CN114181321B - 花鲈fgf6a、fgf6b以及fgf18重组蛋白及其制备方法和应用 - Google Patents
花鲈fgf6a、fgf6b以及fgf18重组蛋白及其制备方法和应用 Download PDFInfo
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- CN114181321B CN114181321B CN202111505223.XA CN202111505223A CN114181321B CN 114181321 B CN114181321 B CN 114181321B CN 202111505223 A CN202111505223 A CN 202111505223A CN 114181321 B CN114181321 B CN 114181321B
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- fgf6b
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Abstract
本发明提供了一种花鲈FGF6A、FGF6B以及FGF18重组蛋白及其制备方法和应用，花鲈FGF6A、FGF6B以及FGF18重组蛋白，包含：FGF6A、FGF6B以及FGF18成熟肽氨基酸序列，以及连接到FGF6A、FGF6B、FGF18成熟肽氨基酸序列的N端的标签肽段，标签肽段包含组氨基酸标签序列和促溶标签序列。本发明还提供了编码该重组蛋白的基因、表达该重组蛋白的载体和重组工程菌。本发明还进一步提供了重组蛋白的制备方法，以及该重组蛋白在花鲈育种的应用。本发明的重组蛋白可通过原核细菌大量诱导表达获取可溶性FGF6A、FGF6B、FGF18重组蛋白，且具有活性，有利于后续工业化提取和纯化。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种大肠杆菌表达系统生产可溶的、有生物活性的花鲈FGF(FGF6A、FGF6B以及FGF18)重组蛋白、制备方法及其在花鲈育种中的应用。
背景技术
花鲈(Lateolabrax maculatus)，俗称海鲈，隶属鲈形目(Perciforms)、鮨科(Serranidae)、花鲈属(Lateolabrax)，其肉质细嫩、味道鲜美、营养价值高，广受消费者喜爱。花鲈为典型的广温、广盐性鱼类，抗逆、抗病能力强，能很好的利用人工配合饲料，在池塘、工厂化、网箱等水体中均可良好生长，具有非常广阔的养殖前景。近年来，我国花鲈养殖年产量均超过15万吨，位居海水养殖鱼类产量前三位，已成为海水养殖支柱产业。然而，花鲈的遗传育种工作起步较晚，目前养殖用亲鱼来源单一且未进行科学选育，累代繁殖后种质退化现象已经显现，良种的缺乏将对花鲈产业的健康可持续发展产生严重的不利影响。急需开展花鲈遗传改良的研究工作，为优质高产的花鲈良种培育提供理论和技术支撑。
生长性状是鱼类遗传改良最有价值的经济性状之一。作为鱼类最大的组织，骨骼肌的生长对鱼类躯体的生长起到了决定性的作用。骨骼肌由多核肌纤维组成，肌纤维的增生取决于卫星细胞，卫星细胞作为动物骨骼肌中的干细胞，被激活后可以进行增殖并分化成肌纤维。因此，骨骼肌细胞的发育分化进程显著影响鱼类的生长表现。成纤维细胞生长因子(Fibroblast growth factors,FGFs)是一类重要的细胞信号蛋白，人体细胞中存在22个该家族成员，其功能主要包括参与细胞增殖、分化、迁移、凋亡等多种生物学过程。研究表明，在哺乳动物中，FGFs作为一种促分裂原素参与骨骼肌细胞的发育调控。在信号传导过程中，FGFs结合并激活跨膜的受体蛋白—成纤维细胞生长因子受体(Fibroblast growthfactor receptors,FGFRs)，进而将信号传递到细胞内发挥作用。据报道，在哺乳动物中FGF6及FGF18能与FGFR4结合，参与骨骼肌细胞增殖、分化过程。FGFs-FGFR通路调控骨骼肌细胞增殖、分化的分子机制研究仍处于探索阶段，在鱼类中的相关研究仍属空白。
发明内容
针对现有技术中的不足，本发明的目的是提供一种花鲈FGF6A、FGF6B以及FGF18重组蛋白及其制备方法和应用。
为达到上述目的，本发明的解决方案是：
一种花鲈FGF6A、FGF6B以及FGF18重组蛋白，其包括：FGF6A、FGF6B以及FGF18成熟肽氨基酸序列，以及连接到FGF6A、FGF6B以及FGF18成熟肽氨基酸序列的N端的标签肽段。
其中，标签肽段包括组氨基酸标签序列和促溶标签序列。
作为本发明的优选实施例，FGF6A、FGF6B以及FGF18成熟肽氨基酸序列分别为SEQID NO.1、SEQ ID NO.2和SEQ ID NO.3。
作为本发明的优选实施例，促溶标签序列为小泛素相关修饰蛋白(SUMO)序列。
作为本发明的优选实施例，SUMO氨基酸序列为SEQ ID NO.4。
作为本发明的优选实施例，组氨基酸标签序列为六聚组氨酸(His6)。
作为本发明的优选实施例，花鲈FGF6A、FGF6B以及FGF18重组蛋白的氨基酸序列分别为SEQ ID NO.5、SEQ ID NO.6和SEQ ID NO.7。
编码上述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的基因，其包括：编码FGF6A、FGF6B以及FGF18成熟肽氨基酸的基因序列，以及连接到FGF6A、FGF6B以及FGF18成熟肽氨基酸基因序列的5'端的标签肽段的编码基因序列，标签肽段的编码基因序列包括组氨基酸标签编码基因序列和促溶标签编码基因序列。
作为本发明的优选实施例，编码FGF6A、FGF6B以及FGF18成熟肽氨基酸对应的核苷酸序列分别为SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10。
作为本发明的优选实施例，编码FGF6A、FGF6B以及FGF18重组蛋白对应的核苷酸序列分别为SEQ ID NO.11、SEQ ID NO.12和SEQ ID NO.13。
一种用于表达上述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的表达载体，其包含花鲈FGF6A、FGF6B以及FGF18重组蛋白的编码基因和骨架质粒，骨架质粒为pET-28a(+)。
一种用于表达上述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的重组工程菌，其包括上述的表达载体。
作为本发明的优选实施例，重组工程菌的宿主菌选自BL21(DE3)。
一种花鲈FGF6A、FGF6B以及FGF18重组蛋白的制备方法，其包括如下步骤：
1)构建上述的FGF6A、FGF6B以及FGF18重组蛋白编码基因，然后连到到骨架质粒上构建得到FGF6A、FGF6B以及FGF18重组蛋白的表达载体；
2)将表达载体转化到宿主菌中，诱导表达FGF6A、FGF6B以及FGF18重组蛋白；
3)基于FGF6A、FGF6B以及FGF18重组蛋白中的组氨酸标签亲和层析分离得到FGF6A、FGF6B以及FGF18重组蛋白。
作为本发明的优选实施例，步骤1)中，编码FGF6A、FGF6B以及FGF18成熟肽氨基酸对应的核苷酸序列分别为SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10。
作为本发明的优选实施例，步骤1)中，骨架质粒为pET-28a(+)。
作为本发明的优选实施例，步骤2)中，宿主菌选自BL21(DE3)。
一种上述的花鲈FGF6A、FGF6B以及FGF18重组蛋白在花鲈育种中的应用。
由于采用上述方案，本发明的有益效果是：
本发明首次构建了花鲈FGF6A、FGF6B以及FGF18的原核表达载体，并且将该载体转染BL21(DE3)大肠杆菌，得到的pET-28a(+)-SUMO-FGF-BL21(DE3)表达菌株，能大量诱导表达获取可溶的重组蛋白。通过蛋白质标签亲和纯化后，可得到重组蛋白。本发明的方法能够得到氨基酸序列与FGF6A、FGF6B以及FGF18成熟肽一致的花鲈FGF6A、FGF6B以及FGF18重组蛋白，该花鲈FGF6A、FGF6B以及FGF18重组蛋白能够用于花鲈生长相关研究。另外，本发明的重组蛋白可通过原核细菌大量诱导表达获取可溶性FGF6A、FGF6B、FGF18重组蛋白，且具有活性，有利于后续工业化提取和纯化。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。而且在整个附图中，用相同的参考符号表示相同的部件。在附图中：
图1为花鲈FGF基因开放阅读框(ORF)克隆；其中，A、B、C分别为FGF6A、FGF6B、FGF18基因ORF核苷酸序列以及翻译的氨基酸序列，灰色区域为FGF6A、FGF6B以及FGF18成熟肽氨基酸序列。
图2为花鲈FGF6A、FGF6B以及FGF18重组蛋白基因序列及翻译的氨基酸序列；其中：深灰色区域：His6标签；中灰色区域：SUMO标签；浅灰色区域：花鲈FGF6A、FGF6B以及FGF18成熟肽。
图3为FGF6A、FGF6B以及FGF18重组蛋白表达、纯化SDS-PAGE凝胶电泳图；其中，M：蛋白分子量标准；1：IPTG诱导前细菌总蛋白；2：0.15mmol/L IPTG 20℃诱导16h后细菌总蛋白；3：诱导后细菌裂解液沉淀；4：诱导后细菌裂解液上清；5：TED-Ni亲和纯化流穿液；6-7：洗杂液；8-9：洗脱液(含目的蛋白)。
图4为花鲈FGF6A、FGF6B以及FGF18重组蛋白刺激诱导分化的骨骼肌细胞3d后细胞早期分化标记基因myog的mRNA相对表达水平；其中，A：FGF6A基因；B：FGF6B基因；C：FGF18基因。
具体实施方式
本发明提供了一种花鲈FGF6A、FGF6B以及FGF18重组蛋白及其制备方法和应用。
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
实施例1：花鲈FGF6A、FGF6B以及FGF18表达载体pET-28a(+)-SUMO-FGF的构建
以花鲈肌肉cDNA为模板，使用引物FGF6A-F/R、FGF6B-F/R、FGF18-F/R(生工，上海)和试剂2×Phanta Max Master Mix(诺唯赞，南京)进行PCR扩增出花鲈FGF6A、FGF6B以及FGF18基因的开放阅读框(ORF)的DNA序列。通过TOPO连接将其连入pCE2 TA/Blunt Zero载体(诺唯赞，南京)转染DH5α大肠杆菌，氨苄固体培养基筛选单菌落，桑格测序、NCBI BLAST鉴定序列是否无误。以上述花鲈FGF6A、FGF6B以及FGF18单克隆菌液为模板，使用引物PE-FGF-F/R和2×Phanta Max Master Mix进行PCR扩增，得到带有同源臂的FGF6A、FGF6B以及FGF18成熟肽对应的双链DNA片段；同时将pET-28a(+)-SUMO载体用限制性核酸内切酶BamHI和Xho I(NEB，美国)双酶切线性化。使用FastPure Gel DNA Extraction Mini Kit(诺唯赞，南京)将PCR产物以及双酶切产物胶回收，用无缝克隆试剂盒(碧云天，上海)连接插入片段和线性载体，转染DH5α感受态大肠杆菌，氨苄固体培养基筛选单菌落，桑格测序确认无误后，使用质粒小提中量试剂盒(天根，北京)抽提质粒，即为花鲈FGF6A、FGF6B以及FGF18原核表达载体。
表1载体构建所使用的引物序列图表
注：引物小写字母示同源臂，用于同源重组连接构建原核表达载体
实施例2：花鲈FGF6A、FGF6B以及FGF18大肠杆菌表达菌株的制备
将表达载体pET-28a(+)-SUMO-FGF转染BL21(DE3)感受态大肠杆菌(昂羽，上海)，用卡那霉素抗性LB固体培养基37℃培养过夜筛选单菌落，即为花鲈FGF6A、FGF6B以及FGF18原核表达工程菌pET-28a(+)-SUMO-FGF-BL21(DE3)。挑取单菌落在10mL卡那霉素抗性LB液体培养基中37℃，220转/分培养过夜，然后按照1:100的比例接种在100mL卡那霉素抗性LB液体培养基中，37℃，220转/分培养至OD600到0.4-0.5，然后加入IPTG使其终浓度为0.15mmol/L(取出50mL菌液作为诱导前的对照)，20℃，120转/分培养12h后4000×g离心10min收集菌体，弃上清，菌体用5mL非变性裂菌缓冲液(500mmol/L NaCl，20mmol/L PB，pH为7.4)重悬，加入终浓度1mg/mL溶菌酶，冰上裂解30min，60W超声破碎15min，超5s停5s，12000×g离心10min分别收集上清和沉淀，沉淀用5mL非变性裂菌缓冲液重悬，分别取80μL样品和20μL 5×蛋白上样缓冲液混匀，金属浴95℃、10min，进行SDS-PAGE验证蛋白是否正确表达(图3中样品1-4)。
实施例3：花鲈FGF6A、FGF6B以及FGF18重组蛋白可溶表达、蛋白纯化
按照1:100的比例接种在500mL卡那霉素抗性LB液体培养基中，37℃，220转/分培养至OD600到0.4-0.5，然后加入IPTG使其终浓度为0.15mmol/L，20℃，120转/分培养12h后4000×g离心10min收集菌体，弃上清，菌体用50mL非变性裂菌缓冲液重悬，加入终浓度1mg/mL溶菌酶，冰上裂解30min，60W超声破碎15min，超5s停5s，12000×g离心10min弃沉淀，保留上清，用0.45μm或0.22μm滤膜过滤除去不溶的微颗粒，使用TED-Ni填料(爱必信，上海)进行His标签亲和纯化，得到FGF6A、FGF6B以及FGF18重组蛋白(图3中样品8-9)，可用于花鲈活体注射或者刺激离体细胞。
实施例4：FGF6A、FGF6B以及FGF18重组蛋白刺激花鲈骨骼肌细胞，q-PCR检测细胞早期分化标记基因myog的mRNA相对表达量。
采集花鲈侧线以上、第一背鳍以下的肌肉，体外培养花鲈骨骼肌细胞。待细胞长好后，用含有20％FBS、1％(青霉素、链霉素、庆大霉素)的L15培养基将细胞浓度稀释为5×105细胞/mL，然后铺在六孔板中，每个孔加入1mL细胞稀释液和1mL的L15培养基，放在25℃的培养箱中，培养过夜后，换成2mL的无FBS的L15培养基饥饿12h，然后再加入含2％马血清、1％(青霉素、链霉素、庆大霉素)的L15培养基培养基诱导细胞分化，向处理组中每孔分别加入FGF6A、FGF6B以及FGF18蛋白，终浓度为4nmol/mL，对照组加入等浓度的空载蛋白，在培养箱中培养3d，每天更换一次培养基和FGF。3d后，收集细胞，使用TRIzol试剂(诺唯赞，南京)提取细胞总RNA。使用逆转录试剂盒(诺唯赞，南京)合成cDNA。然后，使用SYBR Green试剂盒(诺唯赞，南京)对3个重复样品进行实时定量PCR，以花鲈α-tublin作为内参基因，采用2-△△CT算法估算肌细胞早期分化标记基因myog的mRNA相对表达量。结果如图4所示。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
序列表
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Thr Asp Ala Thr Ser Leu Glu Lys Arg Trp Glu Thr Leu Phe Ser Arg
130 135 140
Ser Val Leu Gly Ile Ser Gly Glu Lys Pro Glu Leu Asn Trp Glu Ser
145 150 155 160
Asp Tyr Leu Leu Gly Ile Lys Arg Val Arg Arg Leu Tyr Cys Asn Val
165 170 175
Gly Ile Gly Phe His Leu Gln Val Leu Pro Asp Gly Gly Ile Asn Gly
180 185 190
Ala His Asn Glu Asn Gln Tyr Ser Leu Ile Glu Ile Ser Thr Val Glu
195 200 205
Arg Gly Val Val Ser Leu Tyr Gly Val Lys Ser Glu Leu Phe Val Ala
210 215 220
Met Asn Ser Arg Gly Arg Leu Tyr Gly Thr Thr Val Phe His Asp Glu
225 230 235 240
Cys Lys Phe Lys Glu Ser Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu
245 250 255
Ser Leu Val Tyr Arg Gly Ser Tyr Ile Ala Leu Ser Lys His Gly Arg
260 265 270
Val Lys Arg Gly Asn Lys Ala Thr Thr Ala Met Thr Val Thr His Phe
275 280 285
Leu Pro Arg Ile
290
<210> 6
<211> 291
<212> PRT
<213> 人工序列(Artficial Sequence)
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu
20 25 30
Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn
35 40 45
Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys
50 55 60
Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly
65 70 75 80
Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln
85 90 95
Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile
100 105 110
Glu Ala His Arg Glu Gln Ile Gly Gly Tyr Pro Ile Pro Ser Arg Thr
115 120 125
Asn Ala Thr Leu Met Glu Lys Lys Trp Glu Thr Leu Phe Ser Arg Ser
130 135 140
Tyr Leu Gly Ile Thr Gly Ser Lys Ser Glu Leu Asn Trp Glu Ser Asp
145 150 155 160
Tyr Leu Gln Gly Ile Lys Arg Val Arg Arg Leu Tyr Cys Asn Val Gly
165 170 175
Ile Gly Phe His Leu Gln Val Leu Pro Asp Gly Arg Ile Ser Gly Ala
180 185 190
His Ser Glu Asn Gln Tyr Ser Leu Ile Glu Ile Ser Thr Val Asp Arg
195 200 205
Gly Val Ile Ser Leu Phe Gly Val Arg Ser Glu Leu Phe Val Ala Met
210 215 220
Asn Ser Arg Gly Arg Leu Tyr Gly Thr Arg Val Phe Val Asp Glu Cys
225 230 235 240
Lys Phe Lys Glu Thr Leu Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser
245 250 255
Phe Val Tyr Lys Gly Phe Tyr Ile Ala Leu Ser Lys His Gly Arg Val
260 265 270
Lys Arg Gly Asn Lys Ala Thr Thr Val Met Thr Val Thr His Phe Leu
275 280 285
Pro Arg Leu
290
<210> 7
<211> 300
<212> PRT
<213> 人工序列(Artficial Sequence)
<400> 7
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Ser Asp Ser Glu Val Asn Gln Glu
20 25 30
Ala Lys Pro Glu Val Lys Pro Glu Val Lys Pro Glu Thr His Ile Asn
35 40 45
Leu Lys Val Ser Asp Gly Ser Ser Glu Ile Phe Phe Lys Ile Lys Lys
50 55 60
Thr Thr Pro Leu Arg Arg Leu Met Glu Ala Phe Ala Lys Arg Gln Gly
65 70 75 80
Lys Glu Met Asp Ser Leu Arg Phe Leu Tyr Asp Gly Ile Arg Ile Gln
85 90 95
Ala Asp Gln Thr Pro Glu Asp Leu Asp Met Glu Asp Asn Asp Ile Ile
100 105 110
Glu Ala His Arg Glu Gln Ile Gly Gly Val Asn Phe Ser Val His Val
115 120 125
Glu Asn Gln Thr Gln Val Arg Asp Thr Met Ser Arg Arg His His Arg
130 135 140
Val Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Val Gln Val Leu
145 150 155 160
Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly Asp Lys Tyr Ala Gln
165 170 175
Leu Val Val Glu Ala Asp Thr Phe Gly Ser Gln Val Arg Ile Arg Gly
180 185 190
Lys Glu Thr Asn Phe Tyr Leu Cys Met Asn Arg Arg Gly Lys Leu Val
195 200 205
Gly Lys Lys Ala Ser Asn Arg Ser Ala Asp Cys Val Phe Val Glu Lys
210 215 220
Val Leu Glu Asn His Tyr Thr Ala Leu Met Ser Ala Arg Tyr Thr Gly
225 230 235 240
Trp Tyr Val Gly Phe Thr Lys Arg Gly Arg Pro Arg Arg Gly Pro His
245 250 255
Thr Leu Pro Asn Gln Gln Asp Val His Phe Met Lys Arg Phe Pro Pro
260 265 270
Gly Glu Gln Pro Asp Leu Thr Thr Pro Phe Arg Phe Thr Thr Val Ser
275 280 285
Lys Arg Gly Lys Arg Val Arg Ala Thr Gly Pro Arg
290 295 300
<210> 8
<211> 516
<212> DNA
<213> 人工序列(Artficial Sequence)
<400> 8
tacccgctgc cgagcggcag gaccgacgca acttcgctgg agaaacgatg ggagaccctg 60
ttctcccgct ccgtgctggg gatctccggg gagaaaccgg agctcaactg ggagagcgac 120
tatctgctgg gcatcaagag agtgcggcgg ctctactgca acgtgggcat cgggtttcac 180
ctccaggtcc tccccgacgg cgggataaac ggtgcacata atgaaaacca gtacagtcta 240
atagagatct ccacggtgga gagaggagtg gtgagcctgt atggggtgaa gagtgagctg 300
tttgtcgcaa tgaacagccg cgggaggtta tacggaacga cagtcttcca tgacgagtgc 360
aagttcaagg agagcttgct cccgaacaac tacaacgcct acgagtctct ggtttacaga 420
ggctcctaca tagcactcag caagcatggc cgcgtgaaga ggggcaacaa ggccaccact 480
gccatgactg taacgcactt cctaccccga atatga 516
<210> 9
<211> 513
<212> DNA
<213> 人工序列(Artficial Sequence)
<400> 9
tatccgattc cgagcaggac taatgcgact ttaatggaaa agaagtggga gacgctcttc 60
tcccgctcct acctgggtat aaccgggtcg aaatcggagc tgaactggga gagtgactat 120
ttgcagggca tcaaaagagt gcggcggctc tactgcaacg tgggcattgg gtttcacctg 180
caggtgctcc cggatggcag gataagcggt gcacacagtg agaaccaata cagtctaata 240
gaaatctcca ccgtggaccg aggagtgatc agcctgttcg gggtgaggag cgagctgttt 300
gtcgcaatga acagcagggg aaggttatac ggaacgagag tcttcgtgga tgagtgcaag 360
ttcaaggaga ctttgctgcc caacaattac aacgcctacg agtcttttgt ttacaagggc 420
ttctatattg ccctcagcaa gcatggccgc gtaaagagag gcaacaaggc caccaccgtc 480
atgactgtca cacatttcct cccacgacta tga 513
<210> 10
<211> 540
<212> DNA
<213> 人工序列(Artficial Sequence)
<400> 10
gtcaacttca gcgtgcatgt ggagaaccag acgcaggtgc gagacaccat gagtcggcga 60
caccaccggg tctaccagct ctacagccgc accagtggca aacacgtcca ggtgctggga 120
cgcagaataa gcgctcgagg agaagatgga gacaaatatg cccagctcgt agtggaggcc 180
gatacctttg gtagccaggt gagaatccgg ggcaaagaaa ccaatttcta cctgtgcatg 240
aaccgccgcg gaaagctggt tggaaagaag gccagtaatc gaagcgccga ctgcgtcttt 300
gtggaaaagg ttctggaaaa ccactacacg gctctgatgt cggcgcgcta cacaggatgg 360
tacgtgggct tcaccaagag aggccgccct cgccgtggcc cccacacgct ccccaaccag 420
caggacgtac acttcatgaa gcgcttcccg cccggggagc agcccgacct caccacgccc 480
ttccgcttca ccaccgtcag caagcggggc aagagggtgc gcgctactgg gccccgctag 540
<210> 11
<211> 879
<212> DNA
<213> 人工序列(Artficial Sequence)
<400> 11
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggctagca tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa 120
gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc 180
aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt 240
aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc 300
cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt 360
ggatacccgc tgccgagcgg caggaccgac gcaacttcgc tggagaaacg atgggagacc 420
ctgttctccc gctccgtgct ggggatctcc ggggagaaac cggagctcaa ctgggagagc 480
gactatctgc tgggcatcaa gagagtgcgg cggctctact gcaacgtggg catcgggttt 540
cacctccagg tcctccccga cggcgggata aacggtgcac ataatgaaaa ccagtacagt 600
ctaatagaga tctccacggt ggagagagga gtggtgagcc tgtatggggt gaagagtgag 660
ctgtttgtcg caatgaacag ccgcgggagg ttatacggaa cgacagtctt ccatgacgag 720
tgcaagttca aggagagctt gctcccgaac aactacaacg cctacgagtc tctggtttac 780
agaggctcct acatagcact cagcaagcat ggccgcgtga agaggggcaa caaggccacc 840
actgccatga ctgtaacgca cttcctaccc cgaatataa 879
<210> 12
<211> 876
<212> DNA
<213> 人工序列(Artficial Sequence)
<400> 12
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggctagca tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa 120
gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc 180
aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt 240
aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc 300
cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt 360
ggatatccga ttccgagcag gactaatgcg actttaatgg aaaagaagtg ggagacgctc 420
ttctcccgct cctacctggg tataaccggg tcgaaatcgg agctgaactg ggagagtgac 480
tatttgcagg gcatcaaaag agtgcggcgg ctctactgca acgtgggcat tgggtttcac 540
ctgcaggtgc tcccggatgg caggataagc ggtgcacaca gtgagaacca atacagtcta 600
atagaaatct ccaccgtgga ccgaggagtg atcagcctgt tcggggtgag gagcgagctg 660
tttgtcgcaa tgaacagcag gggaaggtta tacggaacga gagtcttcgt ggatgagtgc 720
aagttcaagg agactttgct gcccaacaat tacaacgcct acgagtcttt tgtttacaag 780
ggcttctata ttgccctcag caagcatggc cgcgtaaaga gaggcaacaa ggccaccacc 840
gtcatgactg tcacacattt cctcccacga ctataa 876
<210> 13
<211> 903
<212> DNA
<213> 人工序列(Artficial Sequence)
<400> 13
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggctagca tgtcggactc agaagtcaat caagaagcta agccagaggt caagccagaa 120
gtcaagcctg agactcacat caatttaaag gtgtccgatg gatcttcaga gatcttcttc 180
aagatcaaaa agaccactcc tttaagaagg ctgatggaag cgttcgctaa aagacagggt 240
aaggaaatgg actccttaag attcttgtac gacggtatta gaattcaagc tgatcagacc 300
cctgaagatt tggacatgga ggataacgat attattgagg ctcacagaga acagattggt 360
ggagtcaact tcagcgtgca tgtggagaac cagacgcagg tgcgagacac catgagtcgg 420
cgacaccacc gggtctacca gctctacagc cgcaccagtg gcaaacacgt ccaggtgctg 480
ggacgcagaa taagcgctcg aggagaagat ggagacaaat atgcccagct cgtagtggag 540
gccgatacct ttggtagcca ggtgagaatc cggggcaaag aaaccaattt ctacctgtgc 600
atgaaccgcc gcggaaagct ggttggaaag aaggccagta atcgaagcgc cgactgcgtc 660
tttgtggaaa aggttctgga aaaccactac acggctctga tgtcggcgcg ctacacagga 720
tggtacgtgg gcttcaccaa gagaggccgc cctcgccgtg gcccccacac gctccccaac 780
cagcaggacg tacacttcat gaagcgcttc ccgcccgggg agcagcccga cctcaccacg 840
cccttccgct tcaccaccgt cagcaagcgg ggcaagaggg tgcgcgctac tgggccccgc 900
taa 903
Claims (12)
1.一种花鲈FGF6A、FGF6B以及FGF18重组蛋白，其特征在于：包括：FGF6A、FGF6B以及FGF18成熟肽氨基酸序列，以及连接到所述FGF6A、FGF6B以及FGF18成熟肽氨基酸序列的N端的标签肽段；
所述标签肽段包括六聚组氨酸标签和促溶标签小泛素相关修饰蛋白（SUMO）；
所述FGF6A、FGF6B以及FGF18成熟肽氨基酸序列分别为SEQ ID NO.1、SEQ ID NO.2和SEQ ID NO.3；
所述小泛素相关修饰蛋白（SUMO）的氨基酸序列为SEQ ID NO.4。
2.根据权利要求1所述的花鲈FGF6A、FGF6B以及FGF18重组蛋白，其特征在于：所述花鲈FGF6A、FGF6B以及FGF18重组蛋白的氨基酸序列分别为SEQ ID NO.5、SEQ ID NO.6和SEQ IDNO.7。
3.编码如权利要求1或2所述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的基因，其特征在于：包括：编码FGF6A、FGF6B以及FGF18成熟肽氨基酸的基因序列，以及连接到所述FGF6A、FGF6B以及FGF18成熟肽氨基酸基因序列的5'端的标签肽段的编码基因序列，所述标签肽段的编码基因序列包括组氨基酸标签编码基因序列和促溶标签编码基因序列。
4.根据权利要求3所述的基因，其特征在于：所述编码FGF6A、FGF6B以及FGF18成熟肽氨基酸对应的核苷酸序列分别为SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10。
5.根据权利要求3所述的基因，其特征在于：所述编码FGF6A、FGF6B以及FGF18重组蛋白对应的核苷酸序列分别为SEQ ID NO.11、SEQ ID NO.12和SEQ ID NO.13。
6.一种用于表达如权利要求1或2所述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的表达载体，其特征在于：包含如权利要求5所述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的编码基因和骨架质粒，所述骨架质粒为pET-28a（+）。
7.一种用于表达如权利要求1或2所述的花鲈FGF6A、FGF6B以及FGF18重组蛋白的重组工程菌，其特征在于：包括如权利要求6所述的表达载体。
8.根据权利要求7所述的重组工程菌，其特征在于：所述重组工程菌的宿主菌选自BL21（DE3）。
9.一种花鲈FGF6A、FGF6B以及FGF18重组蛋白的制备方法，其特征在于：其包括如下步骤：
1）构建如权利要求5所述的FGF6A、FGF6B以及FGF18重组蛋白编码基因，然后连到到骨架质粒上构建得到FGF6A、FGF6B以及FGF18重组蛋白的表达载体；
2）将所述表达载体转化到宿主菌中，诱导表达FGF6A、FGF6B以及FGF18重组蛋白；
3）基于所述FGF6A、FGF6B以及FGF18重组蛋白中的组氨酸标签亲和层析分离得到FGF6A、FGF6B以及FGF18重组蛋白。
10.根据权利要求9所述的制备方法，其特征在于：步骤1）中，所述编码FGF6A、FGF6B以及FGF18成熟肽氨基酸对应的核苷酸序列分别为SEQ ID NO.8、SEQ ID NO.9和SEQ IDNO.10。
11.根据权利要求9所述的制备方法，其特征在于：步骤1）中，所述骨架质粒为pET-28a（+）。
12.根据权利要求9所述的制备方法，其特征在于：步骤2）中，所述宿主菌选自BL21（DE3）。
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