸盐而成为培养基中的优势工程菌构建过程图如图1所示，大肠杆菌 BL21(DE3)(pGEX-2T-for-Linker-ptx)由于插入了融合基因(甲酰胺酶基因 +Linker+亚磷酸脱氢酶基因)，所以改造后的大肠杆菌BL21(DE3)(pGEX-2T- for-Linker-ptx)既能分解甲酰胺成NH4 +，又能氧化亚磷酸盐成磷酸盐，从而能为大肠杆菌BL21(DE3)(pGEX-2T-for-Linker-ptx)提供生长利用的氮源和磷源，而其他杂菌微生物由于缺乏同时拥有这两条途径，所以不能生长。
实施例5
重组大肠杆菌BL21(DE3)(pGEX-2T-for-Linker-ptx)在特定MOPS培养基中的生长。
将获得的重组表达菌株BL21(DE3)(pGEX-2T-for-Linker-ptx)进行诱导培养，具体过程如下：
将BL21(DE3)(pGEX-2T-for-Linker-ptx)按照体积比1：100接种到含有30mL的LB培养基，37℃、180rpm过夜培养16小时；进行诱导培养，取1 mL过夜培养的重组大肠杆菌接种到装有100mL新鲜的LB培养基，于37℃、 180rpm培养重组菌浓度至OD600＝0.6，降温至20℃后，加入终浓度为0.2mM 的IPTG进行诱导16h后，4℃、8000rpm、离心5min收集菌体；
用生理盐水(4℃预冷)悬浮收集的菌体，4℃、8000rpm、离心5min收集菌体，并重复此步骤2次，洗除残留的LB培养基；再把菌体加入到含有甲酰胺(200mM)和亚磷酸盐(1.32mM)的基础MOPS培养基，使菌体OD600为0.1，加入终浓度为0.2mM的IPTG，于30℃、180rpm继续培养重组大肠杆菌。
考察重组菌在含有甲酰胺和亚磷酸盐的MOPS培养基中的生长情况，重组菌BL21(DE3)(pGEX-2T-for-Linker-ptx)在含有甲酰胺(200mM)和亚磷酸盐(1.32mM)的MOPS培养基中的生长曲线图如图2所示，由图2可知重组菌BL21(DE3)(pGEX-2T-for-Linker-ptx)能在该培养基中生长，到第三天后，菌浓度达到最大值，A600为2.223；而对照菌株BL21(DE3)在该MOPS培养基基本没有生长。
为验证该菌在MOPS培养基中合成外源基因的表达能力，利用绿色荧光蛋白(GFP)基因对重组菌表达外源基因的能力进行验证。把pET-28a-GFP质粒转化入BL21(DE3)(pGEX-2T-for-Linker-ptx)，形成重组菌，并且命名为 BL21(DE3)(pGEX-2T-for-Linker-ptx+pET-28a-GFP)，得到的重组菌在含甲酰胺和亚磷酸盐的MOPS培养基中诱导表达GFP。然后利用荧光倒置显微镜，激发波长为488nm，发射波长为507nm对培养发酵后的重组菌进行观察，从图3中可以看出，重组菌能在MOPS培养基正常生长，并且能表达外源绿色荧光蛋白基因。
序列表
<110> 华南理工大学
<120> 一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌及其构建方法与应用
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<213> 类芽孢杆菌(Paenibacillus pasadenensis. CS0611)
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Gly Cys Thr Thr Gly Ala Ala Cys Ala Ala Ala Thr Cys Gly Cys Cys
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Gly Gly Ala Cys Gly Gly Cys Gly Thr Cys Gly Thr Cys Ala Thr Cys
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Gly Gly Cys Cys Thr Cys Gly Cys Gly Cys Ala Gly Cys Thr Gly Cys
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Ala Gly Cys Thr Gly Cys Cys Gly Gly Cys Cys Gly Thr Cys Gly Ala
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Gly Cys Gly Gly Cys Gly Cys Ala Gly Gly Cys Gly Ala Gly Gly Cys
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Gly Cys Ala Thr Cys Gly Cys Gly Gly Ala Gly Ala Thr Gly Ala Cys
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Gly Gly Cys Cys Ala Ala Gly Gly Cys Gly Cys Gly Gly Ala Ala Gly
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Gly Gly Cys Thr Cys Gly Cys Gly Cys Thr Cys Gly Ala Thr Gly Gly
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Ala Thr Cys Thr Gly Ala Thr Cys Gly Thr Ala Thr Thr Thr Cys Cys
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Cys Gly Ala Ala Thr Ala Thr Thr Cys Gly Cys Thr Gly Cys Ala Thr
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Gly Gly Cys Cys Thr Ala Thr Cys Gly Ala Thr Gly Ala Ala Thr Ala
195 200 205
Cys Cys Gly Ala Thr Cys Cys Gly Gly Cys Gly Cys Thr Cys Ala Thr
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Gly Thr Gly Cys Cys Gly Gly Gly Thr Cys Gly Ala Cys Gly Gly Gly
225 230 235 240
Cys Cys Gly Gly Ala Gly Gly Thr Cys Gly Ala Gly Cys Thr Gly Thr
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Gly Gly Cys Gly Gly Gly Ala Gly Gly Cys Cys Thr Gly Cys Cys Gly
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Cys Gly Ala Gly Cys Ala Thr Cys Gly Cys Ala Thr Cys Thr Gly Gly
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Gly Gly Cys Thr Gly Cys Thr Thr Cys Ala Gly Cys Ala Thr Cys Ala
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Thr Gly Gly Ala Gly Cys Thr Cys Ala Ala Thr Cys Cys Gly Gly Ala
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Cys Gly Gly Cys Ala Ala Thr Cys Cys Gly Thr Ala Cys Ala Ala Cys
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Ala Cys Gly Gly Gly Gly Cys Thr Cys Ala Thr Cys Ala Thr Cys Gly
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Ala Cys Gly Ala Cys Gly Ala Ala Gly Gly Cys Gly Gly Ala Ala Thr
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Cys Cys Gly Gly Cys Thr Gly Ala Ala Gly Thr Ala Cys Cys Gly Cys
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Ala Ala Gly Cys Thr Gly Cys Ala Thr Cys Cys Gly Thr Gly Gly Gly
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Thr Gly Cys Cys Gly Gly Thr Gly Gly Ala Gly Cys Cys Gly Thr Gly
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Gly Gly Ala Gly Cys Cys Gly Gly Gly Cys Gly Ala Thr Cys Thr Cys
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Gly Gly Cys Ala Thr Cys Cys Cys Gly Ala Thr Gly Thr Gly Cys Gly
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Ala Cys Gly Gly Gly Cys Cys Gly Ala Ala Cys Gly Gly Cys Ala Gly
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Cys Cys Gly Gly Thr Thr Gly Gly Cys Gly Cys Thr Cys Gly Thr Cys
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Ala Thr Cys Thr Gly Cys Cys Ala Thr Gly Ala Cys Gly Gly Cys Ala
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Thr Gly Thr Thr Cys Cys Cys Gly Gly Ala Ala Ala Thr Gly Gly Cys
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Gly Cys Gly Cys Gly Ala Ala Thr Gly Cys Gly Cys Cys Thr Ala Thr
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Cys Thr Cys Gly Gly Thr Gly Cys Gly Gly Ala Cys Ala Thr Cys Ala
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Thr Gly Cys Thr Gly Cys Gly Cys Ala Cys Gly Gly Cys Cys Gly Gly
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Cys Thr Ala Thr Ala Cys Gly Gly Cys Thr Cys Cys Gly Ala Thr Cys
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Cys Gly Cys Cys Ala Thr Gly Cys Ala Thr Gly Gly Cys Ala Gly Gly
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Ala Thr Gly Thr Ala Cys Ala Cys Cys Gly Cys Cys Thr Cys Gly Gly
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Thr Cys Thr Gly Cys Cys Thr Gly Ala Gly Thr Gly Gly Cys Ala Gly
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Cys Gly Ala Cys Gly Gly Cys Gly Thr Cys Thr Thr Cys Gly Ala Cys
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Thr Cys Gly Ala Thr Gly Gly Gly Cys Gly Ala Gly Gly Cys Gly Ala
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Thr Gly Ala Thr Cys Gly Thr Cys Gly Gly Cys Thr Thr Cys Gly Ala
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Cys Gly Gly Cys Ala Thr Gly Ala Cys Gly Cys Thr Cys Gly Thr Cys
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Cys Ala Cys Gly Gly Cys Gly Gly Cys Gly Gly Ala Cys Gly Gly Cys
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Cys Cys Gly Ala Cys Gly Ala Gly Ala Thr Cys Gly Thr Gly Gly Cys
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Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly Cys Gly Gly Cys Cys Gly
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Thr Cys Gly Cys Thr Cys Gly Thr Cys Cys Gly Cys Gly Ala Gly Gly
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Cys Gly Cys Gly Cys Cys Gly Cys Ala Thr Cys Thr Gly Gly Gly Gly
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Cys Gly Thr Gly Gly Ala Gly Ala Ala Cys Ala Ala Cys Cys Thr Gly
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Thr Ala Cys Cys Ala Gly Cys Thr Cys Gly Gly Cys Cys Ala Thr Cys
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Gly Cys Gly Gly Cys Thr Ala Cys Gly Thr Cys Gly Cys Gly Gly Thr
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Gly Cys Ala Gly Gly Gly Cys Gly Gly Cys Gly Cys Ala Gly Gly Cys
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Gly Ala Cys Thr Gly Thr Cys Cys Gly Thr Ala Cys Ala Cys Cys Thr
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Ala Cys Ala Thr Gly Cys Ala Thr Gly Ala Thr Cys Thr Cys Gly Cys
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Cys Gly Cys Cys Gly Gly Cys Cys Gly Cys Thr Ala Cys Cys Gly Gly
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Cys Thr Gly Cys Cys Thr Thr Gly Gly Gly Ala Gly Gly Ala Thr Gly
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Ala Gly Gly Thr Gly Cys Thr Cys Ala Thr Cys Ala Ala Gly Gly Ala
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Cys Gly Gly Cys Ala Cys Gly Ala Gly Cys Gly Ala Gly Gly Gly Gly
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Thr Thr Cys Cys Cys Gly Cys Cys Gly Cys Cys Gly Gly Ala Gly Cys
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Gly Gly Cys Gly Ala Thr Ala Thr Gly Gly Cys Gly Gly Ala Gly Cys
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Gly Ala Ala Gly Gly Gly Ala Gly Gly Cys Gly Cys Gly Gly Cys Gly
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<213> 肺炎克雷伯菌(Klebsiella pneumonia. OU7)
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Ala Thr Gly Cys Thr Gly Cys Cys Gly Ala Ala Ala Cys Thr Cys Gly
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Thr Thr Ala Thr Ala Ala Cys Thr Cys Ala Cys Cys Gly Ala Gly Thr
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Ala Cys Ala Cys Gly Ala Thr Gly Ala Gly Ala Thr Cys Cys Thr Gly
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Cys Ala Ala Cys Thr Gly Cys Thr Gly Gly Cys Gly Cys Cys Ala Cys
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Ala Thr Thr Gly Cys Gly Ala Gly Cys Thr Gly Gly Thr Gly Ala Cys
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Cys Ala Ala Cys Cys Ala Gly Ala Cys Cys Gly Ala Cys Ala Gly Cys
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Ala Cys Gly Cys Thr Gly Ala Cys Gly Cys Gly Cys Gly Ala Gly Gly
100 105 110
Ala Ala Ala Thr Thr Cys Thr Gly Cys Gly Cys Cys Gly Cys Thr Gly
115 120 125
Thr Cys Gly Cys Gly Ala Thr Gly Cys Thr Cys Ala Gly Gly Cys Gly
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Ala Thr Gly Ala Thr Gly Gly Cys Gly Thr Thr Cys Ala Thr Gly Cys
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Cys Cys Gly Ala Thr Cys Gly Gly Gly Thr Cys Gly Ala Thr Gly Cys
165 170 175
Ala Gly Ala Cys Thr Thr Thr Cys Thr Thr Cys Ala Ala Gly Cys Cys
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Thr Ala Gly Thr Cys Gly Gly Cys Thr Gly Cys Gly Cys Gly Cys Thr
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Cys Ala Ala Gly Gly Gly Cys Thr Thr Cys Gly Ala Cys Ala Ala Thr
225 230 235 240
Thr Thr Cys Gly Ala Thr Gly Thr Gly Gly Ala Cys Gly Cys Cys Thr
245 250 255
Gly Thr Ala Cys Thr Gly Cys Cys Cys Gly Cys Gly Gly Gly Gly Thr
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Cys Thr Gly Gly Cys Thr Gly Ala Cys Cys Thr Thr Cys Gly Thr Gly
275 280 285
Cys Cys Thr Gly Ala Thr Cys Thr Gly Thr Thr Gly Ala Cys Gly Gly
290 295 300
Thr Cys Cys Cys Gly Ala Cys Thr Gly Cys Cys Gly Ala Gly Cys Thr
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Gly Gly Cys Gly Ala Thr Cys Gly Gly Ala Cys Thr Gly Gly Cys Gly
325 330 335
Gly Thr Gly Gly Gly Gly Cys Thr Gly Gly Gly Gly Cys Gly Gly Cys
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Ala Thr Cys Thr Gly Cys Gly Gly Gly Cys Ala Gly Cys Ala Gly Ala
355 360 365
Thr Gly Cys Gly Thr Thr Cys Gly Thr Cys Cys Gly Cys Thr Cys Thr
370 375 380
Gly Gly Cys Gly Ala Gly Thr Thr Cys Cys Ala Gly Gly Gly Cys Thr
385 390 395 400
Gly Gly Cys Ala Ala Cys Cys Ala Cys Ala Gly Thr Thr Cys Thr Ala
405 410 415
Cys Gly Gly Cys Ala Cys Gly Gly Gly Gly Cys Thr Gly Gly Ala Thr
420 425 430
Ala Ala Cys Gly Cys Thr Ala Cys Gly Gly Thr Cys Gly Gly Cys Ala
435 440 445
Thr Cys Cys Thr Thr Gly Gly Cys Ala Thr Gly Gly Gly Cys Gly Cys
450 455 460
Cys Ala Thr Cys Gly Gly Ala Cys Thr Gly Gly Cys Cys Ala Thr Gly
465 470 475 480
Gly Cys Thr Gly Ala Gly Cys Gly Cys Thr Thr Gly Cys Ala Gly Gly
485 490 495
Gly Ala Thr Gly Gly Gly Gly Cys Gly Cys Gly Ala Cys Cys Cys Thr
500 505 510
Gly Cys Ala Gly Thr Ala Cys Cys Ala Cys Gly Ala Gly Gly Cys Gly
515 520 525
Ala Ala Gly Gly Cys Thr Cys Thr Gly Gly Ala Thr Ala Cys Ala Cys
530 535 540
Ala Ala Ala Cys Cys Gly Ala Gly Cys Ala Ala Cys Gly Gly Cys Thr
545 550 555 560
Cys Gly Gly Cys Cys Thr Gly Cys Gly Cys Cys Ala Gly Gly Thr Gly
565 570 575
Gly Cys Gly Thr Gly Cys Ala Gly Cys Gly Ala Ala Cys Thr Cys Thr
580 585 590
Thr Cys Gly Cys Cys Ala Gly Cys Thr Cys Gly Gly Ala Cys Thr Thr
595 600 605
Cys Ala Thr Cys Cys Thr Gly Cys Thr Gly Gly Cys Gly Cys Thr Thr
610 615 620
Cys Cys Cys Thr Thr Gly Ala Ala Thr Gly Cys Cys Gly Ala Thr Ala
625 630 635 640
Cys Cys Cys Ala Gly Cys Ala Thr Cys Thr Gly Gly Thr Cys Ala Ala
645 650 655
Cys Gly Cys Cys Gly Ala Gly Cys Thr Gly Cys Thr Thr Gly Cys Cys
660 665 670
Cys Thr Cys Gly Thr Ala Cys Gly Gly Cys Cys Gly Gly Gly Cys Gly
675 680 685
Cys Thr Cys Thr Gly Cys Thr Thr Gly Thr Ala Ala Ala Cys Cys Cys
690 695 700
Cys Thr Gly Thr Cys Gly Thr Gly Gly Thr Thr Cys Gly Gly Thr Ala
705 710 715 720
Gly Thr Gly Gly Ala Thr Gly Ala Ala Gly Cys Cys Gly Cys Cys Gly
725 730 735
Thr Gly Cys Thr Cys Gly Cys Gly Gly Cys Gly Cys Thr Thr Gly Ala
740 745 750
Gly Cys Gly Ala Gly Gly Cys Cys Ala Gly Cys Thr Cys Gly Gly Cys
755 760 765
Gly Gly Gly Thr Ala Thr Gly Cys Gly Gly Cys Gly Gly Ala Thr Gly
770 775 780
Thr Ala Thr Thr Cys Gly Ala Ala Ala Thr Gly Gly Ala Ala Gly Ala
785 790 795 800
Cys Thr Gly Gly Gly Cys Thr Cys Gly Cys Gly Cys Gly Gly Ala Cys
805 810 815
Cys Gly Gly Cys Cys Gly Cys Gly Gly Cys Thr Gly Ala Thr Cys Gly
820 825 830
Ala Thr Cys Cys Thr Gly Cys Gly Cys Thr Gly Cys Thr Cys Gly Cys
835 840 845
Gly Cys Ala Thr Cys Cys Gly Ala Ala Thr Ala Cys Gly Cys Thr Gly
850 855 860
Thr Thr Cys Ala Cys Thr Cys Cys Gly Cys Ala Cys Ala Thr Ala Gly
865 870 875 880
Gly Gly Thr Cys Gly Gly Cys Ala Gly Thr Gly Cys Gly Cys Gly Cys
885 890 895
Gly Gly Thr Gly Cys Gly Cys Cys Thr Gly Gly Ala Gly Ala Thr Thr
900 905 910
Gly Ala Ala Cys Gly Thr Thr Gly Thr Gly Cys Ala Gly Cys Gly Cys
915 920 925
Ala Gly Ala Ala Cys Ala Thr Cys Ala Thr Cys Cys Ala Gly Gly Thr
930 935 940
Ala Thr Thr Gly Gly Cys Ala Gly Gly Thr Gly Cys Gly Cys Gly Cys
945 950 955 960
Cys Cys Ala Ala Thr Cys Ala Ala Cys Gly Cys Thr Gly Cys Gly Ala
965 970 975
Ala Cys Cys Gly Thr Cys Thr Gly Cys Cys Cys Ala Ala Gly Gly Cys
980 985 990
Cys Gly Ala Gly Cys Cys Thr Gly Cys Cys Gly Cys Ala Thr Gly Thr
995 1000 1005
<210> 3
<211> 2049
<212> PRT
<213> 融合基因(for-Linker-ptx)
<400> 3
Ala Thr Gly Ala Ala Cys Gly Gly Ala Cys Thr Gly Gly Gly Cys Gly
1 5 10 15
Gly Cys Thr Thr Gly Ala Ala Cys Ala Ala Ala Thr Cys Gly Cys Cys
20 25 30
Gly Gly Ala Cys Gly Gly Cys Gly Thr Cys Gly Thr Cys Ala Thr Cys
35 40 45
Gly Gly Cys Cys Thr Cys Gly Cys Gly Cys Ala Gly Cys Thr Gly Cys
50 55 60
Ala Gly Cys Thr Gly Cys Cys Gly Gly Cys Cys Gly Thr Cys Gly Ala
65 70 75 80
Gly Ala Cys Gly Cys Cys Gly Gly Ala Gly Cys Ala Gly Cys Thr Gly
85 90 95
Gly Cys Gly Gly Cys Gly Cys Ala Gly Gly Cys Gly Ala Gly Gly Cys
100 105 110
Gly Cys Ala Thr Cys Gly Cys Gly Gly Ala Gly Ala Thr Gly Ala Cys
115 120 125
Gly Gly Cys Cys Ala Ala Gly Gly Cys Gly Cys Gly Gly Ala Ala Gly
130 135 140
Gly Gly Cys Thr Cys Gly Cys Gly Cys Thr Cys Gly Ala Thr Gly Gly
145 150 155 160
Ala Thr Cys Thr Gly Ala Thr Cys Gly Thr Ala Thr Thr Thr Cys Cys
165 170 175
Cys Gly Ala Ala Thr Ala Thr Thr Cys Gly Cys Thr Gly Cys Ala Thr
180 185 190
Gly Gly Cys Cys Thr Ala Thr Cys Gly Ala Thr Gly Ala Ala Thr Ala
195 200 205
Cys Cys Gly Ala Thr Cys Cys Gly Gly Cys Gly Cys Thr Cys Ala Thr
210 215 220
Gly Thr Gly Cys Cys Gly Gly Gly Thr Cys Gly Ala Cys Gly Gly Gly
225 230 235 240
Cys Cys Gly Gly Ala Gly Gly Thr Cys Gly Ala Gly Cys Thr Gly Thr
245 250 255
Gly Gly Cys Gly Gly Gly Ala Gly Gly Cys Cys Thr Gly Cys Cys Gly
260 265 270
Cys Gly Ala Gly Cys Ala Thr Cys Gly Cys Ala Thr Cys Thr Gly Gly
275 280 285
Gly Gly Cys Thr Gly Cys Thr Thr Cys Ala Gly Cys Ala Thr Cys Ala
290 295 300
Thr Gly Gly Ala Gly Cys Thr Cys Ala Ala Thr Cys Cys Gly Gly Ala
305 310 315 320
Cys Gly Gly Cys Ala Ala Thr Cys Cys Gly Thr Ala Cys Ala Ala Cys
325 330 335
Ala Cys Gly Gly Gly Gly Cys Thr Cys Ala Thr Cys Ala Thr Cys Gly
340 345 350
Ala Cys Gly Ala Cys Gly Ala Ala Gly Gly Cys Gly Gly Ala Ala Thr
355 360 365
Cys Cys Gly Gly Cys Thr Gly Ala Ala Gly Thr Ala Cys Cys Gly Cys
370 375 380
Ala Ala Gly Cys Thr Gly Cys Ala Thr Cys Cys Gly Thr Gly Gly Gly
385 390 395 400
Thr Gly Cys Cys Gly Gly Thr Gly Gly Ala Gly Cys Cys Gly Thr Gly
405 410 415
Gly Gly Ala Gly Cys Cys Gly Gly Gly Cys Gly Ala Thr Cys Thr Cys
420 425 430
Gly Gly Cys Ala Thr Cys Cys Cys Gly Ala Thr Gly Thr Gly Cys Gly
435 440 445
Ala Cys Gly Gly Gly Cys Cys Gly Ala Ala Cys Gly Gly Cys Ala Gly
450 455 460
Cys Cys Gly Gly Thr Thr Gly Gly Cys Gly Cys Thr Cys Gly Thr Cys
465 470 475 480
Ala Thr Cys Thr Gly Cys Cys Ala Thr Gly Ala Cys Gly Gly Cys Ala
485 490 495
Thr Gly Thr Thr Cys Cys Cys Gly Gly Ala Ala Ala Thr Gly Gly Cys
500 505 510
Gly Cys Gly Cys Gly Ala Ala Thr Gly Cys Gly Cys Cys Thr Ala Thr
515 520 525
Cys Thr Cys Gly Gly Thr Gly Cys Gly Gly Ala Cys Ala Thr Cys Ala
530 535 540
Thr Gly Cys Thr Gly Cys Gly Cys Ala Cys Gly Gly Cys Cys Gly Gly
545 550 555 560
Cys Thr Ala Thr Ala Cys Gly Gly Cys Thr Cys Cys Gly Ala Thr Cys
565 570 575
Cys Gly Cys Cys Ala Thr Gly Cys Ala Thr Gly Gly Cys Ala Gly Gly
580 585 590
Thr Gly Ala Cys Gly Ala Ala Cys Cys Ala Gly Gly Cys Gly Cys Ala
595 600 605
Cys Gly Cys Cys Thr Thr Thr Thr Gly Cys Ala Ala Cys Cys Thr Gly
610 615 620
Ala Thr Gly Thr Ala Cys Ala Cys Cys Gly Cys Cys Thr Cys Gly Gly
625 630 635 640
Thr Cys Thr Gly Cys Cys Thr Gly Ala Gly Thr Gly Gly Cys Ala Gly
645 650 655
Cys Gly Ala Cys Gly Gly Cys Gly Thr Cys Thr Thr Cys Gly Ala Cys
660 665 670
Thr Cys Gly Ala Thr Gly Gly Gly Cys Gly Ala Gly Gly Cys Gly Ala
675 680 685
Thr Gly Ala Thr Cys Gly Thr Cys Gly Gly Cys Thr Thr Cys Gly Ala
690 695 700
Cys Gly Gly Cys Ala Thr Gly Ala Cys Gly Cys Thr Cys Gly Thr Cys
705 710 715 720
Cys Ala Cys Gly Gly Cys Gly Gly Cys Gly Gly Ala Cys Gly Gly Cys
725 730 735
Cys Cys Gly Ala Cys Gly Ala Gly Ala Thr Cys Gly Thr Gly Gly Cys
740 745 750
Cys Gly Gly Cys Gly Ala Gly Gly Thr Gly Cys Gly Gly Cys Cys Gly
755 760 765
Thr Cys Gly Cys Thr Cys Gly Thr Cys Cys Gly Cys Gly Ala Gly Gly
770 775 780
Cys Gly Cys Gly Cys Cys Gly Cys Ala Thr Cys Thr Gly Gly Gly Gly
785 790 795 800
Cys Gly Thr Gly Gly Ala Gly Ala Ala Cys Ala Ala Cys Cys Thr Gly
805 810 815
Thr Ala Cys Cys Ala Gly Cys Thr Cys Gly Gly Cys Cys Ala Thr Cys
820 825 830
Gly Cys Gly Gly Cys Thr Ala Cys Gly Thr Cys Gly Cys Gly Gly Thr
835 840 845
Gly Cys Ala Gly Gly Gly Cys Gly Gly Cys Gly Cys Ala Gly Gly Cys
850 855 860
Gly Ala Cys Thr Gly Thr Cys Cys Gly Thr Ala Cys Ala Cys Cys Thr
865 870 875 880
Ala Cys Ala Thr Gly Cys Ala Thr Gly Ala Thr Cys Thr Cys Gly Cys
885 890 895
Cys Gly Cys Cys Gly Gly Cys Cys Gly Cys Thr Ala Cys Cys Gly Gly
900 905 910
Cys Thr Gly Cys Cys Thr Thr Gly Gly Gly Ala Gly Gly Ala Thr Gly
915 920 925
Ala Gly Gly Thr Gly Cys Thr Cys Ala Thr Cys Ala Ala Gly Gly Ala
930 935 940
Cys Gly Gly Cys Ala Cys Gly Ala Gly Cys Gly Ala Gly Gly Gly Gly
945 950 955 960
Thr Thr Cys Cys Cys Gly Cys Cys Gly Cys Cys Gly Gly Ala Gly Cys
965 970 975
Gly Gly Cys Gly Ala Thr Ala Thr Gly Gly Cys Gly Gly Ala Gly Cys
980 985 990
Gly Ala Ala Gly Gly Gly Ala Gly Gly Cys Gly Cys Gly Gly Cys Gly
995 1000 1005
Cys Gly Ala Gly Gly Thr Gly Gly Cys Gly Gly Thr Gly Gly Cys Thr
1010 1015 1020
Cys Gly Gly Gly Cys Gly Gly Thr Gly Gly Thr Gly Gly Gly Thr Cys
1025 1030 1035 1040
Gly Ala Thr Gly Cys Thr Gly Cys Cys Gly Ala Ala Ala Cys Thr Cys
1045 1050 1055
Gly Thr Thr Ala Thr Ala Ala Cys Thr Cys Ala Cys Cys Gly Ala Gly
1060 1065 1070
Thr Ala Cys Ala Cys Gly Ala Thr Gly Ala Gly Ala Thr Cys Cys Thr
1075 1080 1085
Gly Cys Ala Ala Cys Thr Gly Cys Thr Gly Gly Cys Gly Cys Cys Ala
1090 1095 1100
Cys Ala Thr Thr Gly Cys Gly Ala Gly Cys Thr Gly Gly Thr Gly Ala
1105 1110 1115 1120
Cys Cys Ala Ala Cys Cys Ala Gly Ala Cys Cys Gly Ala Cys Ala Gly
1125 1130 1135
Cys Ala Cys Gly Cys Thr Gly Ala Cys Gly Cys Gly Cys Gly Ala Gly
1140 1145 1150
Gly Ala Ala Ala Thr Thr Cys Thr Gly Cys Gly Cys Cys Gly Cys Thr
1155 1160 1165
Gly Thr Cys Gly Cys Gly Ala Thr Gly Cys Thr Cys Ala Gly Gly Cys
1170 1175 1180
Gly Ala Thr Gly Ala Thr Gly Gly Cys Gly Thr Thr Cys Ala Thr Gly
1185 1190 1195 1200
Cys Cys Cys Gly Ala Thr Cys Gly Gly Gly Thr Cys Gly Ala Thr Gly
1205 1210 1215
Cys Ala Gly Ala Cys Thr Thr Thr Cys Thr Thr Cys Ala Ala Gly Cys
1220 1225 1230
Cys Thr Gly Cys Cys Cys Thr Gly Ala Gly Cys Thr Gly Cys Gly Thr
1235 1240 1245
Gly Thr Ala Gly Thr Cys Gly Gly Cys Thr Gly Cys Gly Cys Gly Cys
1250 1255 1260
Thr Cys Ala Ala Gly Gly Gly Cys Thr Thr Cys Gly Ala Cys Ala Ala
1265 1270 1275 1280
Thr Thr Thr Cys Gly Ala Thr Gly Thr Gly Gly Ala Cys Gly Cys Cys
1285 1290 1295
Thr Gly Thr Ala Cys Thr Gly Cys Cys Cys Gly Cys Gly Gly Gly Gly
1300 1305 1310
Thr Cys Thr Gly Gly Cys Thr Gly Ala Cys Cys Thr Thr Cys Gly Thr
1315 1320 1325
Gly Cys Cys Thr Gly Ala Thr Cys Thr Gly Thr Thr Gly Ala Cys Gly
1330 1335 1340
Gly Thr Cys Cys Cys Gly Ala Cys Thr Gly Cys Cys Gly Ala Gly Cys
1345 1350 1355 1360
Thr Gly Gly Cys Gly Ala Thr Cys Gly Gly Ala Cys Thr Gly Gly Cys
1365 1370 1375
Gly Gly Thr Gly Gly Gly Gly Cys Thr Gly Gly Gly Gly Cys Gly Gly
1380 1385 1390
Cys Ala Thr Cys Thr Gly Cys Gly Gly Gly Cys Ala Gly Cys Ala Gly
1395 1400 1405
Ala Thr Gly Cys Gly Thr Thr Cys Gly Thr Cys Cys Gly Cys Thr Cys
1410 1415 1420
Thr Gly Gly Cys Gly Ala Gly Thr Thr Cys Cys Ala Gly Gly Gly Cys
1425 1430 1435 1440
Thr Gly Gly Cys Ala Ala Cys Cys Ala Cys Ala Gly Thr Thr Cys Thr
1445 1450 1455
Ala Cys Gly Gly Cys Ala Cys Gly Gly Gly Gly Cys Thr Gly Gly Ala
1460 1465 1470
Thr Ala Ala Cys Gly Cys Thr Ala Cys Gly Gly Thr Cys Gly Gly Cys
1475 1480 1485
Ala Thr Cys Cys Thr Thr Gly Gly Cys Ala Thr Gly Gly Gly Cys Gly
1490 1495 1500
Cys Cys Ala Thr Cys Gly Gly Ala Cys Thr Gly Gly Cys Cys Ala Thr
1505 1510 1515 1520
Gly Gly Cys Thr Gly Ala Gly Cys Gly Cys Thr Thr Gly Cys Ala Gly
1525 1530 1535
Gly Gly Ala Thr Gly Gly Gly Gly Cys Gly Cys Gly Ala Cys Cys Cys
1540 1545 1550
Thr Gly Cys Ala Gly Thr Ala Cys Cys Ala Cys Gly Ala Gly Gly Cys
1555 1560 1565
Gly Ala Ala Gly Gly Cys Thr Cys Thr Gly Gly Ala Thr Ala Cys Ala
1570 1575 1580
Cys Ala Ala Ala Cys Cys Gly Ala Gly Cys Ala Ala Cys Gly Gly Cys
1585 1590 1595 1600
Thr Cys Gly Gly Cys Cys Thr Gly Cys Gly Cys Cys Ala Gly Gly Thr
1605 1610 1615
Gly Gly Cys Gly Thr Gly Cys Ala Gly Cys Gly Ala Ala Cys Thr Cys
1620 1625 1630
Thr Thr Cys Gly Cys Cys Ala Gly Cys Thr Cys Gly Gly Ala Cys Thr
1635 1640 1645
Thr Cys Ala Thr Cys Cys Thr Gly Cys Thr Gly Gly Cys Gly Cys Thr
1650 1655 1660
Thr Cys Cys Cys Thr Thr Gly Ala Ala Thr Gly Cys Cys Gly Ala Thr
1665 1670 1675 1680
Ala Cys Cys Cys Ala Gly Cys Ala Thr Cys Thr Gly Gly Thr Cys Ala
1685 1690 1695
Ala Cys Gly Cys Cys Gly Ala Gly Cys Thr Gly Cys Thr Thr Gly Cys
1700 1705 1710
Cys Cys Thr Cys Gly Thr Ala Cys Gly Gly Cys Cys Gly Gly Gly Cys
1715 1720 1725
Gly Cys Thr Cys Thr Gly Cys Thr Thr Gly Thr Ala Ala Ala Cys Cys
1730 1735 1740
Cys Cys Thr Gly Thr Cys Gly Thr Gly Gly Thr Thr Cys Gly Gly Thr
1745 1750 1755 1760
Ala Gly Thr Gly Gly Ala Thr Gly Ala Ala Gly Cys Cys Gly Cys Cys
1765 1770 1775
Gly Thr Gly Cys Thr Cys Gly Cys Gly Gly Cys Gly Cys Thr Thr Gly
1780 1785 1790
Ala Gly Cys Gly Ala Gly Gly Cys Cys Ala Gly Cys Thr Cys Gly Gly
1795 1800 1805
Cys Gly Gly Gly Thr Ala Thr Gly Cys Gly Gly Cys Gly Gly Ala Thr
1810 1815 1820
Gly Thr Ala Thr Thr Cys Gly Ala Ala Ala Thr Gly Gly Ala Ala Gly
1825 1830 1835 1840
Ala Cys Thr Gly Gly Gly Cys Thr Cys Gly Cys Gly Cys Gly Gly Ala
1845 1850 1855
Cys Cys Gly Gly Cys Cys Gly Cys Gly Gly Cys Thr Gly Ala Thr Cys
1860 1865 1870
Gly Ala Thr Cys Cys Thr Gly Cys Gly Cys Thr Gly Cys Thr Cys Gly
1875 1880 1885
Cys Gly Cys Ala Thr Cys Cys Gly Ala Ala Thr Ala Cys Gly Cys Thr
1890 1895 1900
Gly Thr Thr Cys Ala Cys Thr Cys Cys Gly Cys Ala Cys Ala Thr Ala
1905 1910 1915 1920
Gly Gly Gly Thr Cys Gly Gly Cys Ala Gly Thr Gly Cys Gly Cys Gly
1925 1930 1935
Cys Gly Gly Thr Gly Cys Gly Cys Cys Thr Gly Gly Ala Gly Ala Thr
1940 1945 1950
Thr Gly Ala Ala Cys Gly Thr Thr Gly Thr Gly Cys Ala Gly Cys Gly
1955 1960 1965
Cys Ala Gly Ala Ala Cys Ala Thr Cys Ala Thr Cys Cys Ala Gly Gly
1970 1975 1980
Thr Ala Thr Thr Gly Gly Cys Ala Gly Gly Thr Gly Cys Gly Cys Gly
1985 1990 1995 2000
Cys Cys Cys Ala Ala Thr Cys Ala Ala Cys Gly Cys Thr Gly Cys Gly
2005 2010 2015
Ala Ala Cys Cys Gly Thr Cys Thr Gly Cys Cys Cys Ala Ala Gly Gly
2020 2025 2030
Cys Cys Gly Ala Gly Cys Cys Thr Gly Cys Cys Gly Cys Ala Thr Gly
2035 2040 2045
Thr

Claims (9)

1.一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，包括如下步骤：
（1）设计引物，PCR扩增含Linker序列的甲酰胺酶基因
上游引物A1(5'-CGC GGATCC GATGAACGGACTGGGCGGCTTGAAC-3')，下划线斜体为BamH I酶切点；下游引物A2(5'- CGACCCACCACCGCCCGAGCCACCGCCACCTCGCGCCGCGCCTCCCTTCGC -3')，下划线为Linker序列；
以类芽孢杆菌Paenibacillus pasadenensis. CS0611的基因组为模板，克隆出1041bp的含Linker序列的甲酰胺酶基因序列for-Linker；然后，以A1和B1（5'-TATAACGAGTTTCGGCAGCATCGACCCACCACCGCCCGAGCCA-3'）扩增for-Linker片段；所述类芽孢杆菌Paenibacillus pasadenensis. CS0611的含Linker序列的甲酰胺酶基因序列如序列表SEQ1所示，片段长度为1041 bp，含Linker序列，编码347个氨基酸；所述类芽孢杆菌Paenibacillus pasadenensis. CS0611于2014年10月8日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO：M2014458；
（2）设计引物，PCR扩增亚磷酸脱氢酶基因
上游引物B2（5'-TGGCTCGGGCGGTGGTGGGTCGATGCTGCCGAAACTCGTTATA-3'），下划线为Linker的部分DNA；下游引物B3（5'- CCG GAATTC CGACATGCGGCAGGCTCGGCCTTGGGC-3'），下划线斜体为EcoRⅠ 酶切点；
以肺炎克雷伯菌Klebsiella pneumonia. OU7的基因组为模板，克隆出1008bp的亚磷酸脱氢酶基因序列ptx，得到ptx片段；
（3）重叠PCR扩增得到融合基因
以步骤（1）、步骤（2）扩增得到的for-Linker片段和ptx片段为模板，以A1和引物B3分别为上游、下游引物，扩增得到甲酰胺酶与亚磷酸脱氢酶融合基因for-Linker-ptx；利用BamHI和EcoRⅠ对融合基因for-Linker-ptx进行双酶切，连接入利用相同酶进行酶切的pGEX-2T表达质粒，转化入DH5α，得到阳性克隆子DH5α（pGEX-for-Linker-ptx）；
（4）转化表达重组菌株
从重组大肠杆菌DH5α（pGEX-for-Linker-ptx）中提取重组质粒pGEX-for-Linker-ptx，并将重组质粒pGEX-for-Linker-ptx转化入大肠杆菌表达菌株BL21(DE3)中，得到重组表达菌株BL21(DE3)（pGEX-for-Linker-ptx）；
（5）诱导培养重组大肠杆菌
将得到的重组表达菌株BL21(DE3)（pGEX-for-Linker-ptx）接种到LB培养基中，进行诱导培养后，收集菌体并加入到含有甲酰胺和亚磷酸盐的MOPS培养基中，继续培养得到重组大肠杆菌。
2.根据权利要求1所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，步骤（2）中，所述肺炎克雷伯菌Klebsiella pneumonia. OU7的亚磷酸脱氢酶基因序列如序列表SEQ2所示，片段长度为1008 bp，编码336个氨基酸；所述肺炎克雷伯菌Klebsiella pneumonia. OU7于2017年8月24日保藏于中国典型培养物保藏中心，保藏号为CCTCC NO： M 2017449。
3.根据权利要求1所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，步骤（3）中，所述融合基因for-Linker-ptx的基因序列如序列表SEQ3所示，片段长度为2049 bp，位于5，端的是甲酰胺酶基因，位于3，端的是亚磷酸脱氢酶基因，甲酰胺酶基因片段与亚磷酸脱氢酶基因片段之间有片段长度30 bp的 Linker序列连接。
4.根据权利要求1所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，步骤（1）、（2）、（3）中，所述PCR扩增的条件均为：94℃反应5min；于98℃反应10s、55℃反应5s、72℃反应70s，并循环30次；再于72℃反应7min；最后降温至16℃。
5.根据权利要求1所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，步骤（5）中，所述诱导培养是在LB培养基中，37℃、180rpm培养12~16小时后，再接种于新鲜的LB培养基中，继续于37℃、180rpm培养至重组菌浓度至OD600=0.6，降温至20℃后，加入终浓度为0.2 mM 的异丙基-β-D-硫代半乳糖苷进行诱导 16 h。
6.根据权利要求1所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，步骤（5）中，所述收集菌体是将诱导培养结束后的菌液在4℃、8000rpm、离心5 min后，再用预冷至4℃的盐水悬浮，再次4℃、8000 rpm、离心5 min，重复用盐水悬浮并离心的步骤两次，收集菌体；所述含有甲酰胺和亚磷酸盐的MOPS培养基中，甲酰胺的终浓度为200 mM，亚磷酸盐的终浓度为1.32 mM。
7.根据权利要求1所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的大肠杆菌的构建方法，其特征在于，步骤（5）中，收集的菌体加入到含有甲酰胺和亚磷酸盐的MOPS培养基中后，菌体浓度OD600=0.1~0.15；所述继续培养是在培养基中加入终浓度为0.2 mM的异丙基-β-D-硫代半乳糖苷，并于30℃、180 rpm继续培养84~96小时。
8.由权利要求1~7任一项所述方法构建得到的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌。
9.权利要求8所述的一种表达甲酰胺酶与亚磷酸脱氢酶融合蛋白的重组大肠杆菌应用于大肠杆菌工业发酵合成包括抗体或者有价值的蛋白酶制剂。
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