CN110938656B - 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 - Google Patents
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 Download PDFInfo
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Abstract
本发明公开了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,涉及基因工程技术领域。其包括编码大熊猫FSHα亚基的第一基因和编码大熊猫FSHβ亚基的第二基因,第一基因的序列如SEQ ID NO.1所示,第二基因的序列如SEQ ID NO.2所示。通过构建重组表达大熊猫促卵泡生成素的载体、表达系统可以实现大熊猫FSHα亚基、FSHβ亚基及其亚基复合物的稳定持续活性表达。使用大熊猫源的促卵泡生成素降低了机体产生不良反应的风险,有利于我国珍稀濒危猫科动物如大熊猫,小熊猫等的品种选育、扩繁。此外,该重组制备方法简单、成本低。
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卵泡生成素的载体、表达系统及制备方法。
背景技术
提升大熊猫的繁殖率通常采用异种外源促性腺激素的方法,已有研究表明,长期使用异种外源促性腺激素诱导动物的发情及排卵会对动物产生不良反应,发情效果会下降,严重影响大熊猫的繁殖率。目前,没有有效的方法能解决上述技术问题。
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卵泡生成素的载体、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卵泡生成素的载体,其包括编码大熊猫FSHα亚基的第一基因和编码大熊猫FSHβ亚基的第二基因,第一基因的序列如SEQ ID NO.1所示,第二基因的序列如SEQ ID NO.2所示。
促卵泡生成素(Follicle stimulating hormone,FSH),分子量约为33KD,其由α亚基和β亚基形成的异二聚体组成。
其中α亚基是由120个氨基酸残基构成,α亚基的氨基酸序列参照SEQ ID NO.4所示,而β亚基由约129个氨基酸残基构成,β亚基的氨基酸序列参照SEQ ID NO.5所示。α亚基在物种间具有高度保守性,β亚基具有物种特异性。α亚基与β亚基只有结合在一起才能发挥FSH效应。
FSH在卵巢中的效应包括:促进卵巢上皮组织、性腺等的发育,促进卵泡生长发育,促进雌激素(E)和雌二醇(E2)合成和分泌,参与卵巢中优势卵泡的选择和进一步发育。
FSH在精巢中的效应包括:刺激睾丸曲细精管上皮细胞的发育,促进雄性激素的表达调控,同时分泌抑制素,对精子的正常形成,产生和成熟过程中发挥重要作用。此外,FSH还可以影响睾丸的生精功能。
为解决异种外源促性腺激素的问题,发明人发现采用独立载体表达两个亚基(α亚基和β亚基),在后续转染时存在两个亚基表达量不匹配,进而无法得到具有活性的折叠蛋白。
由于大熊猫的FSHα亚基和FSHβ亚基均存在一定程度的糖基化,因此,采用原核表达的方法无法表达出目的活性的FSH蛋白。
本发明提供了一种重组表达大熊猫促卵泡生成素的载体,该载体包括编码大熊猫FSHα亚基的第一基因和编码大熊猫FSHβ亚基的第二基因。该载体可以用于大熊猫FSHα亚基和大熊猫FSHβ亚基的稳定持续活性表达。表达产物可以用于制备诱导大熊猫发情试剂及促排卵剂,使用大熊猫源的促卵泡生成素降低了机体产生不良反应的风险,有利于我国珍稀濒危动物的品种选育、扩繁。
在本发明应用较佳的实施例中,上述载体还包括连接元件。
在可选的实施方式中,上述连接元件为T2A元件,T2A元件的序列如SEQ ID NO.3所示。T2A元件的氨基酸序列参照SEQ ID NO.6所示。
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FSHα和FSHβ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。
一种重组表达大熊猫促卵泡生成素的载体的构建方法,其包括如下步骤:合成第一基因和第二基因。
第一基因和第二基因分别编码大熊猫的FSHα亚基和FSHβ亚基。T2A元件的两端分别连接第一基因和第二基因。
在可选的实施方式中,构建方法还包括将合成的第一基因和第二基因以及T2A元件连接到质粒pCDH中,构建含有插入序列第一基因、T2A元件和第二基因的重组表达大熊猫促卵泡生成素的载体。
构建方法是将质粒pCDH先进行EcoR I和BamH I双酶切以线性化载体,再进行EcoRI和BamH I双酶切以线性化插入片段,通过T4 DNA ligase酶将线性化的插入片段连接到线性化的pCDH上。
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。合成的第一基因、T2A元件和第二基因两端具有EcoR I和BamH I限制性内切酶位点。在质粒pCDH上也含有EcoR I和BamH I限制性内切酶位点。通过酶切连接的方式将两侧含有粘性末端的第一基因、T2A序列和第二基因连入质粒pCDH相应的酶切位点处,得到pCDH-FSHβ-T2A-FSHα重组表达载体。
含有重组表达大熊猫促卵泡生成素的载体或含有上述构建方法构建的载体的真核细胞表达系统。真核细胞表达系统是将载体转入真核细胞中构建而成。
将重组表达大熊猫促卵泡生成素的载体转入真核细胞中可以表达产生糖基化的FSHα亚基、FSHβ亚基及其亚基复合物。
在可选的实施方式中,真核细胞为CHO-K1细胞中构建而成。在其他实施例中,真核细胞的类别可以根据需要进行选择。
一种在真核细胞表达体系中生产重组大熊猫促卵泡生成素的方法,其包括培养真核细胞表达系统。
在本发明可选的实施方式中,上述将构建的重组表达大熊猫促卵泡生成素的载体转入CHO-K1细胞,构建含有重组表达大熊猫促卵泡生成素的载体的真核细胞表达系统。
为了提升感染效率和成功率,本发明通过慢病毒表达体系感染CHO-K1细胞。慢病毒表达体系选自三质粒系统,包括慢病毒骨架载体pCDH和包装质粒psPAX2和pMD2.G。
将重组表达大熊猫促卵泡生成素的载体-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卵泡生成素的载体的CHO-K1细胞稳定表达系统。
通过慢病毒可以将重组表达大熊猫促卵泡生成素的载体整合到真核细胞基因组上,可实现目的基因长时间稳定的表达。
本发明提供的表达系统可以将大熊猫源FSH分泌到DMEM低糖培养基中,且具有活性。
含有重组表达大熊猫促卵泡生成素的载体的CHO-K1细胞的培养方法包括如下步骤:
(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)吸除培养基,加入适量胰酶/EDTA消化液(0.25%),没过细胞37℃温育2min;(3)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,将混合液转移至15ml无菌离心管中;(4)用吸管轻轻吹打细胞使之重新悬浮,台式离心机1500rpm离心3min;(5)移出上清液,加入培养基5ml,轻轻吹打重悬细胞,1500rpm离心3min;(6)移出上清液,再加入培养基5ml,轻轻吹打重悬,1500rpm离心3min,同时,配制适量含10%胎牛血清(FBS)的完全培养基;(7)移出上清液,加入10ml完全培养基,轻轻吹打并重悬细胞,转移到90-mm培养皿中,置于二氧化碳培养箱中(37℃,5%CO2)培养。
在本发明可选的实施方式中,上述方法还包括将培养的真核细胞表达系统进行分离纯化。
在本发明可选的实施方式中,上述分离纯化的方法包括将培养的真核细胞表达系统进行盐析、离子交换层析分离和亲和层析分离中的至少一种。
本发明具有以下有益效果:
本发明提供了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。通过构建重组表达大熊猫促卵泡生成素的载体、表达系统可以实现大熊猫FSHα亚基、FSHβ亚基及其亚基复合物的稳定持续活性表达。使用大熊猫源的促卵泡生成素降低了机体产生不良反应的风险,有利于我国珍稀濒危猫科动物如大熊猫,小熊猫等的品种选育、扩繁。此外,该重组制备方法简单、成本低。
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FSH的PCR产物电泳胶图;
图2为实施例3中不同浓度重组人FSH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FSH蛋白分别处理表达小鼠FSHR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-FSHβ-T2A-FSHα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;慢病毒体系包装质粒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。
构建方法包括如下步骤:
1、酶切及连接反应:用限制性内切酶EcoR I和BamH I对合成的插入片段和表达载体pCDH质粒(美国SBI公司)进行双酶切反应;按照天根DNA胶回收试剂盒说明书对琼脂糖凝胶电泳分离得到的酶切产物进行纯化回收;将酶切产物按照T4连接酶说明书的体系进行连接反应;将连接产物转化入感受态细胞,于37℃培养箱中倒置培养12-16h。
2、阳性克隆筛选:用表达载体pCDH的通用引物,以上一步所得菌落为模板,按Taq聚合酶说明书的体系进行菌落PCR扩增。电泳检测扩增产物是否正确。取阳性菌落接种到LB液体培养基中于恒温摇床中培养至适宜OD值。
3、重组质粒提取、酶切:质粒提取采用天根质粒小提试剂盒按说明书进行,对得到的质粒选用EcoR I和BamH I限制性内切酶鉴定重组质粒插入片段大小是否正确。
4、质粒测序和测序结果分析:选用酶切鉴定正确的重组质粒送到北京华大生物公司测序,利用软件比对分析重组质粒序列。
实施例2
本实施例提供了CHO-K1细胞表达体系的构建方法。
1.在6孔板中培养HEK293T细胞(美国ATCC公司),当细胞长满80%时,进行转染。
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,使用含抗生素和10%血清的生长培养基,并在37℃,5%CO2的培养箱培养细胞生长。
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,经0.45μM滤膜过滤后,于室温下3000g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(美国ATCC公司)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FSH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)注释显示许多基因包括大熊猫促卵泡生成素受体基因(FSHR)等均有待进一步注释。有鉴于大熊猫促卵泡生成素(FSH)依赖结合其特异性受体
(FSHR)发挥生物学效应,由此本实施例通过克隆小鼠FSHR基因,基于大熊猫FSHR和小鼠FSHR均编码具有692个氨基酸的受体蛋白,具有85%的氨基酸序列同源性的特征,本发明将小鼠FSHR用于表达构建真核表达载体(pcDNA3.1-FSHR),实现检测重组FSH蛋白活性。
详细操作如下: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FSHR基因得编码全长约2079bp,小鼠FSH的PCR产物电泳胶图参照图1所示。经BamH I和EcoR I酶切后连接至真核表达载体pcDNA3.1(美国Invitrogen公司),并测序验证表达载体(pcDNA3.1+FSHR)上是否连有小鼠FSHR基因。
本研究克隆所得小鼠FSHR基因cDNA序列参照SEQ ID NO.7所示,氨基酸序列参照SEQ ID NO.8所示。
有研究表明,FSH激素依赖激活其特异性FSHR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FSHR表达质粒(pcDNA3.1+FSHR)与pGL3-CRE-luciferase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FSH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。
细胞转染及荧光素酶活性检测具体包括如下步骤:
转染:当6-cm培养皿中的细胞密度达到70%左右,分别将mFSHR表达质粒和pGL3-CRE-luciferase报告质粒共转染细胞。表达载体用量为400ng每孔,pGL3-CRE-luciferase报告质粒1600ng每孔,转染试剂jetPRIME 2μL每孔,用jetPRIME Buffer配成总体系为200μL的混合物,室温放置10min。将转染混合液加入到6-cm厘米培养皿中,于CO2培养箱中培养培养6h,更换新的含5%FBS的DMEM低糖培养基,继续培养24h。
细胞分至96孔板:用胰蛋白酶消化液将细胞消化,重悬于含0.5%FBS的DMEM-L培养基中,按照每孔180μL(约2×104个细胞)加到96孔板中,培养24h。
配体处理:吸去96孔板中的培养基,每孔加入100μL含不同浓度重组人FSH蛋白的无血清DMEM低糖培养基,培养6h。
裂解细胞:将5×Cell Culture Lysis Buffer细胞裂解液用MilliQ-H2O稀释到1×,吸弃培养基、迅速向每孔加入50μL细胞裂解液。待细胞充分裂解后,每孔取15μL裂解液于测量酶标板中,加入40μL Luciferase底物后混匀,于德国伯托多功能酶标仪LB941中测定荧光数值。
FSH激素对胞内cAMP信号通路的影响参照图2所示,在表达小鼠FSHR的CHO-K1细胞中,重组人FSH处理细胞后,在1ng/ml浓度下即可显著性地激活小鼠FSHR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FSH蛋白的活性检测。
实施例4
在本实例中,我们分别用含2μl,6μl和20μl的大熊猫FSH重组蛋白条件培养基(conditioned medium,CM)处理表达小鼠FSHR的细胞,实验结果参照图3所示,发现条件培养基含量不同的培养液均可激活小鼠FSHR受体,且激活效应与条件培养基含量呈剂量依赖关系,揭示本例所制备的大熊猫FSH重组蛋白具有生物学活性,可特异性激活FSHR受体。
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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Claims (6)
1.一种重组表达大熊猫促卵泡生成素的载体,其特征在于,其包括编码大熊猫FSHα亚基的第一基因和编码大熊猫FSHβ亚基的第二基因,所述第一基因的序列如SEQ ID NO.1所示,所述第二基因的序列如SEQ ID NO.2所示;所述载体还包括连接元件;
所述连接元件为T2A元件,所述T2A元件的序列如SEQ ID NO.3所示;
所述的重组表达大熊猫促卵泡生成素的载体的构建方法包括如下步骤:合成所述第一基因和第二基因;将合成的所述第一基因和第二基因以及T2A元件连接到质粒pCDH中,构建含有插入片段第一基因、T2A元件和第二基因的重组表达大熊猫促卵泡生成素的载体;
所述构建方法是将质粒pCDH先进行EcoR I和BamH I双酶切以线性化载体,再进行EcoRI和BamH I双酶切以线性化插入片段,通过T4 DNA ligase酶将线性化的插入片段连接到线性化的pCDH上。
2.含有权利要求1所述的重组表达大熊猫促卵泡生成素的载体的真核细胞表达系统。
3.根据权利要求2所述的真核细胞表达系统,其特征在于,所述真核细胞表达系统是将所述载体转入真核细胞中构建而成;
所述真核细胞为CHO-K1细胞中构建而成。
4.一种在真核细胞表达体系中生产重组大熊猫促卵泡生成素的方法,其特征在于,其包括培养权利要求2所述的真核细胞表达系统。
5.根据权利要求4所述的方法,其特征在于,将构建的重组表达大熊猫促卵泡生成素的载体转入CHO-K1细胞,构建含有重组表达大熊猫促卵泡生成素的载体的真核细胞表达系统。
6.根据权利要求4所述的方法,其特征在于,所述方法还包括将培养的真核细胞表达系统进行分离纯化;
所述分离纯化的方法包括将培养的真核细胞表达系统进行盐析、离子交换层析分离和亲和层析分离中的至少一种。
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Cloning and sequence analysis of FSH and LH in the giant panda (Ailuropoda melanoleuca);Ming-JuanLiao 等;《Animal Reproduction Science》;20030515;第77卷(第1-2期);摘要,第107-116页 * |
大熊猫等濒危动物FSH/LH和GH/GHR基因的克隆及其表达研究;廖鸣娟;《中国优秀博硕士学位论文全文数据库 (博士) 基础科学辑》;20031215(第 04 期);摘要,第二章第1小节至第3小节,图2 * |
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