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。
技术实现要素：
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paenibacilluspasadenensis.cs0611的含linker序列的甲酰胺酶基因for-linker和肺炎克雷伯菌klebsiellapneumonia.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
以类芽孢杆菌paenibacilluspasadenensis.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-19tsimple上，再将重组载体转化到大肠杆菌dh5α，得到含有重组载体pmd-19tsimple-for-linker的重组大肠杆菌dh5α(pmd-19tsimple-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
以肺炎克雷伯菌klebsiellapneumonia.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bamhi和ecorⅰ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paenibacilluspasadenensis.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paenibacilluspasadenensis.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cctccno：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klebsiellapneumonia.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klebsiellapneumonia.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cctccno：m2017449。
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℃、180rpm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℃、8000rpm、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hpo42-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paenibacilluspasadenensis.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cctccno：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klebsiellapneumonia.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cctccno：m2017449，且肺炎克雷伯菌klebsiellapneumonia.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paenibacilluspasadenensis.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paenibacilluspasadenensis.cs0611基因组进行提取；
以提取的paenibacilluspasadenensis.cs0611基因组为模板，以
pcr所用的酶试剂为takara公司的
pcr反应液组成(25μl)
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klebsiellapneumonia.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klebsiellapneumonia.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
pcr所用的酶试剂为takara公司的
pcr反应液组成(25μl)
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
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ecori进行双酶切，上游引物下划线斜体为bamhi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：
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thermofisherscientific公司的t4dnaligase，连接体系如下：
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890μllb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1ml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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cysalacysglycysthrglyalacysglycysglycysglyalagly
114011451150
glyalaalaalathrthrcysthrglycysglycyscysglycysthr
115511601165
glythrcysglycysglyalathrglycysthrcysalaglyglycys
117011751180
glyalathrglyalathrglyglycysglythrthrcysalathrgly
1185119011951200
cyscyscysglyalathrcysglyglyglythrcysglyalathrgly
120512101215
cysalaglyalacysthrthrthrcysthrthrcysalaalaglycys
122012251230
cysthrglycyscyscysthrglyalaglycysthrglycysglythr
123512401245
glythralaglythrcysglyglycysthrglycysglycysglycys
125012551260
thrcysalaalaglyglyglycysthrthrcysglyalacysalaala
1265127012751280
thrthrthrcysglyalathrglythrglyglyalacysglycyscys
128512901295
thrglythralacysthrglycyscyscysglycysglyglyglygly
130013051310
thrcysthrglyglycysthrglyalacyscysthrthrcysglythr
131513201325
glycyscysthrglyalathrcysthrglythrthrglyalacysgly
133013351340
glythrcyscyscysglyalacysthrglycyscysglyalaglycys
1345135013551360
thrglyglycysglyalathrcysglyglyalacysthrglyglycys
136513701375
glyglythrglyglyglyglycysthrglyglyglyglycysglygly
138013851390
cysalathrcysthrglycysglyglyglycysalaglycysalagly
139514001405
alathrglycysglythrthrcysglythrcyscysglycysthrcys
141014151420
thrglyglycysglyalaglythrthrcyscysalaglyglyglycys
1425143014351440
thrglyglycysalaalacyscysalacysalaglythrthrcysthr
144514501455
alacysglyglycysalacysglyglyglyglycysthrglyglyala
146014651470
thralaalacysglycysthralacysglyglythrcysglyglycys
147514801485
alathrcyscysthrthrglyglycysalathrglyglyglycysgly
149014951500
cyscysalathrcysglyglyalacysthrglyglycyscysalathr
1505151015151520
glyglycysthrglyalaglycysglycysthrthrglycysalagly
152515301535
glyglyalathrglyglyglyglycysglycysglyalacyscyscys
154015451550
thrglycysalaglythralacyscysalacysglyalaglyglycys
155515601565
glyalaalaglyglycysthrcysthrglyglyalathralacysala
157015751580
cysalaalaalacyscysglyalaglycysalaalacysglyglycys
1585159015951600
thrcysglyglycyscysthrglycysglycyscysalaglyglythr
160516101615
glyglycysglythrglycysalaglycysglyalaalacysthrcys
162016251630
thrthrcysglycyscysalaglycysthrcysglyglyalacysthr
163516401645
thrcysalathrcyscysthrglycysthrglyglycysglycysthr
165016551660
thrcyscyscysthrthrglyalaalathrglycyscysglyalathr
1665167016751680
alacyscyscysalaglycysalathrcysthrglyglythrcysala
168516901695
alacysglycyscysglyalaglycysthrglycysthrthrglycys
170017051710
cyscysthrcysglythralacysglyglycyscysglyglyglycys
171517201725
glycysthrcysthrglycysthrthrglythralaalaalacyscys
173017351740
cyscysthrglythrcysglythrglyglythrthrcysglyglythr
1745175017551760
alaglythrglyglyalathrglyalaalaglycyscysglycyscys
176517701775
glythrglycysthrcysglycysglyglycysglycysthrthrgly
178017851790
alaglycysglyalaglyglycyscysalaglycysthrcysglygly
179518001805
cysglyglyglythralathrglycysglyglycysglyglyalathr
181018151820
glythralathrthrcysglyalaalaalathrglyglyalaalagly
1825183018351840
alacysthrglyglyglycysthrcysglycysglycysglyglyala
184518501855
cyscysglyglycyscysglycysglyglycysthrglyalathrcys
186018651870
glyalathrcyscysthrglycysglycysthrglycysthrcysgly
187518801885
cysglycysalathrcyscysglyalaalathralacysglycysthr
189018951900
glythrthrcysalacysthrcyscysglycysalacysalathrala
1905191019151920
glyglyglythrcysglyglycysalaglythrglycysglycysgly
192519301935
cysglyglythrglycysglycyscysthrglyglyalaglyalathr
194019451950
thrglyalaalacysglythrthrglythrglycysalaglycysgly
195519601965
cysalaglyalaalacysalathrcysalathrcyscysalaglygly
197019751980
thralathrthrglyglycysalaglyglythrglycysglycysgly
1985199019952000
cyscyscysalaalathrcysalaalacysglycysthrglycysgly
200520102015
alaalacyscysglythrcysthrglycyscyscysalaalaglygly
202020252030
cyscysglyalaglycyscysthrglycyscysglycysalathrgly
203520402045
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