CN114957442B - 一种具有促进大菱鲆卵巢发育功能的重组蛋白 - Google Patents
一种具有促进大菱鲆卵巢发育功能的重组蛋白 Download PDFInfo
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Abstract
本发明提供一种具有促进大菱鲆卵巢发育的重组蛋白，所提供的重组蛋白的氨基酸序列为SEQ ID NO:1或SEQ ID NO:2；改造后的重组蛋白的氨基酸序列为SEQ ID NO:6或SEQ ID NO:8。本发明所提供的重组蛋白可用于促进大菱鲆性腺发育。本发明采用重叠PCR技术将大菱鲆重组促性腺激素α亚基与β亚基进行拼接，采用毕赤酵母表达体系进行重组促性腺激素的表达，真核表达体系能进行复杂的翻译后修饰，有益于重组蛋白的活性。本发明成功表达的大菱鲆重组促性腺激素融合蛋白经过卵母细胞体外培养，可明显促进大菱鲆卵母细胞的成熟。
Description
技术领域
本发明属于海水鱼类养殖技术领域，具体涉及一种具有促进大菱鲆卵巢发 育功能的重组蛋白。
背景技术
大菱鲆(Scophthalmus maximus)是当前欧洲的重要海水养殖鱼类之一，自 1992年大菱鲆被引进中国以来，在水产科研工作者和养殖企业的共同努力下， 经过近30年发展，大菱鲆在工业化养殖领域取得了重大成效，产量稳定在5万 吨以上，年产值逾40亿元，从而成为我国水产养殖业一个新的经济增长点。
大菱鲆在人工养殖条件下，不能自发性产卵受精，在其繁殖过程中需要通 过光照和温度调控，促进卵巢的发育，而后通过注射相关生殖调控激素促进卵 母细胞细胞成熟后，通过人工挤卵的方式获取成熟卵子，进行体外受精孵化。 研究表明，卵母细胞的最后成熟和排卵受促性腺激素(卵泡刺激素FSH和黄体 生成素LH)直接调控，其中FSH刺激卵巢组织分泌产生雌二醇促进卵母细胞 成熟、LH促进卵母细胞由卵巢壁排入卵巢腔，因此，注射特异性促性腺激素 (FSH和LH)是稳定批量获取高质量成熟卵子的关键。
目前在大菱鲆苗种繁育和雌性亲鱼生殖调控实际生产过程中，并未研发出 针对大菱鲆雌性亲鱼的特异性促性腺激素蛋白，通常采用鱼类通用型催产催熟 激素对卵子进行催熟和催产，虽然能批量获取成熟卵子，但卵子质量和效果不 稳定，存在批次间差异，同时非特异性催产催熟激素也严重影响大菱鲆雌性亲 鱼卵巢发育，缩短雌性亲鱼生殖年限。同高等哺乳动物不同，促性腺激素是由 两个亚基形成的结构异二聚体糖蛋白，即由一个共有α亚基(CGα)与一个特 异性β(FSHβ、LHβ)亚基组成的异二聚体，特异性的β亚基赋予其功能特异 性。由于促性腺激素的特异性，两种亚基结合时才会产生生物学效应。而且鱼 类存在种属间差异，如何实现大菱鲆α亚基(CGα)与β亚基(FSHβ、LHβ) 连接后的高效表达，纯获得高活性的蛋白，进而用于亲鱼催产催熟，是目前急 需突破的技术瓶颈。
发明内容
本发明的目的是提供一种具有促进大菱鲆卵巢发育的重组蛋白，该重组蛋 白能够有效的促进大菱鲆卵巢发育，从而弥补现有技术的不足。
本发明首先提供一种重组蛋白，所述的重组蛋白为CF或CL， CF的氨基酸序列如下：
MVTATTTMGSVRSAVLSFLLLSFFLHIADSYPNIDASNMGCEECKLRKNS LFSGDHPVYQCMGCCFSKAYPTPLKTIKTMMIPKNITSEATCCVAKNSYESVF QTDVGVGIKVRNHTECHCSTCFFHKIMQLVVMAAVLAMAGTGQGCSLGCKL ANITLRVESCGVTEVIETTECSGLCHNQDPNYIGNGDMDEQKICNGDWSYEA KHINGCPVAARYPVASNCRCTTCDEDSTYCGRTPRYMPSCFSR(SEQ ID NO:1)；
CL的氨基酸序列如下：
MVTATTTMGSVRSAVLSFLLLSFFLHIADSYPNIDASNMGCEECKLRKNSLF SGDHPVYQCMGCCFSKAYPTPLKTIKTMMIPKNITSEATCCVAKNSYESVFQT DVGVGIKVRNHTECHCSTCFFHKIMMIHLTLLLGASFSVWPLAPAAALELPPC ELVNMTLSLEKEGCPRCHMVETTICSGHCRTKEPSIIFPHLKVYQHVCTYREL HYRTVQLPDCPAGVDPSVSYPEALSCHCNLCVTNMADCIRHEHREPDVCDN DLTFHV(SEQ ID NO:2)；
编码上述重组蛋白的基因，其中编码CF重组蛋白的基因，其序列如下：
ATGGTAACTGCTACAACCACAATGGGCTCAGTGAGATCAGCTGTACTGTCT TTTCTTCTGTTGTCTTTTTTTCTTCACATAGCTGATTCTTACCCCAACATCGA CGCATCAAACATGGGCTGTGAGGAGTGCAAGCTGAGAAAGAACTCTCTTT TCTCCGGGGACCATCCAGTCTACCAGTGCATGGGCTGCTGCTTCTCCAAAG CGTACCCGACACCACTCAAGACAATAAAGACAATGATGATCCCAAAGAAC ATCACCTCGGAGGCGACATGCTGTGTTGCAAAGAACAGCTATGAGTCAGT CTTTCAGACAGATGTGGGTGTCGGCATAAAAGTGAGAAACCACACGGAAT GCCACTGCAGCACCTGTTTTTTTCACAAGATAATGCAGCTGGTTGTCATGG CAGCAGTGCTGGCAATGGCGGGGACGGGACAGGGCTGCAGCCTCGGCTG CAAACTGGCCAACATCACTCTCCGCGTGGAGAGCTGCGGCGTCACCGAGG TGATCGAGACCACCGAGTGCAGCGGACTGTGCCACAACCAGGATCCCAAC TACATCGGCAATGGCGACATGGACGAACAGAAGATCTGCAACGGGGACTG GTCCTACGAGGCGAAGCACATTAACGGATGTCCGGTGGCCGCCAGGTACC CAGTGGCCTCAAACTGCAGGTGTACCACATGCGATGAAGACAGCACGTAC TGCGGACGCACTCCCAGATACATGCCCAGCTGCTTTTCCCGC(SEQ ID NO:3)；
其中编码CL重组蛋白的基因，其序列如下：
ATGGTAACTGCTACAACCACAATGGGCTCAGTGAGATCAGCTGTACTG TCTTTTCTTCTGTTGTCTTTTTTTCTTCACATAGCTGATTCTTACCCCAACAT CGACGCATCAAACATGGGCTGTGAGGAGTGCAAGCTGAGAAAGAACTCTC TTTTCTCCGGGGACCATCCAGTCTACCAGTGCATGGGCTGCTGCTTCTCCA AAGCGTACCCGACACCACTCAAGACAATAAAGACAATGATGATCCCAAAG AACATCACCTCGGAGGCGACATGCTGTGTTGCAAAGAACAGCTATGAGTC AGTCTTTCAGACAGATGTGGGTGTCGGCATAAAAGTGAGAAACCACACGG AATGCCACTGCAGCACCTGTTTTTTTCACAAGATAATGATGATCCATCTGAC GCTGCTCCTCGGGGCCTCGTTCTCCGTTTGGCCCTTGGCTCCTGCAGCGGC GTTGGAGCTGCCGCCCTGCGAGCTCGTCAACATGACGCTGTCTCTGGAGA AGGAAGGCTGTCCCCGGTGTCACATGGTGGAGACGACCATCTGCAGCGGC CACTGCAGAACCAAGGAACCCTCCATCATCTTTCCGCACTTGAAAGTGTAC CAGCACGTGTGCACGTACCGCGAGCTGCACTACAGGACGGTGCAGCTGCC GGACTGTCCCGCGGGCGTCGACCCCAGCGTGTCGTACCCGGAGGCGCTGA GCTGCCACTGCAACCTGTGCGTCACCAACATGGCGGACTGCATCCGACAC GAACACCGGGAGCCGGACGTCTGCGACAACGACCTCACGTTCCACGTG (SEQ IDNO:4)。
对上述的重组蛋白CF或CL进行改造，改造后的重组蛋白，其氨基酸序列 分别为SEQ ID NO:6和SEQ ID NO:8；其编码基因的核苷酸序列为SEQ ID NO:5和SEQ ID NO:7。
本发明还提供用于重组表达上述重组蛋白的工程菌株；
作为实施例的具体记载，所述的工程菌株为毕赤酵母GS115；
本发明所提供的重组蛋白可用于促进大菱鲆性腺发育。
本发明将大菱鲆促性腺激素α亚基与β亚基进行重组拼接，采用毕赤酵母 表达体系进行重组促性腺激素的表达。本发明成功表达的大菱鲆重组促性腺激 素融合蛋白经过卵母细胞体外培养，可明显促进大菱鲆卵母细胞的成熟。
附图说明
图1：目的基因扩增图，其中图a中1、2、3、4分别为LHβ、FSHβ、 CGα、18S；图b中CL表示CGα与LHβ融合片段，CF表示CGα与FSHβ融合 片段；
图2：双酶切质粒图，其中M为DL5000 DNA Marker，1为未酶切 pPICZA质粒，2为双酶切pPICZA质粒。
图3：阳性克隆菌落PCR验证图，其中M为DL2000 DNA Marker，1-5为 pPICZA-CL，6-9为pPICZA-CF。
图4：双酶切重组质粒图，其中(a)为双酶切pPICZA-CL，其中M为 DL5000 DNAMarker，1为双酶切pPICZA-CL，2为单酶切pPICZA-CL，3为 未酶切质粒。(b)为双酶切pPICZA-CF，其中M为DL2000 DNA Marker，1为 双酶切pPICZA-CF，2为未酶切质粒。
图5：大菱鲆纯化后重组促性腺激素SDS-PAGE图，
图6：大菱鲆卵母细胞体外成熟过程图，其中a:未成熟；b：半成熟-I；c： 半成熟-II；d:完全成熟；
图7：大菱鲆重组促性腺激素卵泡体外培养效果图，其中横坐标表示不同 培养时间，纵坐标表示卵母细胞GVBD，不同颜色的柱表示不同的激素；以空 白DMEM、生理盐水(Nacl)为阴性对照，孕酮(DHP)为阳性对照。
具体实施方式
本发明将大菱鲆促性腺激素(gonadtrophin honrmones，Gths)的CGα亚基分 别与β亚基(LHβ、FSHβ)进行融合拼接，克隆获取CL(CGα+LHβ)和CF (CGα+FSHβ)二聚体，并且与真核表达载体pPZICa成功拼接，构建重组真核 表达载体pPZICa-CL、pPZICa-CF；将重组表达载体pPZICa-CL、pPZICa-CF 成功导入GS115菌株中，构建重组菌株；进行诱导表达，明确了表达时间和诱 导剂浓度，大量培养后经过蛋白纯化与SDS-PAGE，获得高活性促性腺激素重组蛋白rCL、rCF；对雌性亲鱼卵泡进行体外孵育，结果表明融合蛋白对卵子具 有催熟和催产效果。
下面结合实施例和附图对本发明进行详细的描述。
实施例1：大菱鲆促性腺激素基因的获取
1)由大菱鲆垂体组织制备cDNA，采用引物的信息如下：
LHβ-F1：5′-ATGATGATCCATCTGACGC-3′、
LHβ-R1：5′-CACGTGGAACGTGAGGTCG-3′，
FSHβ-F1：5′-ATGCAGCTGGTTGTCATG-3′、
FSHβ-R1：5′-GTTGCAGATCTTCTGTTC-3′，
CGα-F1：5′-ATGGTAACTGCTACAACCAC-3′、
CGα-R1：5′-TATCTTGTGAAAAAAACAGG-3′。
从中克隆得到大菱鲆促性腺激素蛋白的编码序列，使用高保真酶进行PCR 扩增。PCR反应体系如下：
LHβPCR反应液：
PCR反应程序：94℃2min，(98℃10s 58℃15s 72℃40s)35个循 环，72℃10min。PCR产物采用1％琼脂糖凝胶验证，后取10μl送至北京擎科 生物科技有限公司进行测序。
FSHβPCR反应液：
PCR反应程序同LHβPCR反应。
PCR反应程序同LHβPCR反应。
采用重叠pcr(overlap pcr)技术扩增融合基因序列，以LHβ、CGα为模 板,CL-F1：5′-CCTGTTTTTTTCACAAGATATGATGATCCATCTGACGC-3′、CL- R1：5′-GCGTCAGATGGATCATCATCATATCTTGTGAAAAAAACAGG-3′PCR 扩增CL(CGα-LHβ)，以FSHβ、CGα为PCR反应模板，CF-F1：5′-CCTGTTTTTTTCACAAGATATGCAGCTGGTTGTCATG-3′、CF-R1：5′- CATGACAACCAGCTGCATATCTTGTGAAAAAAACAGG-3′扩增CF(CGα- FSHβ)片段，
pcr反应体系如下：
CL PCR反应液：
CF PCR反应液：
PCR反应程序同LHβPCR反应。PCR产物采用SteadyPure Agarose Gel DNAPurification Kit(AG21006)回收备用。
本步骤成功进行后，得到两段编码大菱鲆促性腺激素的编码序列，该碱基 序列是序列表中的SEQ ID NO:3、SEQ ID NO:4。由该碱基序列翻译得到大菱 鲆促性腺激素蛋白，氨基酸序列分别是序列表中的SEQ ID NO:1、SEQ ID NO:2。
实施例2、构建蛋白成熟肽重组表达质粒及工程菌株
1)对CF、CL蛋白基因序列翻译所得的氨基酸序列进行生物信息学分 析，通过与其它物种的类似功能蛋白的结构进行比较后，确定该蛋白需表达的 成熟肽。再对该蛋白成熟肽所对应的核酸序列通过DNA MAN软件进行分析， 确定核酸序列上所含有的内切酶的酶切位点。选用合适的质粒为表达该蛋白成 熟肽所需的载体，
2)将该蛋白成熟肽对应核酸序列上所含的酶切位点与该载体上的多克隆 位点上的酶切位点进行比对，选择表达载体上含有而目的核酸序列上没有的 NotI和EcoR I位点作为构建质粒的酶切位点。使用实施例1中获得的CL、CF 的PCR产物为模板，使用的引物的信息如下：
CL-F2：5′- AGAGAGGCTGAAGCTGAATTCATGGTAACTGCTACAACCACAATGG-3′、 CL-R2：5′-TGTTCTAGAAAGCTGGCGGCCGCCACGTGGAACGTGAGGTCG- 3′，CF-F2： 5′-AGAGAGGCTGAAGCTGAATTCATGGTAACTGCTACAACCACAATGG-3′、 CF-R2： 5′-TGTTCTAGAAAGCTGGCGGCCGCCGTTGCAGATCTTCTGTTC-3′。
用高保真酶Polymerase进行PCR扩增，反应体系、程序同上。产物采用 SteadyPureAgarose Gel DNA Purification Kit回收后，采用超微量分光光度计测 量其浓度，命名为CL、CF备用。
3)将含有pPICZA表达载体的甘油菌进行活化后扩大培养，采用Steady Pure质粒DNA提取试剂盒提取质粒，所提取质粒采用1％琼脂糖凝胶电泳验 证，以及超微量紫外分光光度计测定其浓度后，取10μl送至北京擎科生物科 技有限公司进行测序。根据本实验目的基因CL、CF序列上的酶切位点，以及 pPICZA表达载体的酶切位点，选用EcoRⅠ、NotⅠ作为限制性酶切位点。
双酶切pPICZA表达载体，酶切体系如下：
4)采用ClonExpress II One Step Cloning Kit(诺维赞，南京)进行重组载 体构建
重组反应的反应体系如下：
轻轻抽吸混匀后短暂离心，收集反应液至管底。37℃反应30min后，4℃ 冷却备用。
5)将感受态细胞DH5α提前置于冰上融化，取10μl重组产物与100μl感 受态细胞DH5α轻轻混匀，冰上30min、42℃热激45s、冰上冷却2min，后 加入900μlSOC培养基，37℃200rpm培养1h，1h后5000rpm离心5min,保 留100μl上清重悬菌体，涂布于LB(zeocin)平板37℃倒置培养12h-16h， 观察结果。观察过夜培养的LB(zeocin)平板，挑取单克隆于1ml LB(zeocin) 液体培养基中进行菌落PCR验证，200μl LB(zeocin)液体培养基5000rpm 10min，离心弃上清，适量无菌水清洗后，采用200μl无菌水悬浮菌体，95℃， 10min,冷却后作为菌落PCR模板，以通用引物5AOX、3AOX进行PCR扩 增，PCR反应程序同上。将阳性克隆扩大培养后提取重组pPICZA-CL、 pPICZA-CF质粒，采用超微量紫外分光光度计测定其浓度备用。
6)将构建成功的真核表达载体pPICZA-CL、pPICZA-CF，采用Quick& Easy YeastTransformation Mix(TAKARA)导入到毕赤酵母GS115菌株中，方法 如下：
将实验室保存的GS115菌株在YPD固体培养基活化。
活化后的GS115菌株挑取单克隆，采用300微升无菌水重悬，3000g离心 3min后弃上清，备用。
将YeastmakerTM Carrier DNA(10mg/ml)95℃变性5min后至于冰上备 用，
转化液配置：
100μl转化液重悬GS115酵母细胞，45℃孵育70min，使用无菌水进行 10倍稀释涂布于YPDZ平板，30℃培养，直至长出单克隆菌落。
将上述提取的大菱鲆促性腺激素活性蛋白的重组表达质粒转化到毕赤酵母GS115中，分别以载体引物和基因特异性引物进行PCR双向筛选。筛选出的阳 性克隆经序列鉴定后作为工程菌株。
实施例3：获取重组大菱鲆促性腺激素
1)将实施例2种阳性重组菌株GS115-pPZICa-CL、GS115-pPZICa-CF，取8 μl接种至5mlYPDZ液体培养基中，30℃200rpm，培养24h；取500ulYPDZ菌 液，接种至30ml BMGY培养基中，30℃200rpm，培养24h后测量OD值， 5000rpm 10min离心收集菌体；将菌体采用1ml BMMY培养基重选，取500ul 重悬菌液，加入至30mlBMMY培养基30℃200rpm培养。每隔24h补加甲醇 至终浓度为0.1％、0.5％、1％。每隔24h吸取1ml菌液，12000rpm 5min离心后 收集上清用于SDS-PAGE。
2)上清采用TCA法沉淀蛋白，流程如下：
吸取900μl上清移入1.5ml离心管，加入100μl 100％TCA，颠倒混匀后置 于冰上30min(可过夜)。15000g离心10min后可见棕黑色沉淀，弃上清，离 心管倒置吸水纸上，除尽残液。沉淀加入80μl 0.1M氢氧化钠溶液充分溶解， 用于SDS-PAGE；取溶解后的液体20μl与5μl 5*loading buffer混匀后，金属浴 95℃10min，冷却后上样；吸取20μl进行点样，电压设置为120V 45min，电 泳结束后采用考马斯亮蓝染液染色，摇床染色30min后进行脱色直至条带清 晰，拍照保存。
3)依照(2)诱导表达结果，选用最优表达条件(30℃、0.5％甲醇、72 h)扩大培养，将培养后的菌液GS115-pPZICa-CL、GS115-pPZICa-CF分别 8000rpm 4℃离心30min，收集发酵上清。
4)蛋白粗提：
(a)用硫酸铵沉淀蛋白法收集上清中的蛋白，即上清与饱和硫酸铵溶液 (SAS)按照1：1比例4℃搅拌6h，保证上清中的蛋白充分沉淀且不影响其 活性。
(b)将上述液体8000rpm 4℃离心30min后弃上清保留沉淀，沉淀采用适 量1*PBS缓冲液悬浮。
(c)透析除盐
根据rCL、rCF氨基酸大小选用3500分子量透析袋，首先进行透析袋前处 理：将透析袋剪成15cm小段，使用2％(W/V)碳酸氢钠和1mmol/L EDTA- Na2(pH8.0)将透析袋持续煮沸10min，使用蒸馏水彻底清洗透析袋后将透析袋 置于1mmol/L EDTA(pH8.0)中将其煮沸10min，冷却后置于4℃，备用。
使用透析袋前将透析袋内装满水后排出，彻底清洗干净。将PBS悬浮的粗 蛋白溶液分别加入到透析袋中，使用透析夹将两端夹住，保证内部液体不流 出。置于1L PBS缓冲液中进行透析，透析装置采用磁力搅拌器4℃进行24h 搅拌，实验过程中需更换透析液。透析结束后将透析样品使用移液枪转移至 15ml离心管中，-20℃备用。
5)重组蛋白rCL、rCF带有组氨酸(6*His)标签，因此选用镍离子亲和层 析柱，纯化柱以及试剂为HisTALON Superflow Cartridg(TAKARA)
纯化流程为：
(a)配置Wash Buffer：将9.34ml Equilibration Buffer与660μl ElutionBuffer 充分混匀。
(b)采用10ml Equilibration Buffer活化镍离子亲和层析柱。
(c将(4)透析后的蛋白粗体样品分别过柱纯化。
(d)采用8ml Equilibration Buffer与7ml Wash Buffer清洗层析柱。
(e)采用8ml Elution Buffer洗脱目的蛋白，收集流出液，用于测量rCL、 rCF蛋白质含量。
其中纯化后rCL、rCF最终蛋白浓度分别为：2.17μg/ml、2.48μg/ml。
选取大菱鲆雌性亲鱼，解剖后分离大菱鲆卵母细胞，挑选大小均一的卵粒 以每孔50粒的密度接种到24孔板中，使用DMEM高糖细胞培养液进行培 养，以生理盐水、空白DMEM培养基为阴性对照，以孕酮(DHP)为阳性对照， 分别加入不同浓度的重组蛋白rCL、rCF培养卵母细胞，于1、3、6、9、12、 15、18、24h八个时间点观察大菱鲆卵母细胞成熟率。
如图6所示。卵母细胞成熟过程分为四种阶段：未成熟卵母细胞；半成熟- I阶段卵母细胞；半成熟-II阶段卵母细胞；完全成熟卵母细胞。成熟率以100 个未成熟卵母细胞中经过处理后达到完全成熟阶段卵母细胞个数计算统计。
如图7结果所示：rCL和rCF处理处理15小时后才能发挥对卵母细胞促 成熟作用，但促熟率未超过20％，且随着处理进程进行，其促熟效果不稳定。
实施例4：对重组蛋白进行序列优化
在分析了rCL、rCF的基础上行，将CGα分别截取21和24个核苷酸序列 后，得到编码大菱鲆促性腺激素的改造后的序列。
将编码rCF重组蛋白的基因进行改造，改造后的基因命名为rCsF，其核苷 酸序列如下：
ATGGTAACTGCTACAACCACAATGGGCTCAGTGAGATCAGCTGTACTG TCTTTTCTTCTGTTGTCTTTTTTTCTTCACATAGCTGATTCTTACCCCAACAT CGAGGAGTGCAAGCTGAGAAAGAACTCTCTTTTCTCCGGGGACCATCCAG TCTACCAGTGCATGGGCTGCTGCTTCTCCAAAGCGTACCCGACACCACTCA AGACAATAAAGACAATGATGATCCCAAAGAACATCACCTCGGAGGCGACA TGCTGTGTTGCAAAGAACAGCTATGAGTCAGTCTTTCAGACAGATGTGGGT GTCGGCATAAAAGTGAGAAACCACACGGAATGCCACTGCAGCACCTGTTT TTTTCACAAGATAATGCAGCTGGTTGTCATGGCAGCAGTGCTGGCAATGGC GGGGACGGGACAGGGCTGCAGCCTCGGCTGCAAACTGGCCAACATCACT CTCCGCGTGGAGAGCTGCGGCGTCACCGAGGTGATCGAGACCACCGAGTG CAGCGGACTGTGCCACAACCAGGATCCCAACTACATCGGCAATGGCGACA TGGACGAACAGAAGATCTGCAACGGGGACTGGTCCTACGAGGCGAAGCA CATTAACGGATGTCCGGTGGCCGCCAGGTACCCAGTGGCCTCAAACTGCA GGTGTACCACATGCGATGAAGACAGCACGTACTGCGGACGCACTCCCAGA TACATGCCCAGCTGCTTTTCCCGC(SEQ ID NO:5)；
其编码蛋白的氨基酸序列如下：
MVTATTTMGSVRSAVLSFLLLSFFLHIADSYPNIEECKLRKNSLFSGDHPV YQCMGCCFSKAYPTPLKTIKTMMIPKNITSEATCCVAKNSYESVFQTDVGVGI KVRNHTECHCSTCFFHKIMQLVVMAAVLAMAGTGQGCSLGCKLANITLRVE SCGVTEVIETTECSGLCHNQDPNYIGNGDMDEQKICNGDWSYEAKHINGCPV AARYPVASNCRCTTCDEDSTYCGRTPRYMPSCFSR(SEQ ID NO:6)。
将编码rCL重组蛋白进行改造，改造后的基因命名为rCsL，其核苷酸序列 如下：
ATGGTAACTGCTACAACCACAATGGGCTCAGTGAGATCAGCTGTACTG TCTTTTCTTCTGTTGTCTTTTTTTCTTCACATAGCTGATTCTTACCCCAACAT CGACGCATCAAACATGGGCTGTGAGGAGTGCAAGCTGAGAAAGAACTCTC ACCAGTGCATGGGCTGCTGCTTCTCCAAAGCGTACCCGACACCACTCAAG ACAATAAAGACAATGATGATCCCAAAGAACATCACCTCGGAGGCGACATG CTGTGTTGCAAAGAACAGCTATGAGTCAGTCTTTCAGACAGATGTGGGTGT CGGCATAAAAGTGAGAAACCACACGGAATGCCACTGCAGCACCTGTTTTT TTCACAAGATAATGATGATCCATCTGACGCTGCTCCTCGGGGCCTCGTTCTC CGTTTGGCCCTTGGCTCCTGCAGCGGCGTTGGAGCTGCCGCCCTGCGAGC TCGTCAACATGACGCTGTCTCTGGAGAAGGAAGGCTGTCCCCGGTGTCAC ATGGTGGAGACGACCATCTGCAGCGGCCACTGCAGAACCAAGGAACCCTC CATCATCTTTCCGCACTTGAAAGTGTACCAGCACGTGTGCACGTACCGCGA GCTGCACTACAGGACGGTGCAGCTGCCGGACTGTCCCGCGGGCGTCGACC CCAGCGTGTCGTACCCGGAGGCGCTGAGCTGCCACTGCAACCTGTGCGTC ACCAACATGGCGGACTGCATCCGACACGAACACCGGGAGCCGGACGTCTG CGACAACGACCTCACGTTCCACGTG(SEQ ID NO:7)。
其编码蛋白的氨基酸序列如下：
MVTATTTMGSVRSAVLSFLLLSFFLHIADSYPNIDASNMGCEECKLRKNS HQCMGCCFSKAYPTPLKTIKTMMIPKNITSEATCCVAKNSYESVFQTDVGVGI KVRNHTECHCSTCFFHKIMMIHLTLLLGASFSVWPLAPAAALELPPCELVNMT LSLEKEGCPRCHMVETTICSGHCRTKEPSIIFPHLKVYQHVCTYRELHYRTVQ LPDCPAGVDPSVSYPEALSCHCNLCVTNMADCIRHEHREPDVCDNDLTFHV (SEQ ID NO:8)。
按照实施例3的方法表达重组促性腺激素rCsL、rCsF，最终蛋白浓度分别 为3.27μg/ml、4.56μg/ml。
实施例5：大菱鲆重组促性腺激素功能验证-体外细胞培养
按照实施例3的方法，选取大菱鲆雌性亲鱼，解剖后分离大菱鲆卵母细 胞，挑选大小均一的卵粒以每孔50粒的密度接种到24孔板中，使用DMEM 高糖细胞培养液进行培养，以生理盐水、空白DMEM培养基为阴性对照，以 孕酮(DHP)为阳性对照，分别加入不同浓度的重组蛋白rCL、rCF、rCsL、rCsF 培养卵母细胞，于1、3、6、9、12、15、18、24h八个时间点观察大菱鲆卵母 细胞发育情况，通过观察卵母细胞生发泡破裂(GVBD)过程，统计卵母细胞 成熟率。
由图7可知，所得重组蛋白能够显著促进大菱鲆卵母细胞成熟，在卵母细 胞体外培养15h时重组促性腺激素rCL、rCF、rCsL、rCsF促进GVBD现象显 著高于DHP组,同时rCsL、rCsF促成熟率高于rCL、rCF。表明重组蛋白 rCL、rCF、rCsL、rCsF具有明显的生物学效应，可显著促进大菱鲆卵母细胞成 熟，其中rCsL、rCsF效果更佳。
序列表
<110> 中国水产科学研究院黄海水产研究所
<120> 一种具有促进大菱鲆卵巢发育功能的重组蛋白
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 249
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
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Asn Ile Asp Ala Ser Asn Met Gly Cys Glu Glu Cys Lys Leu Arg Lys
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Asn Ser Leu Phe Ser Gly Asp His Pro Val Tyr Gln Cys Met Gly Cys
50 55 60
Cys Phe Ser Lys Ala Tyr Pro Thr Pro Leu Lys Thr Ile Lys Thr Met
65 70 75 80
Met Ile Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys
85 90 95
Asn Ser Tyr Glu Ser Val Phe Gln Thr Asp Val Gly Val Gly Ile Lys
100 105 110
Val Arg Asn His Thr Glu Cys His Cys Ser Thr Cys Phe Phe His Lys
115 120 125
Ile Met Gln Leu Val Val Met Ala Ala Val Leu Ala Met Ala Gly Thr
130 135 140
Gly Gln Gly Cys Ser Leu Gly Cys Lys Leu Ala Asn Ile Thr Leu Arg
145 150 155 160
Val Glu Ser Cys Gly Val Thr Glu Val Ile Glu Thr Thr Glu Cys Ser
165 170 175
Gly Leu Cys His Asn Gln Asp Pro Asn Tyr Ile Gly Asn Gly Asp Met
180 185 190
Asp Glu Gln Lys Ile Cys Asn Gly Asp Trp Ser Tyr Glu Ala Lys His
195 200 205
Ile Asn Gly Cys Pro Val Ala Ala Arg Tyr Pro Val Ala Ser Asn Cys
210 215 220
Arg Cys Thr Thr Cys Asp Glu Asp Ser Thr Tyr Cys Gly Arg Thr Pro
225 230 235 240
Arg Tyr Met Pro Ser Cys Phe Ser Arg
245
<210> 2
<211> 268
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Val Thr Ala Thr Thr Thr Met Gly Ser Val Arg Ser Ala Val Leu
1 5 10 15
Ser Phe Leu Leu Leu Ser Phe Phe Leu His Ile Ala Asp Ser Tyr Pro
20 25 30
Asn Ile Asp Ala Ser Asn Met Gly Cys Glu Glu Cys Lys Leu Arg Lys
35 40 45
Asn Ser Leu Phe Ser Gly Asp His Pro Val Tyr Gln Cys Met Gly Cys
50 55 60
Cys Phe Ser Lys Ala Tyr Pro Thr Pro Leu Lys Thr Ile Lys Thr Met
65 70 75 80
Met Ile Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys
85 90 95
Asn Ser Tyr Glu Ser Val Phe Gln Thr Asp Val Gly Val Gly Ile Lys
100 105 110
Val Arg Asn His Thr Glu Cys His Cys Ser Thr Cys Phe Phe His Lys
115 120 125
Ile Met Met Ile His Leu Thr Leu Leu Leu Gly Ala Ser Phe Ser Val
130 135 140
Trp Pro Leu Ala Pro Ala Ala Ala Leu Glu Leu Pro Pro Cys Glu Leu
145 150 155 160
Val Asn Met Thr Leu Ser Leu Glu Lys Glu Gly Cys Pro Arg Cys His
165 170 175
Met Val Glu Thr Thr Ile Cys Ser Gly His Cys Arg Thr Lys Glu Pro
180 185 190
Ser Ile Ile Phe Pro His Leu Lys Val Tyr Gln His Val Cys Thr Tyr
195 200 205
Arg Glu Leu His Tyr Arg Thr Val Gln Leu Pro Asp Cys Pro Ala Gly
210 215 220
Val Asp Pro Ser Val Ser Tyr Pro Glu Ala Leu Ser Cys His Cys Asn
225 230 235 240
Leu Cys Val Thr Asn Met Ala Asp Cys Ile Arg His Glu His Arg Glu
245 250 255
Pro Asp Val Cys Asp Asn Asp Leu Thr Phe His Val
260 265
<210> 3
<211> 747
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggtaactg ctacaaccac aatgggctca gtgagatcag ctgtactgtc ttttcttctg 60
ttgtcttttt ttcttcacat agctgattct taccccaaca tcgacgcatc aaacatgggc 120
tgtgaggagt gcaagctgag aaagaactct cttttctccg gggaccatcc agtctaccag 180
tgcatgggct gctgcttctc caaagcgtac ccgacaccac tcaagacaat aaagacaatg 240
atgatcccaa agaacatcac ctcggaggcg acatgctgtg ttgcaaagaa cagctatgag 300
tcagtctttc agacagatgt gggtgtcggc ataaaagtga gaaaccacac ggaatgccac 360
tgcagcacct gtttttttca caagataatg cagctggttg tcatggcagc agtgctggca 420
atggcgggga cgggacaggg ctgcagcctc ggctgcaaac tggccaacat cactctccgc 480
gtggagagct gcggcgtcac cgaggtgatc gagaccaccg agtgcagcgg actgtgccac 540
aaccaggatc ccaactacat cggcaatggc gacatggacg aacagaagat ctgcaacggg 600
gactggtcct acgaggcgaa gcacattaac ggatgtccgg tggccgccag gtacccagtg 660
gcctcaaact gcaggtgtac cacatgcgat gaagacagca cgtactgcgg acgcactccc 720
agatacatgc ccagctgctt ttcccgc 747
<210> 4
<211> 804
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atggtaactg ctacaaccac aatgggctca gtgagatcag ctgtactgtc ttttcttctg 60
ttgtcttttt ttcttcacat agctgattct taccccaaca tcgacgcatc aaacatgggc 120
tgtgaggagt gcaagctgag aaagaactct cttttctccg gggaccatcc agtctaccag 180
tgcatgggct gctgcttctc caaagcgtac ccgacaccac tcaagacaat aaagacaatg 240
atgatcccaa agaacatcac ctcggaggcg acatgctgtg ttgcaaagaa cagctatgag 300
tcagtctttc agacagatgt gggtgtcggc ataaaagtga gaaaccacac ggaatgccac 360
tgcagcacct gtttttttca caagataatg atgatccatc tgacgctgct cctcggggcc 420
tcgttctccg tttggccctt ggctcctgca gcggcgttgg agctgccgcc ctgcgagctc 480
gtcaacatga cgctgtctct ggagaaggaa ggctgtcccc ggtgtcacat ggtggagacg 540
accatctgca gcggccactg cagaaccaag gaaccctcca tcatctttcc gcacttgaaa 600
gtgtaccagc acgtgtgcac gtaccgcgag ctgcactaca ggacggtgca gctgccggac 660
tgtcccgcgg gcgtcgaccc cagcgtgtcg tacccggagg cgctgagctg ccactgcaac 720
ctgtgcgtca ccaacatggc ggactgcatc cgacacgaac accgggagcc ggacgtctgc 780
gacaacgacc tcacgttcca cgtg 804
<210> 5
<211> 726
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atggtaactg ctacaaccac aatgggctca gtgagatcag ctgtactgtc ttttcttctg 60
ttgtcttttt ttcttcacat agctgattct taccccaaca tcgaggagtg caagctgaga 120
aagaactctc ttttctccgg ggaccatcca gtctaccagt gcatgggctg ctgcttctcc 180
aaagcgtacc cgacaccact caagacaata aagacaatga tgatcccaaa gaacatcacc 240
tcggaggcga catgctgtgt tgcaaagaac agctatgagt cagtctttca gacagatgtg 300
ggtgtcggca taaaagtgag aaaccacacg gaatgccact gcagcacctg tttttttcac 360
aagataatgc agctggttgt catggcagca gtgctggcaa tggcggggac gggacagggc 420
tgcagcctcg gctgcaaact ggccaacatc actctccgcg tggagagctg cggcgtcacc 480
gaggtgatcg agaccaccga gtgcagcgga ctgtgccaca accaggatcc caactacatc 540
ggcaatggcg acatggacga acagaagatc tgcaacgggg actggtccta cgaggcgaag 600
cacattaacg gatgtccggt ggccgccagg tacccagtgg cctcaaactg caggtgtacc 660
acatgcgatg aagacagcac gtactgcgga cgcactccca gatacatgcc cagctgcttt 720
tcccgc 726
<210> 6
<211> 242
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Val Thr Ala Thr Thr Thr Met Gly Ser Val Arg Ser Ala Val Leu
1 5 10 15
Ser Phe Leu Leu Leu Ser Phe Phe Leu His Ile Ala Asp Ser Tyr Pro
20 25 30
Asn Ile Glu Glu Cys Lys Leu Arg Lys Asn Ser Leu Phe Ser Gly Asp
35 40 45
His Pro Val Tyr Gln Cys Met Gly Cys Cys Phe Ser Lys Ala Tyr Pro
50 55 60
Thr Pro Leu Lys Thr Ile Lys Thr Met Met Ile Pro Lys Asn Ile Thr
65 70 75 80
Ser Glu Ala Thr Cys Cys Val Ala Lys Asn Ser Tyr Glu Ser Val Phe
85 90 95
Gln Thr Asp Val Gly Val Gly Ile Lys Val Arg Asn His Thr Glu Cys
100 105 110
His Cys Ser Thr Cys Phe Phe His Lys Ile Met Gln Leu Val Val Met
115 120 125
Ala Ala Val Leu Ala Met Ala Gly Thr Gly Gln Gly Cys Ser Leu Gly
130 135 140
Cys Lys Leu Ala Asn Ile Thr Leu Arg Val Glu Ser Cys Gly Val Thr
145 150 155 160
Glu Val Ile Glu Thr Thr Glu Cys Ser Gly Leu Cys His Asn Gln Asp
165 170 175
Pro Asn Tyr Ile Gly Asn Gly Asp Met Asp Glu Gln Lys Ile Cys Asn
180 185 190
Gly Asp Trp Ser Tyr Glu Ala Lys His Ile Asn Gly Cys Pro Val Ala
195 200 205
Ala Arg Tyr Pro Val Ala Ser Asn Cys Arg Cys Thr Thr Cys Asp Glu
210 215 220
Asp Ser Thr Tyr Cys Gly Arg Thr Pro Arg Tyr Met Pro Ser Cys Phe
225 230 235 240
Ser Arg
<210> 7
<211> 780
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
atggtaactg ctacaaccac aatgggctca gtgagatcag ctgtactgtc ttttcttctg 60
ttgtcttttt ttcttcacat agctgattct taccccaaca tcgacgcatc aaacatgggc 120
tgtgaggagt gcaagctgag aaagaactct caccagtgca tgggctgctg cttctccaaa 180
gcgtacccga caccactcaa gacaataaag acaatgatga tcccaaagaa catcacctcg 240
gaggcgacat gctgtgttgc aaagaacagc tatgagtcag tctttcagac agatgtgggt 300
gtcggcataa aagtgagaaa ccacacggaa tgccactgca gcacctgttt ttttcacaag 360
ataatgatga tccatctgac gctgctcctc ggggcctcgt tctccgtttg gcccttggct 420
cctgcagcgg cgttggagct gccgccctgc gagctcgtca acatgacgct gtctctggag 480
aaggaaggct gtccccggtg tcacatggtg gagacgacca tctgcagcgg ccactgcaga 540
accaaggaac cctccatcat ctttccgcac ttgaaagtgt accagcacgt gtgcacgtac 600
cgcgagctgc actacaggac ggtgcagctg ccggactgtc ccgcgggcgt cgaccccagc 660
gtgtcgtacc cggaggcgct gagctgccac tgcaacctgt gcgtcaccaa catggcggac 720
tgcatccgac acgaacaccg ggagccggac gtctgcgaca acgacctcac gttccacgtg 780
<210> 8
<211> 260
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Val Thr Ala Thr Thr Thr Met Gly Ser Val Arg Ser Ala Val Leu
1 5 10 15
Ser Phe Leu Leu Leu Ser Phe Phe Leu His Ile Ala Asp Ser Tyr Pro
20 25 30
Asn Ile Asp Ala Ser Asn Met Gly Cys Glu Glu Cys Lys Leu Arg Lys
35 40 45
Asn Ser His Gln Cys Met Gly Cys Cys Phe Ser Lys Ala Tyr Pro Thr
50 55 60
Pro Leu Lys Thr Ile Lys Thr Met Met Ile Pro Lys Asn Ile Thr Ser
65 70 75 80
Glu Ala Thr Cys Cys Val Ala Lys Asn Ser Tyr Glu Ser Val Phe Gln
85 90 95
Thr Asp Val Gly Val Gly Ile Lys Val Arg Asn His Thr Glu Cys His
100 105 110
Cys Ser Thr Cys Phe Phe His Lys Ile Met Met Ile His Leu Thr Leu
115 120 125
Leu Leu Gly Ala Ser Phe Ser Val Trp Pro Leu Ala Pro Ala Ala Ala
130 135 140
Leu Glu Leu Pro Pro Cys Glu Leu Val Asn Met Thr Leu Ser Leu Glu
145 150 155 160
Lys Glu Gly Cys Pro Arg Cys His Met Val Glu Thr Thr Ile Cys Ser
165 170 175
Gly His Cys Arg Thr Lys Glu Pro Ser Ile Ile Phe Pro His Leu Lys
180 185 190
Val Tyr Gln His Val Cys Thr Tyr Arg Glu Leu His Tyr Arg Thr Val
195 200 205
Gln Leu Pro Asp Cys Pro Ala Gly Val Asp Pro Ser Val Ser Tyr Pro
210 215 220
Glu Ala Leu Ser Cys His Cys Asn Leu Cys Val Thr Asn Met Ala Asp
225 230 235 240
Cys Ile Arg His Glu His Arg Glu Pro Asp Val Cys Asp Asn Asp Leu
245 250 255
Thr Phe His Val
260
Claims (8)
1.一种重组蛋白，其特征在于，所述的重组蛋白的氨基酸序列为SEQ ID NO:6或SEQ IDNO:8。
2.一种基因，其特征在于，所述的基因用于编码权利要求1所述的重组蛋白。
3.如权利要求2所述的基因，其特征在于，所述的基因，其中编码氨基酸序列为SEQ IDNO:6的重组蛋白的基因，其核苷酸序列为SEQ ID NO:5；编码氨基酸序列为SEQ ID NO:8的重组蛋白的基因，其核苷酸序列为SEQ ID NO:7。
4.一种重组表达载体，其特征在于，所述的重组表达载体为插入有权利要求3所述的基因的表达载体。
5.一种重组工程菌，其特征在于，所述的重组工程菌为携带有权利要求4所述的重组表达载体的工程菌株。
6.一种制备权利要求1所述的重组蛋白的方法，其特征在于，所述的方法是使用权利要求5所述的重组工程菌来发酵制备重组蛋白。
7.权利要求1所述的重组蛋白在制备促进大菱鲆性腺发育的制品中的应用。
8.一种用于促进大菱鲆性腺发育的制品，其特征在于，所述的制品中包含有药理有效浓度的权利要求1所述的重组蛋白。
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| CN101067118A (zh) * | 2007-04-24 | 2007-11-07 | 新疆农业科学院微生物应用研究所 | 一种重组牛促卵泡激素在毕赤酵母中的表达 |
| CN102225968A (zh) * | 2011-05-09 | 2011-10-26 | 中山大学 | 重组花鳗鲡促卵泡刺激素FSHβα及其制备方法和应用 |
| CN107540748A (zh) * | 2017-09-15 | 2018-01-05 | 北京伟杰信生物科技有限公司 | 长效重组猪fsh融合蛋白及其制备方法与应用 |
| CN110938656A (zh) * | 2019-12-24 | 2020-03-31 | 中国大熊猫保护研究中心 | 重组表达大熊猫促卵泡生成素的载体、表达系统及制备方法 |
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| JPH1036399A (ja) * | 1996-07-24 | 1998-02-10 | Denki Kagaku Kogyo Kk | 新規な性腺刺激ホルモン及びその製造法 |
| CN101067118A (zh) * | 2007-04-24 | 2007-11-07 | 新疆农业科学院微生物应用研究所 | 一种重组牛促卵泡激素在毕赤酵母中的表达 |
| CN102225968A (zh) * | 2011-05-09 | 2011-10-26 | 中山大学 | 重组花鳗鲡促卵泡刺激素FSHβα及其制备方法和应用 |
| CN107540748A (zh) * | 2017-09-15 | 2018-01-05 | 北京伟杰信生物科技有限公司 | 长效重组猪fsh融合蛋白及其制备方法与应用 |
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