CN108264549A - 重组绒促性素rhCG的制备方法及应用 - Google Patents
重组绒促性素rhCG的制备方法及应用 Download PDFInfo
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- CN108264549A CN108264549A CN201810271691.7A CN201810271691A CN108264549A CN 108264549 A CN108264549 A CN 108264549A CN 201810271691 A CN201810271691 A CN 201810271691A CN 108264549 A CN108264549 A CN 108264549A
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Abstract
本发明涉及重组绒促性素rhCG的制备方法,利用哺乳动物真核表达***表达重组绒促性素rhCG;所述重组绒促性素rhCG包含α和β亚基,二者通过范德华力连接。本发明的重组绒促性素rhCG用于鱼类的催产,比目前生产用的尿源hCG质量更稳定、纯度更高、杂质更少,催产效果更好,可替代尿源hCG在鱼类繁殖中的应用。本发明还涉及重组hCG在淡水鱼人工繁殖领域的应用。
Description
技术领域
本发明涉及生物医药和动物繁殖技术领域,具体地说,涉及重组绒促性素rhCG的制备方法及应用。
背景技术
鱼类繁殖与所处的生态环境密切相关,一般情况下,生态因素,如营养、温度、光照、水流等,是鱼类性腺发育的决定因素。然而人工繁殖条件下,池塘中缺乏江河具备的综合生态条件(如水的流速,水位的骤变等),有的家鱼性腺不能发育成熟,如青鱼亲鱼性腺一般很难达到Ⅳ期末;即使性腺发育成熟的家鱼,在池塘中也不能自行***,须人工注射催产剂方能***受精。人工注射催产剂的目的是:一方面使某些在人工条件下性腺不能自行成熟的鱼类(如青鱼)发育成熟以产卵***;另一方面也促使能产卵产精的鱼类集中同期***产精便于统一的繁殖管理。
适时催产,合理使用催产剂,对鱼类繁殖至关重要。目前生产上常用的催产剂主要有人工合成类似物(LRH-A)、鱼脑垂体(PG)、人绒毛膜***(绒促性素,hCG)和地欧酮(DOM)。不同种类的亲鱼对不同催产剂的敏感性差异很大,在实际应用中需选择对该亲鱼敏感的催产剂,或者将几种催产剂配合使用,效果比单一催产剂好。
hCG是一种由人胎盘合体滋养层细胞分泌的糖蛋白激素,包括α和β两个非共价键结合的亚基。hCG促进雌性生物***的发育成熟和黄体的形成,促进雄性生物***的生成。hCG在人医临床上常用于辅助生殖治疗,治疗***不育。hCG在动物繁殖生产上常用于猪、牛、羊的同期发情和超数***。hCG在鱼类繁殖方面主要用于鱼类的人工催熟催产、***产精,是鱼类人工繁殖的首选催产剂之一。
目前商品化hCG主要是从孕妇尿中提取纯化的,但是大规模收集孕妇尿难度大、成本高,纯化得到的hCG产品批次差异大,且存在尿液供体带来的安全风险。此外hCG对光线、温度和酸度等条件极为敏感,收集的尿液若不及时提取,很容易失去生物学活性,造成资源的浪费。因此,人们已经利用基因工程技术开发新型人用重组hCG产品,如默克雪兰诺的Ovidrel(重组人绒促性素注射液)、丽珠的注射用绒促性素(丽珠目前是尿源hCG),重组hCG产品质量相对均一、纯度高、杂质少、安全系数大,逐渐取代人用尿源hCG产品。
目前重组蛋白常用的表达***主要有:大肠杆菌、酵母、昆虫细胞和哺乳动物细胞。每种表达***都有各自的优势和不足,需根据目的蛋白和表达***的特点,选择合适的表达***。大肠杆菌表达***生产成本低、产量高、表达流程简单快速,但因缺乏蛋白质翻译后的加工机制,如糖基化,正确折叠等,不适合表达复杂和大多数动物细胞蛋白。酵母表达***兼有原核和高等真核表达***的优点,收获的蛋白具有一定的翻译后加工能力,但是仍存在不足,如容易形成蛋白多聚体和过度糖基化,因此更适合表达简单的真核生物蛋白。昆虫表达***可以表达高等真核生物蛋白,能对蛋白进行正确折叠和修饰,但蛋白含量低,难以形成种子批。哺乳动物细胞表达***能对蛋白进行正确折叠和复杂的糖基化修饰,适合表达复杂高等真核生物蛋白;但是表达流程复杂,周期长,成本较高。在实际应用中,应根据不同的表达要求,选用适合的表达***和策略。hCG是一种由人胎盘合体滋养层细胞分泌的糖蛋白激素,包括α和β两个非共价键结合的亚基。理论上用哺乳动物细胞来表达生产生殖激素蛋白,其空间结构和糖基化更接近天然产物,可保证其生理活性。
目前动保行业应用的hCG仍主要从孕妇尿液中提取,尚无重组hCG产品的相关报道。
发明内容
本发明的目的是提供重组绒促性素rhCG的制备方法。
本发明的另一目的是提供重组绒促性素rhCG的应用。
本发明的再一目的是提供一种鱼类催产的方法。
本发明的构思如下:利用基因重组技术能够大规模地获得重组hCG产品,且质量相对均一、纯度高、杂质少、安全系数大。哺乳动物细胞表达***,与其他表达***相比,能够获得在分子结构、理化特性和生物学功能方面最接近于天然的高等生物蛋白分子。
为了实现本发明目的,本发明提供重组绒促性素rhCG的制备方法,利用哺乳动物真核表达***表达重组绒促性素rhCG;所述重组绒促性素rhCG包含α和β亚基,二者通过范德华力连接。
其中,所述α亚基:
i)由SEQ ID NO:1所示的氨基酸序列构成的蛋白;或
ii)SEQ ID NO:1所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由i)衍生的蛋白;或
iii)与SEQ ID NO:1所示氨基酸序列同源性在90%以上的,且具有同等功能的氨基酸序列构成的蛋白。
所述β亚基:
i)由SEQ ID NO:3所示的氨基酸序列构成的蛋白;或
ii)SEQ ID NO:3所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由i)衍生的蛋白;或
iii)与SEQ ID NO:3所示氨基酸序列同源性在90%以上的,且具有同等功能的氨基酸序列构成的蛋白。
前述的方法,根据NCBI公布的hCGα和β亚基的基因序列,按照哺乳动物密码子偏爱性进行基因序列优化,优化后的α和β亚基的基因序列分别构建于不同的表达盒,然后连接到同一表达载体上,转化宿主细胞,并在宿主细胞中表达,分离纯化目标蛋白;或者,
优化后的α和β亚基的基因序列分别构建到表达载体上,所得重组载体共同转化宿主细胞,并在宿主细胞中表达,分离纯化目标蛋白。
本发明中,优化后的α和β亚基的基因序列分别如SEQ ID NO:2和4所示。
前述的方法,所述表达载体选自pCG、pcDNA3.1、pMS2-GFP、pCMV5,优选pcDNA3.1。
前述的方法,所述宿主细胞CHO或293细胞。其中,中国仓鼠卵巢细胞(CHO)被美国FDA确认为安全的基因工程受体细胞,是大规模生产医疗药品最常用的表达细胞之一,优选CHO细胞。CHO细胞遗传背景清楚,生理代谢稳定,表达水平相对较高,耐受剪切力,便于大规模培养,是目前重组蛋白最常用的表达***之一。
将上述含重组hCGα和β亚基的重组载体转至宿主表达***中,哺乳动物表达***能够指导蛋白正确折叠,提供复杂的N型糖基化和准确的O型糖基化等多种翻译后加工功能,能获得在分子结构、理化特性和生物学功能方面最接近于天然的蛋白分子。
本发明还提供按照上述方法制备的重组绒促性素rhCG(及其衍生蛋白或经过修饰的蛋白)的以下任一种应用:
1)促进动物繁殖中的应用;
2)治疗动物生殖相关疾病的药物制备中的应用;
3)制备催产剂中的应用。
经过修饰的蛋白,包括所述rhCG经糖基化、聚乙二醇化、乙酰化或与BSA结合等,均属于本发明的保护范围。
经过改造的蛋白,包括所述rhCG与其他蛋白融合构成的不改变所述rhCG蛋白活性的融合蛋白,均属于本发明的保护范围。
前述的应用,所述动物为哺乳动物或鱼类;其中,所述哺乳动物为猪、牛、羊等经济动物,家畜如马或狗。
本发明中,所述鱼类包括淡水或海洋鱼类,优选淡水鱼类,包括四大家鱼(青鱼、草鱼、鲢鱼和鳙鱼)和鲤形目、鲈形目、鲶形目、鲑形目、鲟形目、鲱形目、鳕形目等经济鱼类,更优选鲤形目、鲈形目、鲶形目的经济鱼类。例如,白鲢、乌鳢、黄颡鱼、青鱼等淡水鱼。
所述促进动物繁殖是指促进***成熟,促进同期发情、超数***、催熟催产等。特别适用于鱼类催产。
本发明还提供一种鱼用催产剂,其活性成分中至少含有所述rhCG。此外,还可包含LRH-A(如LRH-A2、LRH-A3)、鱼脑垂体(PG)、地欧酮(DOM)等成分。
本发明还提供鱼类催产的方法,通过给鱼类注射上述催产剂进行催产。
前述的方法,rhCG的注射剂量为200-2000IU/kg体重。对于淡水鱼类rhCG的注射剂量为200-2000IU/kg体重。
优选地,采用两针注射法催产,首次注射的催产剂为LRH-A,注射剂量为1-2μg/kg体重;首次注射10-12h后进行第二次注射,第二次注射的催产剂为LRH-A2-10μg/kg体重、DOM 5-6μg/kg体重和rhCG 400-600IU/kg体重。
对于5kg左右的白鲢亲鱼,催产方法如下:首次注射LRH-A2,剂量为1μg/kg体重;首次注射12h后进行第二次注射,第二次注射LRH-A2 5μg/kg体重、DOM 5μg/kg体重和rhCG500IU/kg体重。
对于1.5kg左右的乌鳢亲鱼,催产方法如下:首次注射LRH-A3,剂量为2μg/kg体重;首次注射12h后进行第二次注射,第二次注射LRH-A3 2μg/kg体重、DOM 5μg/kg体重和rhCG400IU/kg体重。
对于雌鱼100g左右、雄鱼200g左右的黄颡鱼亲鱼,催产方法如下:首次注射LRH-A3,剂量为1μg/kg体重;首次注射10h后进行第二次注射,第二次注射LRH-A310μg/kg体重、DOM 5μg/kg体重和rhCG 600IU/kg体重。
借由上述技术方案,本发明至少具有下列优点及有益效果:
(一)本发明提供的鱼用重组hCG,及其衍生蛋白或经过修饰的蛋白,在哺乳动物细胞-CHO细胞中高效表达,质量相对均一,纯化后的rhCG蛋白纯度达93%,纯度高,安全系数大;活性高,比活可达14300IU/mg。
(二)本发明提供的鱼用重组hCG,及其衍生蛋白或经过修饰的蛋白,能够有效提高对尿源hCG敏感的白鲢的催产效果,结合催产剂LRH-A2和DOM使用,白鲢亲鱼的产卵率和受精率高达95%和96.7%,高于尿源hCG产品的80%和85.6%。
(三)本发明提供的鱼用重组hCG,及其衍生蛋白或经过修饰的蛋白,对尿源hCG不敏感的鲈形目鱼类有良好的催产效果,结合催产剂LRH-A3和DOM使用,提高乌鳢亲鱼的产卵率和受精率,降低效应时间,产卵率和受精率(分别为95%和96.3%)均高于尿源hCG产品(分别为75%和87.3%)。
(四)本发明提供的鱼用重组hCG,及其衍生蛋白或经过修饰的蛋白,对目前尚无有效催产剂催产的鲶形目鱼类亦有良好的催产效果,结合催产剂LRH-A3和DOM使用,能使黄颡鱼的产卵率和受精率提高至90%和85.7%,均高于尿源hCG产品(产卵率和受精率分别为65%和71.2%)。
(五)本发明提供的鱼用重组hCG,及其衍生蛋白或经过修饰的蛋白,对淡水鱼类的催产效果高于尿源hCG产品,可代替尿源hCG产品在淡水鱼类催产中的应用。
附图说明
图1为本发明实施例2中鱼用重组hCG的SDS-PAGE电泳图。其中左图为非还原电泳图,右图为还原电泳图;MK:蛋白Marker;1:澄清发酵液;2:CM FF收集液;3:Phenyl收集液;4:QFF收集液。
图2为本发明实施例2中鱼用重组hCG和尿hCG的SDS-PAGE非还原电泳图。MK:蛋白Marker;1:重组hCG;2:尿hCG。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。若未特别指明,实施例均按照常规实验条件,如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001),或按照制造厂商说明书建议的条件。
实施例4-6分别选取白鲢、乌鳢、黄颡鱼亲鱼进行催产试验,于5月上旬在广东渔场进行。
实施例1重组hCG蛋白表达***的筛选
考察hCG在大肠杆菌(Escherichia coli)-BL21(DE3)、巴斯德毕赤酵母细胞(Pichia pastoris)-GS115和哺乳动物细胞-中国仓鼠卵巢细胞(CHO)的表达情况。在基因库中检索hCGα(GenBank NM_000735.3)和hCGβ(GenBank NM_000737.3)亚基的核苷酸序列并人工合成。将hCGα亚基和β亚基的核苷酸序列分别转入表达载体pET-28a、pPIC9K和pcDNA3.1中。分别将含有α亚基和β亚基的重组载体转入BL21(DE3)、GS115和CHO细胞中。在BL21(DE3)未得到可溶的重组hCG蛋白;GS115细胞中得到重组hCG多聚体,且活性很低;CHO细胞中得到具有活性的重组hCG,但蛋白含量低,需要优化。最终选择CHO细胞作为重组hCG的表达***,并对该表达***进行优化。
实施例2重组hCG蛋白在CHO细胞中的表达和纯化
将实施例1中检索到的hCGα和β亚基的核苷酸序列,根据哺乳动物密码子偏爱性进行密码子优化,优化后α亚基核苷酸序列如SEQ ID NO:2所示,β亚基核苷酸序列如SEQ IDNO:4所示。
将人工合成的hCGα亚基和β亚基的核苷酸序列分别转入表达载体pcDNA3.1中,将重组载体线性化后一起转入CHO细胞中表达重组hCG。
将稳转细胞在发酵罐进行发酵培养,发酵液通过两级深层过滤膜包去除细胞和细胞碎片,然后用0.22μm滤膜过滤,获得澄清的发酵液。澄清的发酵液首先用弱阳离子交换层析(如CM FF,GE Healthcare)纯化:用平衡液(20mM乙酸铵,pH5.0)平衡上样,然后用洗脱液(50Mm PB,3M氯化钠,pH8.5)洗脱、收集洗脱液。其次将弱阳离子交换层析收集液用疏水层析(如Phenyl,GE Healthcare)纯化:用平衡液(50mM PB,3M氯化钠,pH8.5)平衡上样,然后用洗脱液(10mM PB,pH8.5)洗脱、收集洗脱液。最后将疏水层析收集液用强阴离子交换(如QFF,GE Healthcare)层析进一步纯化:用平衡液(50mM PB,pH8.5)平衡上样,然后用洗脱液(50Mm PB,3M氯化钠,pH8.5)洗脱,收集。对纯化的重组hCG(rhCG)进行SDS-PAGE凝胶电泳(图1);纯化后rhCG与尿源hCG进行SDS-PAGE凝胶电泳(图2)。
实施例3重组hCG蛋白的活性检测
通过测定rhCG对未成年小鼠子宫增重的作用,确定重组hCG的活性。参照中国兽药药典“绒促性素生物检定法”,方法如下:分别将hCG标准品和重组hCG样品(预估活性为15000IU/mg)配制成0.625IU/ml、0.375IU/ml、0.225IU/ml高、中、低三个剂量。选取健康合格、出生17-23日、体重9-13g、同一来源的雌性幼小鼠,按体重随机分为6组,每组15只。每日于大致相同时间分别给每鼠皮下注射相应浓度的标准品或重组hCG 0.2ml,每日一次,连续注射3次,于最后一次注射24h后将动物处死,称体重,解剖,摘出子宫,剥离附着的组织,去除卵巢,压干子宫内液,直接称重并换算成每10g体重的子宫重,照生物检定法中的量反应平行线测定法计算重组hCG效价为14300IU/mg,是兽药典规定的尿源hCG(uhCG)效价4500IU/mg的3.2倍。
实施例4重组hCG蛋白在促进白鲢催产中的应用
白鲢,又称鲢子,鲤形目鲤科鲢属鱼类,是我国四大家鱼之一,较宜养殖的优良鱼种之一。性活泼,善跳跃,主食硅藻、绿藻类等浮游植物和轮虫等浮游动物。
选择体重5kg左右,体质健壮、无病无伤、体质丰满的鲢鱼亲鱼,随机分为4组:阴性对照组、尿hCG(uhCG)组、rhCG-1组和rhCG-2组,每组10尾雌鱼5尾雄鱼。培育期间加投适量精料,催产前增加冲水次数,水温25℃时,采用两针注射法催产,依下表时间和剂量注射相应药物,其中雄鱼注射减半,第二次注射结束后,记录效应时间。将发情的雌雄亲鱼人工干法授精,方法如下:擦干鱼体,将待产雌性亲鱼的卵子挤入盆中,马上挤入雄鱼***,用手轻轻搅拌,使精卵充分结合提高受精率。雌鱼6h内未产卵的记为不产,6h内少量产卵记为半产。催产后的亲鱼用20ppm高锰酸钾清洗受伤处,注射3万/尾青霉素消炎。受精卵置于孵化环道中进行人工孵化,12h后计算受精率。
结果如表1所示,白鲢对hCG敏感,单独注射rhCG鲢鱼亲鱼的产卵率和受精率分别为65%和77.3%,但与LRH-A和DOM合用,催产效果更优,其中rhCG-1组雌鱼亲鱼效应时间(7h)与uhCG组(7h)雌鱼相等,均低于阴性对照组(8.5h)和rhCG-2组(8h)。rhCG-1组9尾雌鱼全产,1尾半产,产卵率达到95%,受精率为96.7%,均高于阴性对照组(55%和67.2%)、uhCG组(80%和85.6%)和rhCG-2组。
实施例5重组hCG蛋白在促进乌鳢催产中的应用
乌鳢,俗称黑鱼,鲈形目鳢科鳢属鱼类,肉质细腻、肉味鲜美,生长快、产量高,养殖密度高、效益高,是一种经济价值较高的名贵经济鱼类。性凶猛,以鱼、虾等为食。
选择1.5kg左右,无病、体质健壮、鱼体光滑、鳞片完整无损的乌鳢亲鱼,随机分为4组:阴性对照组、尿hCG(uhCG)组、rhCG-1组和rhCG-2组,每组10尾雌鱼5尾雄鱼。催产前投喂乌鳢喜食的鲜活饵料,如虾、蝌蚪、野杂鱼等,经常冲水,水温25℃时,采用两针注射法催产,依下表时间和剂量注射相应药物,其中雄鱼注射减半,第二次注射结束后,记录效应时间,将发情的雌雄亲鱼人工干法授精,方法如下:擦干鱼体,将待产雌性亲鱼的卵子挤入盆中,马上挤入雄鱼***,用手轻轻搅拌,使精卵充分结合提高受精率。雌鱼6h内未产卵的记为不产,6h内少量产卵记为半产。催产后的亲鱼用20ppm高锰酸钾清洗受伤处,注射3万/尾青霉素消炎。受精卵置于孵化环道中进行人工孵化,12h后计算受精率。
结果如表2所示,单独注射rhCG乌鳢雌鱼亲鱼的效应时间为26h,产卵率和受精率分别为50%和63.5%,乌鳢对hCG不敏感。rhCG与LRH-A和DOM合用对乌鳢亲鱼的催产效果显著,rhCG-1组乌鳢雌鱼亲鱼效应时间(21h)远低于阴性对照组(25h)、uhCG组(22.5h)和rhCG-2组。rhCG-1组10尾雌鱼亲鱼9尾全产,1尾半产,产卵率达到95%,受精率为96.3%,均高于阴性对照组(70%和78.4%)、uhCG组(75%和87.3%)和rhCG-2组。
实施例6重组hCG蛋白在促进黄颡鱼催产中的应用
黄颡鱼,俗称黄腊丁,鲶形目、黄颡鱼属,是一种杂食偏动物性经济鱼类,肉质细嫩鲜美,营养丰富,市场价格高,是一种新兴淡水养殖品种。
选择雌鱼100g、雄鱼200g左右,体质健壮、无病无伤、体质丰满的黄颡鱼亲鱼,随机分为4组:阴性对照组、尿hCG(uhCG)组、rhCG-1组和rhCG-2组,每组10尾雌鱼5尾雄鱼。培育期间加投喂螺蚌等饵料,水温25℃时,采用两针注射法催产,依下表时间和剂量注射相应药物,其中雄鱼注射减半,第二次注射结束后,记录效应时间,将发情的雌鱼亲鱼人工干法授精,方法如下:先杀雄鱼取出***,置于研钵中剪碎,加入***保存液备用。检查雌鱼,将待产雌性亲鱼的卵子挤入盆中,用羽毛将***与卵子混均受精。雌鱼6h内未产卵的记为不产,6h内少量产卵记为半产。催产后的亲鱼用20ppm高锰酸钾清洗受伤处,注射3万/尾青霉素消炎。将受精卵置于孵化道内孵化,12h后计算受精率。
结果如下表3所示,黄颡鱼对各种催产剂均不敏感,单独注射rhCG黄颡鱼亲鱼的效应时间为23.5h,产卵率和受精率分别为40%和61.8%。rhCG与LRH-A和DOM合用对黄颡鱼亲鱼的催产效果显著。rhCG-1组黄颡鱼雌鱼亲鱼效应时间(21.5h)低于阴性对照组(24h)、uhCG组(22.5h)和rhCG-2组,10尾雌鱼亲鱼8尾全产,2尾半产,产卵率和受精率为90%和85.7%,均高于阴性对照组(35%和63.4%)、uhCG组(65%和71.2%)和rhCG-2组。
虽然,上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述,但在本发明基础上,可以对之做一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
序列表
<110> 北京伟杰信生物科技有限公司
<120> 重组绒促性素rhCG的制备方法及应用
<130> KHP181111214.0
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<213> 人(Homo sapiens)
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Met Asp Tyr Tyr Arg Lys Tyr Ala Ala Ile Phe Leu Val Thr Leu Ser
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Val Phe Leu His Val Leu His Ser Ala Pro Asp Val Gln Asp Cys Pro
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Glu Cys Thr Leu Gln Glu Asn Pro Phe Phe Ser Gln Pro Gly Ala Pro
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Ile Leu Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro
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Leu Arg Ser Lys Lys Thr Met Leu Val Gln Lys Asn Val Thr Ser Glu
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Gly Phe Lys Val Glu Asn His Thr Ala Cys His Cys Ser Thr Cys Tyr
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atggactact accggaagta cgccgccatc ttcctggtga ccctgtccgt gttcctgcac 60
gtgctgcact ccgcccctga cgtgcaggac tgccctgagt gcaccctgca ggagaacccc 120
ttcttctccc agcccggagc ccctatcctg cagtgcatgg gctgctgctt ctccagggcc 180
taccctaccc ccctgaggag caagaagacc atgctggtgc agaagaacgt gacctccgag 240
agcacctgct gcgtggccaa gtcctacaac agggtgaccg tgatgggcgg cttcaaggtg 300
gagaaccaca ccgcctgcca ctgctccacc tgctactacc acaagtcc 348
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Met Glu Met Phe Gln Gly Leu Leu Leu Leu Leu Leu Leu Ser Met Gly
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Gly Thr Trp Ala Ser Lys Glu Pro Leu Arg Pro Arg Cys Arg Pro Ile
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Asn Ala Thr Leu Ala Val Glu Lys Glu Gly Cys Pro Val Cys Ile Thr
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Val Asn Thr Thr Ile Cys Ala Gly Tyr Cys Pro Thr Met Thr Arg Val
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Leu Gln Gly Val Leu Pro Ala Leu Pro Gln Val Val Cys Asn Tyr Arg
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gaaggatgcc ccgtgtgcat caccgtgaac accaccatct gcgccggcta ctgccccaca 180
atgaccaggg tgctgcaagg agtgctgcct gccctgcccc aagtggtgtg caactaccgg 240
gacgtgaggt tcgagtccat caggctgcct ggctgcccta ggggagtcaa ccctgtggtc 300
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ggcggaccca aggaccaccc tctgacctgc gacgacccca ggttccaaga ctcctcctcc 420
tccaaggctc cccctccttc cctcccttcc cctagcaggc tgcctggacc ttccgacacc 480
cccattctgc cccag 495
Claims (10)
1.重组绒促性素rhCG的制备方法,其特征在于,利用哺乳动物真核表达***表达重组绒促性素rhCG;所述重组绒促性素rhCG包含α和β亚基,二者通过范德华力连接;
其中,所述α亚基:
i)由SEQ ID NO:1所示的氨基酸序列构成的蛋白;或
ii)SEQ ID NO:1所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由i)衍生的蛋白;或
iii)与SEQ ID NO:1所示氨基酸序列同源性在90%以上的,且具有同等功能的氨基酸序列构成的蛋白;
所述β亚基:
i)由SEQ ID NO:3所示的氨基酸序列构成的蛋白;或
ii)SEQ ID NO:3所示氨基酸序列经取代、缺失和/或添加一个或几个氨基酸且同等功能的由i)衍生的蛋白;或
iii)与SEQ ID NO:3所示氨基酸序列同源性在90%以上的,且具有同等功能的氨基酸序列构成的蛋白。
2.根据权利要求1所述的方法,其特征在于,根据NCBI公布的hCGα和β亚基的基因序列,按照哺乳动物密码子偏爱性进行基因序列优化,优化后的α和β亚基的基因序列分别构建于不同的表达盒,然后连接到同一表达载体上,转化宿主细胞,并在宿主细胞中表达,分离纯化目标蛋白;或者,
将优化后的α和β亚基的基因序列分别构建到表达载体上,所得重组载体共同转化宿主细胞,并在宿主细胞中表达,分离纯化目标蛋白;
其中,优化后的α和β亚基的基因序列分别如SEQ ID NO:2和4所示。
3.根据权利要求2所述的方法,其特征在于,所述表达载体选自pCG、pcDNA3.1、pMS2-GFP、pCMV5,优选pcDNA3.1。
4.根据权利要求2所述的方法,其特征在于,所述宿主细胞CHO或293细胞。
5.权利要求1-4任一项所述方法制备的重组绒促性素rhCG的以下任一种应用:
1)促进动物繁殖中的应用;
2)治疗动物生殖相关疾病的药物制备中的应用;
3)制备催产剂中的应用。
6.根据权利要求5所述的应用,其特征在于,所述动物为哺乳动物或鱼类;其中,所述哺乳动物为猪、牛、羊、马或狗;所述鱼类为淡水或海洋鱼类,优选淡水鱼类。
7.根据权利要求6所述的应用,其特征在于,所述促进动物繁殖是指促进***成熟,促进同期发情、超数***、催熟催产。
8.鱼类催产的方法,其特征在于,通过给鱼类注射催产剂进行催产;其中,所述催产剂的活性成分中至少含有权利要求1-4任一项所述方法制备的重组绒促性素rhCG;
所述鱼类优选淡水鱼类,包括四大家鱼、鲤形目、鲈形目、鲶形目、鲑形目、鲟形目、鲱形目和鳕形目的经济鱼类,更优选鲤形目、鲈形目和鲶形目的经济鱼类。
9.根据权利要求8所述的方法,其特征在于,rhCG的注射剂量为200-2000IU/kg体重。
10.根据权利要求8所述的方法,其特征在于,采用两针注射法催产,首次注射的催产剂为LRH-A,注射剂量为1-2μg/kg体重;首次注射10-12h后进行第二次注射,第二次注射的催产剂为LRH-A 2-10μg/kg体重、DOM 5-6μg/kg体重和rhCG 400-600IU/kg体重。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114349844A (zh) * | 2021-12-29 | 2022-04-15 | 北京伟杰信生物科技有限公司 | 一种兽用重组绒促性素的纯化方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5767251A (en) * | 1983-11-02 | 1998-06-16 | Genzyme Corporation | Recombinant heterodimeric human fertility hormones, and methods, cells, and vectors and DNA for the production thereof |
CN102549011A (zh) * | 2009-10-05 | 2012-07-04 | 辉凌公司 | 包含重组hcg的药物制剂 |
CN103539862A (zh) * | 2013-11-01 | 2014-01-29 | 广州优联康医药科技有限公司 | 一种长效重组促卵泡激素及其应用 |
CN103804498A (zh) * | 2013-11-29 | 2014-05-21 | 广州诺新生物技术有限公司 | 长效重组绒促性素及其应用 |
CN107532172A (zh) * | 2015-04-24 | 2018-01-02 | 辉凌公司 | 产生***的方法 |
-
2018
- 2018-03-29 CN CN201810271691.7A patent/CN108264549A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5767251A (en) * | 1983-11-02 | 1998-06-16 | Genzyme Corporation | Recombinant heterodimeric human fertility hormones, and methods, cells, and vectors and DNA for the production thereof |
CN102549011A (zh) * | 2009-10-05 | 2012-07-04 | 辉凌公司 | 包含重组hcg的药物制剂 |
CN103539862A (zh) * | 2013-11-01 | 2014-01-29 | 广州优联康医药科技有限公司 | 一种长效重组促卵泡激素及其应用 |
CN103804498A (zh) * | 2013-11-29 | 2014-05-21 | 广州诺新生物技术有限公司 | 长效重组绒促性素及其应用 |
CN107532172A (zh) * | 2015-04-24 | 2018-01-02 | 辉凌公司 | 产生***的方法 |
Non-Patent Citations (4)
Title |
---|
SUNG N等: "choriogonadotropin subunit beta 3 precursor [Homo sapiens]", 《GENBANK DATABASE》 * |
WANG H等: "glycoprotein hormones alpha chain isoform 2 precursor [Homo sapiens]", 《GENBANK DATABASE》 * |
刘扬: "CHO细胞表达重组人绒毛膜***性腺激素的调控机理研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
王鸿利,叶裕春主编: "《中华检验医学大辞典》", 31 October 2000, 上海科学技术出版社 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114349844A (zh) * | 2021-12-29 | 2022-04-15 | 北京伟杰信生物科技有限公司 | 一种兽用重组绒促性素的纯化方法 |
CN114349844B (zh) * | 2021-12-29 | 2024-02-13 | 北京伟杰信生物科技有限公司 | 一种兽用重组绒促性素的纯化方法 |
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