CN110938657A - 重组表达大熊猫促黄体生成素的载体、表达系统及制备方法 - Google Patents
重组表达大熊猫促黄体生成素的载体、表达系统及制备方法 Download PDFInfo
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Abstract
本发明公开了重组表达大熊猫促黄体生成素的载体、表达系统及制备方法,涉及基因工程技术领域。其包括编码大熊猫LHβ亚基的第一基因和编码大熊猫LHα亚基的第二基因,第一基因的序列为SEQ ID NO.1所示,第二基因的序列为SEQ ID NO.3所示。通过构建重组表达大熊猫促黄体生成素的载体、表达系统可以实现大熊猫LHβ亚基、LHα亚基及其亚基复合物的稳定持续活性表达。表达产物可以用于诱导大熊猫发情及排卵,使用大熊猫源的促黄体生成素不会诱使机体产生不良反应,有利于大熊猫繁育质量的提升。此外,该重组制备方法简便易行,具有广泛的应用前景。
Description
技术领域
本发明涉及基因工程技术领域,具体而言,涉及重组表达大熊猫促黄体生成素的载体、表达系统及制备方法。
背景技术
大熊猫是我国珍稀濒危动物,繁殖率低下制约着大熊猫种群数量与保育质量。研究表明,大熊猫繁殖率低下可能与促卵泡生成素 (Follicle stimulating hormone,FSH)、促黄体生成素(Luteinizing hormone,LH)等促性腺激素分泌不足有关。
为了提高大熊猫保育质量,多年来我国使用异种外源促性腺激素 (例如:马源促卵泡生成素(FSH),马源促黄体生成素(LH))诱导大熊猫发情及排卵,然而,重复使用异种外源激素可能会诱使机体产生不良反应,进而影响其长期效果。目前,尚未有合适的方法解决上述问题。
鉴于此,特提出本发明。
发明内容
本发明的目的在于提供重组表达大熊猫促黄体生成素的载体、表达系统及制备方法以解决上述技术问题。
本发明是这样实现的:
一种重组表达大熊猫促黄体生成素的载体,其包括编码大熊猫 LHβ亚基的第一基因和编码大熊猫LHα亚基的第二基因,第一基因的序列为SEQ ID NO.1所示,第二基因的序列为SEQ ID NO.3所示。
促黄体生成素(LH)是由α亚基和β亚基形成的异二聚体,其中,α亚基是由120个氨基酸残基构成,其序列参照SEQ ID NO.4所示,而β亚基由约141个氨基酸残基构成,其序列参照SEQ ID NO.5 所示。β亚基需与α亚基协作形成复合物。LH作为胞外信号分子,依赖与其特异性受体(促黄体生成素受体,LHR)发挥生物学活性。 LH相对分子量约为28kDa,LH主要功能是促进性细胞成熟,刺激性激素合成,促进和维持第二性征。
LH作用于卵巢,使卵泡膜产生雌激素,在卵泡发育接近成熟的时候与FSH一起作用于颗粒细胞,促进卵泡的成熟,LH作用于卵细胞周围组织使成熟卵细胞的排出得以顺利进行,参与调控非优势卵泡的闭锁和黄体化。
在睾丸中,LH主要诱导睾酮生成,促进精子成熟。
动物卵巢中含有大量处于不同发育期的卵泡,但并非所有的都能排出体外,约99%都发生闭锁退化,这正是由于FSH和LH共同调控的结果,纵观卵泡发育的整个过程,FSH主要在前期发生作用, LH发挥作用主要在后期,两种激素属于协作关系。在排卵过程中FSH和LH共同激发卵泡内纤溶酶原活化剂,产生大量蛋白溶解酶,使卵泡周围松散;LH突升期,卵泡中的前列腺素制造酶增加,大量前列腺素促使新血管聚集,血液集中,卵泡液增加,卵泡周围平滑肌收缩,将松散的卵子和卵丘细胞排出卵泡。LH不足会降低大熊猫对FSH的反应性,影响卵母细胞发育能力,而添加小剂量的LH,可以补救卵母细胞的发育潜能,使其发育成正常胚胎。
本发明通过构建重组表达大熊猫促黄体生成素的载体可以实现大熊猫LHβ亚基、LHα亚基及其亚基复合物的稳定持续活性表达。表达产物可以用于诱导大熊猫发情及排卵,使用大熊猫源的促黄体生成素不会诱使机体产生不良反应,有利于大熊猫繁育质量的提升。
上述载体还包括连接序列;
优选的,连接序列为T2A序列,T2A的序列为SEQ ID NO.2所示。T2A的氨基酸序列参照SEQ ID NO.6所示。
含有重组表达大熊猫促黄体生成素的载体的宿主细胞表达系统,宿主细胞表达系统是将载体转入宿主细胞中构建而成。宿主细胞为 CHO-K1细胞。CHO-K1细胞株是来自中华仓鼠卵巢细胞,现广泛用于科研用途和工业生产中,将重组表达载体转入CHO-K1细胞可以用于重组大熊猫促黄体生成素的表达。
一种生产重组大熊猫促黄体生成素的方法,其包括如下步骤:培养宿主细胞表达系统。
培养宿主细胞表达系统前还包括载体构建步骤和细胞表达系统的构建,其包括如下步骤:
A:合成第一基因、T2A序列和第二基因;
B:将插入片段第一基因、T2A序列和第二基因连接到慢病毒表达载体质粒pCDH-CMV-MCS-EF1-Puro中,构建含有第一基因、T2A 序列和第二基因的重组表达大熊猫促黄体生成素的载体;
C:将重组表达大熊猫促黄体生成素的载体-CMV-MCS-EF1-Puro 及慢病毒包装质粒共转入HEK293T细胞中,收集纯化重组慢病毒并感染CHO-K1细胞,构建含有表达重组大熊猫促黄体生成素的载体的CHO-K1细胞稳定表达系统。
第一基因编码大熊猫LHβ亚基,第二基因编码大熊猫LHα亚基,第一基因和第二基因可由NCBI上查阅获得。第一基因和T2A 序列和第二基因一起合成,直接整体酶切连接到pCDH载体上。
T2A序列在构建多顺反子载体中具有序列短小、易操作、上下游基因表达平衡性好等优点,利用T2A序列构建的LHβ和LHα共表达载体在转入CHO-K1细胞后能正常翻译。此外,T2A能够在CHO-K1 细胞中发挥自裂解功能。
在本发明应用较佳的实施例中,上述步骤B中质粒 pCDH-CMV-MCS-EF1-Puro先进行EcoR I和BamH I酶双酶切以线性化载体,再进行EcoR I和BamH I酶双酶切以线性化插入片段,通过 T4 DNAligase酶将线性化的插入片段连接到线性化的 pCDH-CMV-MCS-EF1-Puro上。
发明人创造性的发现,若重组表达载体中含有人巨细胞病毒早期转录增强子(human cytomegalovirus immediate early enhancer)元件,则构建的重组载体转入细胞后无法得到具有活性的大熊猫重组LH蛋白,因此,选用了不包含人巨细胞病毒早期转录增强子元件的pCDH质粒作为重组蛋白表达所用载体。
在本发明应用较佳的实施例中,上述重组表达大熊猫促黄体生成素的载体通过慢病毒表达体系制备重组慢病毒感染CHO-K1细胞以获得稳定表达细胞系统;
在本发明应用较佳的实施例中,上述慢病毒表达体系为 pCDH+psPAX2+pMD2.G。
慢病毒与其他病毒工具比较,具有以下优势:
(1)表达时间长:慢病毒通过将外源基因整合到宿主细胞基因组上,可实现目的基因长时间稳定的表达,不随着细胞分裂传代而丢失,是细胞实验的首选;
(2)安全性高:未发现致病性,已被用于CAR-T治疗作用于人体;
(3)免疫原性低:直接注射活体组织不易造成免疫反应,适用于动物实验。
本发明提供的表达系统可以将大熊猫源LH分泌到DMEM低糖培养基中,且具有活性。
在本发明应用较佳的实施例中,上述步骤(4)是在含有嘌呤霉素的培养体系下进行细胞培养。嘌呤霉素用于筛选阳性细胞。
在本发明应用较佳的实施例中,上述嘌呤霉素的浓度为5-10μ g/ml之间。
在本发明应用较佳的实施例中,上述步骤(4)的培养方式包括如下步骤:
(1)培养的CHO-K1细胞每3天传代一次,待细胞密度达到80%左右,用一次性吸管吸出DMEM低糖培养基,缓缓加入PBS没过细胞,轻轻摇晃洗涤90-mm培养皿;(2)尽可能多的吸除培养基,然后加入适量胰酶/EDTA消化液(0.25%),用量以没过细胞为宜;(3) 轻轻摇晃,使TE消化液均匀覆盖皿内所有细胞,37℃温育2min;(4) 在培养皿中添加适量培养基,用一次性吸管沿不同方向吹洗培养皿,使细胞尽可能从板上消化下来,将培养基转移至15ml无菌离心管中; (5)用吸管轻轻吹打细胞使之重新悬浮。用台式离心机1800rpm离心3min;(6)吸弃清液,加入培养基5ml,轻轻吹打重悬细胞,1800 rpm离心3min;(7)吸弃清液,再次加入培养基5ml,轻轻吹打重悬,1800rpm离心3min,同时,配制适量含10%胎牛血清(FBS)的完全培养基;(8)吸弃上清液,加入10ml完全培养基,吹打重悬,转移到90-mm培养皿中,置于二氧化碳培养箱中(37℃,5%CO2) 培养。
在本发明应用较佳的实施例中,上述方法还包括将步骤(4)重组表达的大熊猫促黄体生成素进行分离纯化。
在本发明应用较佳的实施例中,上述分离纯化的方法包括将步骤 (4)的细胞培养液离心,超滤浓缩,得到超滤品。
在本发明应用较佳的实施例中,上述分离纯化的方法还包括将超滤品进行纯化。
在本发明应用较佳的实施例中,上述纯化的方法包括将超滤产品进行盐析、离子交换层析分离和亲和层析分离中的至少一种。
通过分离纯化可以制备得到用于诱导大熊猫发情或排卵的大熊猫源促黄体生成素,满足大熊猫繁育的需求。
本发明具有以下有益效果:
本发明提供了一种重组表达大熊猫促黄体生成素的载体、表达系统及制备方法。通过构建重组表达大熊猫促黄体生成素的载体、表达系统可以实现大熊猫LHβ亚基、LHα亚基及其亚基复合物的稳定持续活性表达。表达产物可以用于诱导大熊猫发情及排卵,使用大熊猫源的促黄体生成素不会诱使机体产生不良反应,有利于大熊猫繁育质量的提升。此外,该重组制备方法简便易行,具有广泛的应用前景。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例3中小鼠LHR的PCR产物电泳胶图;
图2为实施例3中不同浓度重组人LH蛋白(标准品)分别处理转染后的CHO-K1细胞6h的荧光结果图;
图3为实施例4中不同浓度重组大熊猫LH蛋白分别处理转染后的CHO-K1细胞6h的荧光结果图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
实施例1
本实施例提供了pCDH+LHβ-T2A-LHα表达载体的构建方法。所使用的构建材料如下:
菌株:CHO-K1细胞及HEK293T细胞,购自美国ATCC;载体 pcDNA3.1(+),购自美国Invitrogen公司;载体 pCDH-CMV-MCS-EF1-Puro购自美国SBI公司;慢病毒体系质粒psPAX2+pMD2.G购自美国Invitrogen公司。
试剂:第一基因、第二基因和T2A序列由苏州金唯智生物科技有限公司合成获得,引物由北京六合华大基因科技有限公司合成;限制性内切酶、连接酶选自宝日医生物技术(北京)有限公司,转染试剂购自达科为公司。
构建方法:
1.载体和插入片段的酶切反应。
大熊猫LH的α和β亚基基因及连接二者的T2A序列由金唯智公司直接合成,序列两侧人为添加了EcoR I和BamH I酶切位点用于后续亚克隆,利用EcoR I和BamH I酶进行酶切反应,分别得到具有粘性末端的线性化载体和插入片段,反应时间为24h。酶切体系如下:
胶回收试剂盒回收酶切产物。
在进行连接反应前,首先利用胶回收试剂盒进行纯化回收酶切产物,采用生工生物(上海)有限公司的SanPrep柱式DNA胶回收试剂盒进行方法如下:
(1)将插入片段和质粒的酶切产物用1%的琼脂糖凝胶110V电泳至合适位置;
(2)在紫外灯上尽快将目的条带切下,尽量不要切下空白部分 (影响回收效率),放入1.5ml离心管;
(3)根据所切下的琼脂糖凝胶质量加入适量Buffer B2;
(4)将Buffer B2加热到45℃左右使琼脂糖凝胶块融化;
(5)用移液枪将所得溶液转移进干净的吸附柱,8000g离心30 s后吸弃废液;
(6)吸取500μl Wash Solution滴加入吸附柱,8000g离心30s,吸弃废液,重复两次;
(7)将吸附柱,8000g离心2min,尽量除去多余液体;
(8)加入40μl MilliQ-H2O,室温放置3min,8000g离心3min,收集后-20℃保存。
采用T4连接酶连接。本发明使用T4连接酶将插入片段和线性化载体进行连接,连接时间为16小时,其连接体系如下:
2.转化感受态细胞。
(1)取DH5α感受态细胞,迅速置于冰上,冰浴静置融化2~5min,轻弹管壁(2~3次)重悬细胞;(2)添加5μl连接产物,小心吹打混匀,冰浴中静置30min;(3)轻放于45℃水浴热激30s,迅速放回冰浴2min;(4)加入0.9ml无菌冷藏的SOC,混匀,恒温(37℃) 孵育1h;(5)4000rpm离心2min,吸弃多余上清液,保留200μl 将菌体吹散,均匀涂布在含氨苄的琼脂平板表面,待涂布的液体完全被吸收后,倒置恒温(37℃)培养12~16h。
3.挑选阳性克隆。
本实验中,利用pCDH载体的通用引物用于PCR实验,以筛选阳性克隆。配置Taq酶(全式金公司)扩增体系:
挑选阳性克隆包括如下步骤:(1)在超净台中,用无菌牙签沾取菌落,接种在保种板上,将牙签残留菌体洗入对应编号的无菌EP管;
(2)EP管瞬离,然后进行PCR;保种板37℃培养;
(3)完成PCR后,每管加入2μl的6×Loading Buffer,摇匀后进行电泳,确定阳性克隆;
(4)用灭菌牙签从保种板上挑取阳性克隆加入盛有10ml LB (0.3%Amp)培养基,恒温摇床250rpm,37℃,过夜培养。
4.质粒提取
本发明使用天根公司的质粒小提试剂盒进行质粒提取,步骤如下:
(1)吸取500μl的平衡液于吸附柱CP3,8000rpm离心1min,舍弃离心后的废液;
(2)吸取4ml过夜培养的菌液,4000rpm离心3min,吸弃上清;
(3)加入Buffer PⅠ250μl,涡旋震荡使菌体充分散开,加入 Buffer PⅡ250μl,反复轻轻颠倒混匀至液体澄清;
(4)加入Buffer PⅢ300μl,再次颠倒混匀,使Buffer之间充分反应;
(5)然后12000rpm离心15min,吸取650μl上清液加到吸附柱CP3中,8000rpm离1min,舍弃离心出来的废液;
(6)吸取600μl漂洗液PW滴加入离心后的吸附柱,8000rpm 离心1min,再次加入漂洗液PW,离心后舍弃废液;
(7)将吸附柱CP3放回收集管中,8000rpm离心3min;
(8)把吸附柱放入干净的离心管,吸取30μl预热后的 MilliQ-H2O滴加在吸附柱中的膜上,室温放置2min,8000rpm离心 3min,用离心后的质粒溶液再洗一次吸附柱CP3,重复离心一次,最终得到的质粒经过测序进行序列验证。
实施例2
本实施例提供了CHO-K1细胞表达体系的构建方法。
重组病毒包装及靶细胞感染、筛选实验。
(1)在6孔板中培养HEK293T细胞,当细胞长满80%时,进行转染,转染步骤依据试剂盒说明进行。
(4)滴加200μl转染混合液于6孔板中,并摇晃使试剂均匀分布。
(5)4小时后换液,使用含抗生素的生长培养基并在37℃, 5%CO2的培养箱培养生长。
(6)在转染后48时,收集培养液到15-ml无菌、有盖的圆锥离心管中。室温下,3000xg离心15分钟,转移含重组病毒的上清液进入新离心管中。
(7)吸取正常培养密度接近70%的CHO-K1细胞培养皿中的培养基,将上述含病毒的上清液加入至CHO-K1细胞中。
(8)感染48小时后更换含10μg/ml嘌呤霉素的完全培养基继续培养,视细胞状态每几天更换新鲜的嘌呤霉素的培养液,上述重组细胞系即可用于活性蛋白制备及后续功能验证实验。
实施例3
本实施例提供了检测实施例2中重组表达LH活性蛋白的 pGL3-CRE-luciferase萤光素酶报告系统。
至目前,大熊猫基因组(Panda release 92:ailMel1)中序列BLAST 提示大熊猫LHR基因的5端序列存在NNNN缺失,即包含大熊猫 LHR基因的起始密码子的基因组序列存在缺失,表明大熊猫基因组序列有待进一步注释。有鉴于LH依赖结合其特异性受体(LHR) 发挥生物学效应,由此本实施例通过克隆小鼠LHR基因,结合大熊猫LHR和小鼠LHR均编码具有690个氨基酸的受体蛋白,且具有较高的氨基酸序列一致性(87%),提示小鼠LHR可用于表达构建真核表达载体(pcDNA3.1-LHR),实现检测重组LH蛋白活性。
具体实验操作如下:解剖获得C57BL/6小鼠(成都达硕实验动物有限公司)卵巢组织,液氮研磨后,利用RNAzol试剂提取总RNA 并反转录制备cDNA模板,最后以小鼠卵巢组织cDNA文库为模板,通过PCR扩增,扩增小鼠LHR基因得编码全长约2103bp,小鼠LHR 的PCR产物电泳胶图参照图1所示。
经BamHI和EcoR I酶切后连接至真核表达载体pcDNA3.1,并测序验证表达载体(pcDNA3.1+LHR)上是否连有小鼠LHR基因。本研究克隆所得小鼠LHR基因cDNA序列参照SEQID NO.7所示,氨基酸序列参照SEQ ID NO.8所示。
有研究表明,LH激素依赖激活其特异性LHR受体,依赖环磷酸腺苷-蛋白激酶A(cAMP-PKA)通路,改变下游靶蛋白表达谱,发挥生理功能。
本实施例将小鼠LHR表达质粒(pcDNA3.1+LHR)与pGL3-CRE 报告质粒(美国Promega公司)共同转染CHO-K1细胞,用0.1ng/ml、 1ng/ml和10ng/ml的重组人LH蛋白(标准品,美国RD公司)分别处理转染后的CHO-K1细胞6h,经裂解和添加底物孵育后,通过酶标仪测定荧光读数,检测LH激素对胞内cAMP信号通路的影响。
细胞裂解及荧光读数测定具体包括如下步骤:
(1)用MiliQ-H2O将5×Passive Lysis Buffer细胞裂解液稀释5 倍,混合均匀;
(2)将96孔板的培养基吸弃,快速加入50μl稀释后的1×Passive Lysis Buffer细胞裂解液,置于摇床上1小时以充分裂解细胞;
(3)每孔取15μl细胞裂解液,按照Dual-Luciferase Reporter 1000AssaySysterm(Promega)操作步骤加入40μl Luciferase底物后混匀,之后于德国伯托多功能酶标仪LB941中测定荧光素酶活性。
LH激素对胞内cAMP信号通路的影响参照图2所示,在表达小鼠LHR的CHO-K1细胞中,重组人LH处理细胞后,在1ng/ml浓度下即可显著性地激活小鼠LHR受体,并且在10ng/ml浓度下具有更强的激活潜能,揭示该系统可正常工作,可用于测定LH蛋白的活性检测。
实施例4
基于实施例3中的蛋白活性检测体系,本实施例中利用上述报告基因系统检测实施例2中制得的CHO-K1-LH细胞系表达的LH蛋白活性,试验结果参照图3所示,由图3可知,获得的包含大熊猫 LH重组蛋白的条件性培养基(conditioned medium,CM)能够特异性激活鼠LH受体,且随着条件培养基比例增加,对应荧光值强度相应增加,证实其对受体的激活效能呈剂量依赖。本实施例证明稳转 CHO-K1-LH细胞系可以成功表达分泌具有正常活性的重组大熊猫 LH蛋白至培养基中。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
SEQUENCE LISTING
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agccactgct gtgctttcag gaatttgccg aagaaagaac agaatttttc attttccatt 900
tttgaaaact tttccaaaca atgtgaaagc acagttagag aagcgaataa cgagacgctt 960
tattctgcca tctttgagga gaatgaactc agtggctggg attacgatta tgacttctgt 1020
tcacccaaga cactccaatg tactccagaa ccagatgctt tcaatccctg tgaagatatt 1080
atgggctatg ccttccttag ggtgttgatt tggctaatta atatactagc catctttggc 1140
aacttgacag tcctctttgt tctcctgacc agtcgttata aactgacggt gccccgcttc 1200
ctcatgtgta atctctcctt tgcagacttt tgcatggggc tctacctgct gctcattgcc 1260
tcagtagact cccaaacaaa aggccagtac tataaccatg ccatagactg gcagacaggg 1320
agtggctgca gtgcagctgg cttctttact gtgttcgcca gtgaactttc tgtctatacc 1380
cttacagtca tcactctgga aaggtggcac accatcacct atgctgttca gctggaccaa 1440
aagctgaggc tgagacatgc catcccaatt atgctcggag gatggatttt ttctaccctg 1500
atggccacat tgccccttgt gggtgtcagc agttacatga aagtcagcat ctgcctcccc 1560
atggatgtgg aatccactct gtcacaagtc tacatattat ccatcttgct cctcaatgca 1620
gtggcctttg tcgtcatctg tgcttgctac gttaggatat actttgcagt tcaaaatcca 1680
gagctgacgg ctcctaacaa ggacacaaaa attgctaaga agatggccat cctcatcttc 1740
acagacttca catgcatggc acccatctca ttctttgcca tctcagctgc cttcaaagta 1800
ccccttatca ctgtcaccaa ctcaaaagtt ctgctggtcc ttttttatcc tgtcaattct 1860
tgtgccaacc catttctgta cgcagtgttc acgaaggcat ttcagagaga tttctttctc 1920
ttgctgagca gatttggttg ctgtaagcac cgggctgaac tttacagaag gaaggaattt 1980
tctgcatgta ccttcaactc caaaaacggc tttccaagat caagtaagcc ttcccaggct 2040
gccctgaagt tatccatagt gcactgtcaa caacctacac ctccaagagt gttaattcag 2100
taa 2103
<210> 8
<211> 700
<212> PRT
<213> 人工序列
<400> 8
Met Gly Arg Arg Val Pro Ala Leu Arg Gln Leu Leu Val Leu Ala Met
1 5 10 15
Leu Val Leu Lys Gln Ser Gln Leu His Ser Pro Glu Leu Ser Gly Ser
20 25 30
Arg Cys Pro Glu Pro Cys Asp Cys Ala Pro Asp Gly Ala Leu Arg Cys
35 40 45
Pro Gly Pro Arg Ala Gly Leu Ala Arg Leu Ser Leu Thr Tyr Leu Pro
50 55 60
Val Lys Val Ile Pro Ser Gln Ala Phe Arg Gly Leu Asn Glu Val Val
65 70 75 80
Lys Ile Glu Ile Ser Gln Ser Asp Ser Leu Glu Arg Ile Glu Ala Asn
85 90 95
Ala Phe Asp Asn Leu Leu Asn Leu Ser Glu Ile Leu Ile Gln Asn Thr
100 105 110
Lys Asn Leu Leu Tyr Ile Glu Pro Gly Ala Phe Thr Asn Leu Pro Arg
115 120 125
Leu Lys Tyr Leu Ser Ile Cys Asn Thr Gly Ile Arg Thr Leu Pro Asp
130 135 140
Val Ser Lys Ile Ser Ser Ser Glu Phe Asn Phe Ile Leu Glu Ile Cys
145 150 155 160
Asp Asn Leu Tyr Ile Thr Thr Ile Pro Gly Asn Ala Phe Gln Gly Met
165 170 175
Asn Asn Glu Ser Ile Thr Leu Lys Leu Tyr Gly Asn Gly Phe Glu Glu
180 185 190
Val Gln Ser His Ala Phe Asn Gly Thr Thr Leu Ile Ser Leu Glu Leu
195 200 205
Lys Glu Asn Ile Tyr Leu Glu Lys Met His Ser Gly Thr Phe Gln Gly
210 215 220
Ala Thr Gly Pro Ser Ile Leu Asp Val Ser Ser Thr Lys Leu Gln Ala
225 230 235 240
Leu Pro Ser His Gly Leu Glu Ser Ile Gln Thr Leu Ile Ala Thr Ser
245 250 255
Ser Tyr Ser Leu Lys Thr Leu Pro Ser Arg Glu Lys Phe Thr Ser Leu
260 265 270
Leu Val Ala Thr Leu Thr Tyr Pro Ser His Cys Cys Ala Phe Arg Asn
275 280 285
Leu Pro Lys Lys Glu Gln Asn Phe Ser Phe Ser Ile Phe Glu Asn Phe
290 295 300
Ser Lys Gln Cys Glu Ser Thr Val Arg Glu Ala Asn Asn Glu Thr Leu
305 310 315 320
Tyr Ser Ala Ile Phe Glu Glu Asn Glu Leu Ser Gly Trp Asp Tyr Asp
325 330 335
Tyr Asp Phe Cys Ser Pro Lys Thr Leu Gln Cys Thr Pro Glu Pro Asp
340 345 350
Ala Phe Asn Pro Cys Glu Asp Ile Met Gly Tyr Ala Phe Leu Arg Val
355 360 365
Leu Ile Trp Leu Ile Asn Ile Leu Ala Ile Phe Gly Asn Leu Thr Val
370 375 380
Leu Phe Val Leu Leu Thr Ser Arg Tyr Lys Leu Thr Val Pro Arg Phe
385 390 395 400
Leu Met Cys Asn Leu Ser Phe Ala Asp Phe Cys Met Gly Leu Tyr Leu
405 410 415
Leu Leu Ile Ala Ser Val Asp Ser Gln Thr Lys Gly Gln Tyr Tyr Asn
420 425 430
His Ala Ile Asp Trp Gln Thr Gly Ser Gly Cys Ser Ala Ala Gly Phe
435 440 445
Phe Thr Val Phe Ala Ser Glu Leu Ser Val Tyr Thr Leu Thr Val Ile
450 455 460
Thr Leu Glu Arg Trp His Thr Ile Thr Tyr Ala Val Gln Leu Asp Gln
465 470 475 480
Lys Leu Arg Leu Arg His Ala Ile Pro Ile Met Leu Gly Gly Trp Ile
485 490 495
Phe Ser Thr Leu Met Ala Thr Leu Pro Leu Val Gly Val Ser Ser Tyr
500 505 510
Met Lys Val Ser Ile Cys Leu Pro Met Asp Val Glu Ser Thr Leu Ser
515 520 525
Gln Val Tyr Ile Leu Ser Ile Leu Leu Leu Asn Ala Val Ala Phe Val
530 535 540
Val Ile Cys Ala Cys Tyr Val Arg Ile Tyr Phe Ala Val Gln Asn Pro
545 550 555 560
Glu Leu Thr Ala Pro Asn Lys Asp Thr Lys Ile Ala Lys Lys Met Ala
565 570 575
Ile Leu Ile Phe Thr Asp Phe Thr Cys Met Ala Pro Ile Ser Phe Phe
580 585 590
Ala Ile Ser Ala Ala Phe Lys Val Pro Leu Ile Thr Val Thr Asn Ser
595 600 605
Lys Val Leu Leu Val Leu Phe Tyr Pro Val Asn Ser Cys Ala Asn Pro
610 615 620
Phe Leu Tyr Ala Val Phe Thr Lys Ala Phe Gln Arg Asp Phe Phe Leu
625 630 635 640
Leu Leu Ser Arg Phe Gly Cys Cys Lys His Arg Ala Glu Leu Tyr Arg
645 650 655
Arg Lys Glu Phe Ser Ala Cys Thr Phe Asn Ser Lys Asn Gly Phe Pro
660 665 670
Arg Ser Ser Lys Pro Ser Gln Ala Ala Leu Lys Leu Ser Ile Val His
675 680 685
Cys Gln Gln Pro Thr Pro Pro Arg Val Leu Ile Gln
690 695 700
Claims (10)
1.一种重组表达大熊猫促黄体生成素的载体,其特征在于,其包括编码大熊猫LHβ亚基的第一基因和编码大熊猫LHα亚基的第二基因,所述第一基因的序列为SEQ ID NO.1所示,所述第二基因的序列为SEQ ID NO.3所示。
2.根据权利要求1所述的重组表达大熊猫促黄体生成素的载体,其特征在于,所述载体还包括连接序列;
优选的,所述连接序列为T2A序列,所述T2A的序列为SEQ ID NO.2所示。
3.含有权利要求1或2所述的重组表达大熊猫促黄体生成素的载体的宿主细胞表达系统。
4.根据权利要求3所述的宿主细胞表达系统,其特征在于,所述宿主细胞表达系统是将所述载体转入宿主细胞中构建而成;
优选的,所述宿主细胞为CHO-K1细胞。
5.一种生产重组大熊猫促黄体生成素的方法,其特征在于,其包括如下步骤:培养权利要求3或4所述的宿主细胞表达系统。
6.根据权利要求5所述的方法,其特征在于,培养宿主细胞表达系统前还包括载体构建步骤和细胞表达系统的构建,其包括如下步骤:
A:合成第一基因、T2A序列和第二基因;
B:将插入片段第一基因、T2A序列和第二基因一起连接到慢病毒表达载体质粒pCDH-CMV-MCS-EF1-Puro中,构建含有所述第一基因、T2A序列和第二基因的重组表达大熊猫促黄体生成素的载体;
C:将所述重组表达大熊猫促黄体生成素的载体-CMV-MCS-EF1-Puro及慢病毒包装质粒共转入HEK293T细胞中,收集纯化重组慢病毒并感染CHO-K1细胞,构建含有表达重组大熊猫促黄体生成素的载体的CHO-K1细胞稳定表达系统。
7.根据权利要求6所述的方法,其特征在于,所述步骤B中质粒pCDH-CMV-MCS-EF1-Puro先进行EcoR I和BamH I酶双酶切以线性化载体,再进行EcoR I和BamH I酶双酶切以线性化插入片段,通过T4 DNAligase酶将线性化的插入片段连接到线性化的pCDH-CMV-MCS-EF1-Puro上。
8.根据权利要求6所述的方法,其特征在于,所述步骤C中,所述重组表达大熊猫促黄体生成素的载体通过慢病毒表达体系制备重组慢病毒感染CHO-K1细胞以获得稳定表达细胞系统;
优选的,所述慢病毒表达体系为pCDH+psPAX2+pMD2.G。
9.根据权利要求6所述的方法,其特征在于,所述培养宿主细胞表达系统是在含有嘌呤霉素的培养体系下进行CHO-K1细胞培养;
优选的,所述嘌呤霉素的浓度为5-10μg/ml之间;
优选的,所述CHO-K1细胞培养方式包括如下步骤:CHO-K1细胞每3天传代一次,置于二氧化碳培养箱中培养;
优选的,置于37℃,5%CO2二氧化碳培养箱中培养。
10.根据权利要求9所述的方法,其特征在于,所述方法还包括将重组表达的大熊猫促黄体生成素进行分离纯化;
优选的,所述分离纯化的方法包括将细胞培养液离心,超滤浓缩,得到超滤品。
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