CN114957433B - 一种热带爪蛙Fosl1蛋白突变体及其应用 - Google Patents
一种热带爪蛙Fosl1蛋白突变体及其应用 Download PDFInfo
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Abstract
本发明公开了一种热带爪蛙Fosl1蛋白突变体及其应用。本发明利用缺失突变技术构建Fosl1蛋白突变体,筛选到突变体Fosl1‑NT能有效抑制野生型Fosl1蛋白的转录活性,且对Fos家族其他蛋白的功能无显著影响,可用作Fosl1蛋白特异性的显性负效突变体。同时本发明利用所述突变体成功构建了转基因热带爪蛙品系,不但能特异性抑制心肌细胞中Fosl1蛋白的转录活性,而且可以在F0代筛选获得,具有操作简单、构建周期短、效率高等优势。本发明所提供的突变体可用于研究Fosl1蛋白相关疾病的机制,能为筛选Fosl1蛋白相关疾病的药物及靶向干预疗法提供重要的模型工具,具有重要的应用价值。
Description
技术领域
本发明属于基因工程技术领域,具体涉及一种热带爪蛙Fosl1蛋白突变体及其应用。
背景技术
热带爪蛙(Xenopustropicalis)是爪蛙属中唯一具有真正二倍体遗传背景的两栖类模式动物,具有个体小,发育快,体外受精,繁殖量大,培育费用低,早期胚胎透明等特点。目前,具有二倍体遗传背景的热带爪蛙已经发展成为发育生物学、遗传学及再生医学研究领域的理想模式动物,而且在人类疾病模型建立及相关疾病靶向药物的高通量筛选方面显示出极强的应用前景。
Fos家族蛋白包括Fos、FosB、Fos-likeantigen1(Fosl1)及Fos-likeantigen 2(Fosl2)等四个成员,是构成activator protien-1(AP-1)转录因子复合物的重要组成部分。Fos家族蛋白通过与Jun家族蛋白(Jun、JunB、JunD)形成异源二聚体,促进AP-1转录因子复合物的形成与激活。尽管AP-1家族蛋白都含有保守的bZIP结构域(the basic DNA-binding domain and the leucine zipper),但不同构成蛋白的具体结构和生物学功能却不尽相同。既往研究表明,Fos家族蛋白保守的bZIP结构域所对应的蛋白多肽(A-Fos)可通过阻滞靶蛋白与DNA的结合,并抑制靶蛋白之间形成二聚体等方式,进而能有效抑制AP-1转录因子的活性,从而可以作为AP-1的显性负效(dominant-negative,dn)突变体(dnAP-1)(Olive et al.,A dominant negative to activation protein-1(AP1)that abolishesDNA binding and inhibits oncogenesis.J Biol Chem,1997,272:18586-18594)。然而,该dnAP-1突变体(A-Fos)能够抑制所有bZIP家族蛋白,包括Fos家族蛋白(Fos、FosB、Fosl1及Fosl2)及Jun家族蛋白(Jun、JunB及JunD)的功能,不具有选择性。在Fos家族蛋白中,Fosl1在细胞周期、细胞增殖、细胞分化、细胞凋亡等多种生物学过程中具有重要的调控作用。然而,Fosl1蛋白在热带爪蛙中的功能及相应的功能缺失突变体目前仍未见相关报道。因此,研发能特异性抑制Fosl1蛋白功能的dominant-negative突变体(dnFosl1)及相应的热带爪蛙转基因品系,不但有助于阐明Fosl1蛋白的功能,建立Fosl1蛋白相关的疾病动物模型,而且可为Fosl1蛋白相关疾病的机制研究、靶点甄别、药物筛选及靶向干预疗法的建立提供良好的研究模型和工具。
近年来,随着第三代基因编辑技术CRISPR/Cas9系统的发展,为热带爪蛙的基因编辑及利用热带爪蛙建立基因敲除动物模型提供了有效方法。然而,目前利用CRISPR/Cas9系统建立热带爪蛙基因敲除动物模型主要包括3个步骤:1)获得F0代嵌合体;2)F0代嵌合体与野生型交配获得F1代杂合子;3)F1代杂合子自交获得F2代纯合子。即至少需要培育3代,才有可能获得靶基因敲除的纯合子热带爪蛙模型。因此,基于CRISPR/Cas9系统建立基因敲除热带爪蛙模型具有周期过长、耗时耗力等弊端。RNA干扰技术(RNAi)虽然已经在哺乳动物细胞中得到了广泛而有效的应用,但爪蛙模型中因缺少Ago2蛋白,导致RNAi技术并不能在热带爪蛙中有效工作(Lund E,et al.,Limiting Ago protein restricts RNAi andmicroRNA biogenesis during early development in Xenopus laevis.GenesDev.2011,25(11):1121-31)。因此,需要研发一种能够快速、有效建立Fosl1蛋白功能缺失型热带爪蛙模型的技术与方法。
发明内容
本发明的第一个目的在于克服现有技术的缺点与不足,提供一种热带爪蛙Fosl1蛋白突变体。
本发明的第二个目的是提供上述热带爪蛙Fosl1蛋白突变体的应用,即所述突变体在构建Fosl1蛋白功能缺失型热带爪蛙模型中的应用,特别是在构建心肌特异性缺失Fosl1蛋白功能的热带爪蛙模型中的应用。
发明人通过对热带爪蛙Fosl1蛋白的主要功能结构域逐一进行缺失突变,构建了3个突变体:Fosl1-NT、Fosl1ΔDB及Fosl1ΔLZ。并利用报告基因检测系统逐一分析了上述3个突变体对Fos家族蛋白功能的影响,最终发现突变体Fosl1-NT不但能有效抑制Fosl1蛋白的功能,而且对Fos家族其他蛋白(Fos、FosB及Fosl2)的功能并无显著影响。因此,Fosl1-NT可作为热带爪蛙Fosl1蛋白的特异性dominant-negative突变体(dnFosl1)。与以往报道的利用dnAP-1突变体(A-Fos)可抑制所有bZIP家族蛋白的功能不同,本发明所述dnFosl1突变体利用Fosl1蛋白的N端多肽,能特异性的抑制Fosl1蛋白的功能,而不影响Fos家族其他蛋白的功能。利用本发明所述的dnFosl1突变体,结合转基因技术快速建立Fosl1蛋白功能缺失型热带爪蛙模型,具有周期短,效率高等优势,甚至在F0代就有望能筛选到靶蛋白功能缺失型爪蛙品系。所建立的Fosl1蛋白功能缺失型热带爪蛙模型,不但可用于研究Fosl1蛋白相关的疾病机制,而且能为筛选Fosl1蛋白相关疾病的药物及靶向干预疗法提供了重要的模型工具,具有重要的应用价值。
本发明的目的通过以下技术方案实现:
一种热带爪蛙Fosl1蛋白突变体,其氨基酸序列如下所示(亦如SEQ ID NO.1所示):
MYRDFTGAFPQPDSGCSSHGLRNSGSSPILGTSGMGHSVRLNPPPEDAQQKYPVHTSSGQFIPTLNAITSSQDLNWMIQPSVRPLNLPPYQSPRHGVIRNMGGVLSMGRRRNGEHLSPEEEER。
所述的Fosl1蛋白突变体的编码序列如下所示(亦如SEQ ID NO.2所示):
ATGTACAGAGACTTCACTGGAGCCTTCCCTCAGCCAGATTCGGGGTGCAGCAGCCACGGCCTACGGAACTCGGGATCTTCCCCAATTTTAGGAACTTCAGGAATGGGACACAGCGTCAGACTGAACCCACCACCCGAGGACGCGCAGCAGAAGTATCCAGTTCACACATCCTCGGGTCAATTTATCCCAACATTGAATGCTATCACATCAAGTCAGGACCTTAACTGGATGATCCAACCAAGTGTACGCCCCCTGAATTTACCACCATACCAAAGCCCTCGGCATGGGGTAATCCGTAACATGGGAGGAGTCTTGAGTATGGGGCGAAGGAGAAATGGAGAACATCTGTCACCAGAGGAAGAGGAGCGATAG。
上述热带爪蛙Fosl1蛋白突变体、其编码序列、含有其编码序列的重组表达载体或工程菌在制备Fosl1蛋白抑制剂中的应用。
进一步的,在所述的应用中,Fosl1蛋白突变体特异性抑制Fosl1蛋白的转录活性。
上述热带爪蛙Fosl1蛋白突变体、其编码序列、含有其编码序列的重组表达载体或工程菌在构建Fosl1蛋白功能缺失型热带爪蛙模型中的应用。
进一步的,所述的应用的具体操作为:将所述的Fosl1蛋白突变体隆到表达载体中,构建含有Fosl1蛋白突变体的转基因载体,并注射热带爪蛙受精卵的动物极,孵育,筛选成功表达Fosl1蛋白突变体的胚胎,培养至成蛙,获得Fosl1蛋白失活的热带爪蛙品系。
所述的应用优选为在构建心肌细胞特异性失活Fosl1蛋白的热带爪蛙模型中的应用。
进一步的,所述的应用的具体操作为:
S1、根据编码所述的Fosl1蛋白突变体的DNA序列,合成dnFosl1-T2A-EGFP融合DNA片段,并将合成的融合DNA片段克隆到pMlc2-NTR-T2A-Dendra2opt载体的EcoRI与HpaI酶切位点之间,替换原有载体中的NTRopt-T2A-Dendra2opt片段,进而构建pMlc2-dnFosl1-T2A-EGFP转基因载体;其中,所述的融合DNA片段中dnFosl1是Fosl1蛋白突变体,其核苷酸序列如SEQ ID NO.2所示;T2A是连接肽,其核苷酸序列如下所示:GGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA;
S2、将步骤S1所制备的pMlc2-dnFosl1-T2A-EGFP转基因载体与I-SceI归位内切酶共同注射热带爪蛙受精卵的动物极,孵育,筛选心脏特异性表达绿色荧光的F0胚胎,培养至成蛙,获得心肌细胞特异性失活Fosl1蛋白的热带爪蛙品系Tg(Mlc2-dnFosl1-T2A-EGFP)。
所述的共同注射方式为:500ng转基因载体和20U I-SceI归位内切酶用无菌水配置成10μL的注射液,每个受精卵注射2nL。
所述的共同注射用的热带爪蛙受精卵为1细胞期热带爪蛙受精卵。
所述的孵育为孵育胚胎至45期(St45)。
所述的筛选采用荧光体式显微镜实现。
所述的培养所用的培养液为MBS胚胎培养液,优选为0.1×MBS胚胎培养液,配制方法为称取NaCl 25.9g、Hepes 11.9g、NaHCO3 1.0g、KCl 0.4g、MgSO4 0.5g、Ca(NO3)2 0.8g、CaCl2 0.2g,加入800mL去离子水并混匀,pH调至7.4,容量瓶定容至1000mL,高压灭菌后4℃保存备用,使用前稀释50倍。
所述的培养的温度优选为21~25℃,更优选为注射后的12小时内为21℃,然后再调整为25℃。
上述热带爪蛙Fosl1蛋白突变体、其编码序列、含有其编码序列的重组表达载体或工程菌在筛选Fosl1蛋白相关疾病的药物及或制备靶向治疗药物中的应用。
本发明相对于现有技术具有如下的优点及效果:
本发明提供了一个能有效抑制野生型热带爪蛙Fosl1蛋白功能,但对Fos家族其他蛋白转录活性无显著影响的突变体Fosl1-NT,该突变体可用作Fosl1蛋白的显性负效(dominant-negative)突变体(dnFosl1)。本发明所述dnFosl1突变体可利用转基因技术快速建立Fosl1蛋白功能缺失型热带爪蛙模型Tg(Mlc2-dnFosl1-T2A-EGFP),在F1代甚至F0代就可用于相关研究,具有周期短,效率高等优势。所建立的Tg(Mlc2-dnFosl1-T2A-EGFP)热带爪蛙模型,不但可用于研究Fosl1蛋白相关的疾病机制,而且能为筛选Fosl1蛋白相关疾病的药物及靶向干预疗法提供了重要的模型工具,具有重要的应用价值。
附图说明
图1为突变体抑制野生型Fosl1蛋白转录活性的荧光素酶报告基因检测结果图;其中,pAP1-Luc为用于检测Fosl1蛋白转录活性的报告基因载体;pcDNA-Fosl1为热带爪蛙野生型Fosl1基因的过表达载体;pcDNA-Fosl1-NT为突变体Fosl1-NT的过表达载体;pcDNA-Fosl1ΔDB为突变体Fosl1ΔDB的过表达载体;pcDNA-Fosl1ΔLZ为突变体Fosl1ΔLZ的过表达载体;**p<0.01,***p<0.001。
图2为突变体对Fos家族其他蛋白转录活性的荧光素酶报告基因检测结果图;其中,pAP1-Luc为用于检测Fos家族蛋白转录活性的报告基因载体;pcDNA-Fos为热带爪蛙野生型Fos基因的过表达载体;pcDNA-FosB为热带爪蛙野生型FosB基因的过表达载体;pcDNA-Fosl2为热带爪蛙野生型Fosl2基因的过表达载体;pcDNA-Fosl1-NT为突变体Fosl1-NT的过表达载体;pcDNA-Fosl1ΔDB为突变体Fosl1ΔDB的过表达载体;*p<0.05;ns指无显著性差异。
图3为用于在热带爪蛙心肌细胞中特异性过表达突变体dnFosl1的转基因载体pMlc2-dnFosl1-T2A-EGFP的图谱。
图4为发育至St45期的F0代转基因热带爪蛙Tg(Mlc2-dnFosl1-T2A-EGFP)蝌蚪的荧光检测结果图。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
除非另有定义,本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
实施例1:突变体Fosl1-NT的制备及其表达载体构建
1.以野生型热带爪蛙Fosl1基因(XM_002939331.4)的编码区(CDS)为模板,设计特异性PCR引物(FNT-Fw/FNT-Re)扩增编码Fosl1蛋白N端(aa1-123)的DNA序列(Fosl1-NT)。所述PCR引物的序列如下所示:
上述正向引物FNT-Fw序列中,5’端gg是酶切位点保护碱基,下划线字母是KpnI酶切位点序列,方框内的小写字母是人为加入的Kozak信号;上述反向引物FNT-Re序列中,5’端cg是酶切位点保护碱基,下划线字母是EcoRI酶切位点序列,加黑并斜体的字母是人为加入的终止密码子。
2.将扩增的PCR产物用KpnI与EcoRI双酶切,回收带有粘性末端的DNA片段,并连接到pcDNA3.1载体(Invitrogen)的KpnI与EcoRI酶切位点之间,进而构建pcDNA3.1-Fosl1-NT重组载体。
3.将重组载体转化大肠杆菌DH5α,涂平板,挑选阳性克隆,进行DNA测序验证。
上述Fosl1-NT突变体的氨基酸序列如下所示:
MYRDFTGAFPQPDSGCSSHGLRNSGSSPILGTSGMGHSVRLNPPPEDAQQKYPVHTSSGQFIPTLNAITSSQDLNWMIQPSVRPLNLPPYQSPRHGVIRNMGGVLSMGRRRNGEHLSPEEEER(SEQ ID NO.1)。
上述Fosl1-NT突变体的核苷酸序列如下所示:
ATGTACAGAGACTTCACTGGAGCCTTCCCTCAGCCAGATTCGGGGTGCAGCAGCCACGGCCTACGGAACTCGGGATCTTCCCCAATTTTAGGAACTTCAGGAATGGGACACAGCGTCAGACTGAACCCACCACCCGAGGACGCGCAGCAGAAGTATCCAGTTCACACATCCTCGGGTCAATTTATCCCAACATTGAATGCTATCACATCAAGTCAGGACCTTAACTGGATGATCCAACCAAGTGTACGCCCCCTGAATTTACCACCATACCAAAGCCCTCGGCATGGGGTAATCCGTAACATGGGAGGAGTCTTGAGTATGGGGCGAAGGAGAAATGGAGAACATCTGTCACCAGAGGAAGAGGAGCGATAG(SEQ ID NO.2)。
实施例2:突变体Fosl1ΔDB的制备及其表达载体构建
1.以野生型热带爪蛙Fosl1基因(XM_002939331.4)的编码区(CDS)为模板,设计融合PCR引物(FNT-Fw/FΔDB-Re;FΔDB-Fw/FCT-Re)扩增缺失DNA-binding(DB)domain(aa124-147)的缺失型突变体Fosl1ΔDB所对应的编码DNA序列。所述融合PCR引物的序列如下所示:
其中,上述引物FNT-Fw/FΔDB-Re用于扩增位于DNA-binding(DB)domain上游所对应的编码DNA序列(PCR产物1),FΔDB-Fw/FCT-Re引物用于扩增位于DNA-binding(DB)domain下游所对应的编码DNA序列(PCR产物2),并利用Seamless Cloning andAssembly Kit(北京全式金)进行融合PCR,获得缺失编码Fosl1ΔDB突变体的融合DNA片段。
上述正向引物FNT-Fw序列中,5’端gg是酶切位点保护碱基,下划线字母是KpnI酶切位点序列,方框内的小写字母是人为加入的Kozak信号;上述反向引物FCT-Re序列中,5’端cg是酶切位点保护碱基,下划线字母是EcoRI酶切位点序列。
2.将扩增的融合PCR产物,用KpnI与EcoRI双酶切,回收带有粘性末端的DNA片段,并连接到pcDNA3.1载体的KpnI与EcoRI酶切位点之间,进而构建pcDNA3.1-Fosl1ΔDB表达载体。
3.将重组载体转化大肠杆菌HD5a,涂平板,挑选阳性克隆,进行DNA测序验证。
上述Fosl1ΔDB突变体的氨基酸序列如下所示:
MYRDFTGAFPQPDSGCSSHGLRNSGSSPILGTSGMGHSVRLNPPPEDAQQKYPVHTSSGQFIPTLNAITSSQDLNWMIQPSVRPLNLPPYQSPRHGVIRNMGGVLSMGRRRNGEHLSPEEEERYLQAETDKLEEEKSSLQKEIAELQKQKDKLELILEAHQPICKFPDSHHNMQQVDSSRLVKKEPHEESPRGPKVNLPRIELSDTILEPEALHTPTLMKTPSITPFTPNLIFTYPGPQESCSTAHRRLSRSSSSGSSGEQSPCSLSSNSLLTL(SEQ ID NO.3)。
上述Fosl1ΔDB突变体的核苷酸序列如下所示:
ATGTACAGAGACTTCACTGGAGCCTTCCCTCAGCCAGATTCGGGGTGCAGCAGCCACGGCCTACGGAACTCGGGATCTTCCCCAATTTTAGGAACTTCAGGAATGGGACACAGCGTCAGACTGAACCCACCACCCGAGGACGCGCAGCAGAAGTATCCAGTTCACACATCCTCGGGTCAATTTATCCCAACATTGAATGCTATCACATCAAGTCAGGACCTTAACTGGATGATCCAACCAAGTGTACGCCCCCTGAATTTACCACCATACCAAAGCCCTCGGCATGGGGTAATCCGTAACATGGGAGGAGTCTTGAGTATGGGGCGAAGGAGAAATGGAGAACATCTGTCACCAGAGGAAGAGGAGCGATACCTGCAGGCAGAGACAGACAAACTTGAAGAAGAGAAGTCATCCCTCCAGAAAGAAATTGCTGAGCTGCAGAAGCAGAAGGATAAGCTGGAACTCATCCTTGAGGCTCACCAGCCTATATGCAAGTTTCCTGACTCCCATCACAACATGCAGCAAGTGGACTCCTCCAGGCTGGTTAAGAAGGAACCACATGAAGAGTCACCCAGGGGACCTAAAGTCAACCTTCCCAGGATAGAGCTGAGCGACACAATCCTAGAGCCAGAGGCCCTTCACACCCCAACACTCATGAAGACACCATCCATTACTCCTTTTACGCCAAATTTGATATTCACTTATCCTGGTCCACAAGAATCATGTTCTACAGCGCACCGAAGGCTGAGCAGAAGCAGCAGCAGTGGTAGTAGTGGAGAGCAAAGTCCATGTTCTCTAAGTTCTAACAGCCTACTGACTCTTTAG(SEQID NO.4)。
实施例3:突变体Fosl1ΔLZ的制备及其表达载体构建
1.以野生型热带爪蛙Fosl1基因(XM_002939331.4)的编码区(CDS)为模板,设计融合PCR引物(FNT-Fw/FΔLZ-Re;FΔLZ-Fw/FCT-Re)扩增缺失Basic leucine zipper(bZIP)domain(aa149-177)的缺失型突变体Fosl1ΔLZ所对应的编码DNA序列。所述融合PCR引物的序列如下所示:
其中,上述引物FNT-Fw/FΔLZ-Re用于扩增位于Basic leucine zipper(bZIP)domain上游所对应的编码DNA序列(PCR产物1),FΔLZ-Fw/FCT-Re引物用于扩增位于Basicleucine zipper(bZIP)domain下游所对应的编码DNA序列(PCR产物2),并利用Seamless Cloning and Assembly Kit(北京全式金)进行融合PCR,获得缺失编码Fosl1ΔLZ突变体的融合DNA片段。
上述正向引物FNT-Fw序列中,5’端gg是酶切位点保护碱基,下划线字母是KpnI酶切位点序列,方框内的小写字母是人为加入的Kozak信号;上述反向引物FCT-Re序列中,5’端cg是酶切位点保护碱基,下划线字母是EcoRI酶切位点序列。
2.将扩增的融合PCR产物,用KpnI与EcoRI双酶切,回收带有粘性末端的DNA片段,并连接到pcDNA3.1载体的KpnI与EcoRI酶切位点之间,进而构建pcDNA3.1-Fosl1ΔLZ表达载体。
3.将重组载体转化大肠杆菌HD5a,涂平板,挑选阳性克隆,进行DNA测序验证。
上述Fosl1ΔLZ突变体的氨基酸序列如下所示:
MYRDFTGAFPQPDSGCSSHGLRNSGSSPILGTSGMGHSVRLNPPPEDAQQKYPVHTSSGQFIPTLNAITSSQDLNWMIQPSVRPLNLPPYQSPRHGVIRNMGGVLSMGRRRNGEHLSPEEEERRRVRRERNKVAAAKCRNRRKELTDYELILEAHQPICKFPDSHHNMQQVDSSRLVKKEPHEESPRGPKVNLPRIELSDTILEPEALHTPTLMKTPSITPFTPNLIFTYPGPQESCSTAHRRLSRSSSSGSSGEQSPCSLSSNSLLTL(SEQ ID NO.5)。
上述Fosl1ΔLZ突变体的核苷酸序列如下所示:
ATGTACAGAGACTTCACTGGAGCCTTCCCTCAGCCAGATTCGGGGTGCAGCAGCCACGGCCTACGGAACTCGGGATCTTCCCCAATTTTAGGAACTTCAGGAATGGGACACAGCGTCAGACTGAACCCACCACCCGAGGACGCGCAGCAGAAGTATCCAGTTCACACATCCTCGGGTCAATTTATCCCAACATTGAATGCTATCACATCAAGTCAGGACCTTAACTGGATGATCCAACCAAGTGTACGCCCCCTGAATTTACCACCATACCAAAGCCCTCGGCATGGGGTAATCCGTAACATGGGAGGAGTCTTGAGTATGGGGCGAAGGAGAAATGGAGAACATCTGTCACCAGAGGAAGAGGAGCGAAGACGAGTGAGACGTGAGCGGAACAAGGTAGCAGCAGCAAAATGCCGTAATCGTCGCAAGGAGCTAACAGATTACGAACTCATCCTTGAGGCTCACCAGCCTATATGCAAGTTTCCTGACTCCCATCACAACATGCAGCAAGTGGACTCCTCCAGGCTGGTTAAGAAGGAACCACATGAAGAGTCACCCAGGGGACCTAAAGTCAACCTTCCCAGGATAGAGCTGAGCGACACAATCCTAGAGCCAGAGGCCCTTCACACCCCAACACTCATGAAGACACCATCCATTACTCCTTTTACGCCAAATTTGATATTCACTTATCCTGGTCCACAAGAATCATGTTCTACAGCGCACCGAAGGCTGAGCAGAAGCAGCAGCAGTGGTAGTAGTGGAGAGCAAAGTCCATGTTCTCTAAGTTCTAACAGCCTACTGACTCTTTAG(SEQ ID NO.6)。
实施例4:突变体对野生型Fosl1蛋白功能的影响
(1)将实施例1-3所制备的3种突变体的DNA片段分别克隆到pcDNA3.1载体(Invitrogen)的CMV启动子下游的KpnI与EcoRI酶切位点之间,分别构建pcDNA-Fosl1-NT、pcDNA-Fosl1ΔDB、pcDNA-Fosl1ΔLZ表达载体。
(2)将热带爪蛙野生型Fosl1基因(XM_002939331.4)的编码DNA片段克隆到pcDNA3.1载体CMV启动子下游的KpnI与EcoRI酶切位点之间,构建pcDNA-Fosl1表达载体。
(3)利用pAP1-Luc荧光素酶报告基因载体(碧云天)检测实施例1-3所制备的3种突变体对野生型Fosl1蛋白转录活性的影响,具体方案如下:
①实验分为5组,如下所示:
表1实验分组
分组 | 注射质粒 |
1 | pAP1-Luc |
2 | pAP1-Luc+pcDNA-Fosl1 |
3 | pAP1-Luc+pcDNA-Fosl1+pcDNA-Fosl1-NT |
4 | pAP1-Luc+pcDNA-Fosl1+pcDNA-Fosl1ΔDB |
5 | pAP1-Luc+pcDNA-Fosl1+pcDNA-Fosl1ΔLZ |
②将上述每个实验组中的质粒DNA(各500ng,每组总的质粒DNA量为1.5μg,不足质粒DNA用pcDNA3.1补齐)用无菌水配置成10μL的注射液,然后用显微注射仪将2nL的注射液注射到热带爪蛙受精卵(1细胞期)的动物极,注射后的受精卵放置于0.1×MBS胚胎培养液(由5×MBS(配置方法:称取NaCl 25.9g、Hepes 11.9g、NaHCO3 1.0g、KCl 0.4g、MgSO4 0.5g、Ca(NO3)2 0.8g、CaCl2 0.2g,加入800mL去离子水并混匀,pH调至7.4,容量瓶定容至1000mL,高压灭菌后4℃保存备用)稀释而成)中,并在25℃的培养箱中孵育48小时。收集胚胎,利用萤火虫萤光素酶报告基因检测试剂盒(碧云天,RG005)及BioTek Synergy4多功能酶标仪(BioTek)检测荧光素酶的活性。
结果表明,pcDNA-Fosl1-NT及pcDNA-Fosl1ΔDB能有效抑制pcDNA-Fosl1所诱导的荧光素酶的活性(图1),进而说明突变体Fosl1-NT及Fosl1ΔDB的过表达能有效抑制野生型Fosl1蛋白的转录活性,有望成为野生型Fosl1蛋白的dominant-negative突变体(dnFosl1)。
实施例5:突变体对Fos家族其他蛋白功能的影响
(1)将热带爪蛙野生型Fos基因(NM_001016200.2)、FosB基因(XM_002932635.5)及Fosl2基因(XM_002938419.5)的编码DNA片段,分别克隆到pcDNA3.1载体CMV启动子下游的KpnI与EcoRI酶切位点之间,进而构建pcDNA-Fos、pcDNA-FosB及pcDNA-Fosl2等3个表达载体。
(2)利用pAP1-Luc荧光素酶报告基因载体(碧云天)检测实施例4所筛选的能有效抑制野生型Fosl1蛋白功能的2种突变体(Fosl1-NT及Fosl1ΔDB)对Fos家族其他蛋白功能的影响,具体方案如下:
①实验分为10组,如下所示:
表2实验分组
分组 | 注射质粒 |
1 | pAP1-Luc |
2 | pAP1-Luc+pcDNA-Fos |
3 | pAP1-Luc+pcDNA-Fos+pcDNA-Fosl1-NT |
4 | pAP1-Luc+pcDNA-Fos+pcDNA-Fosl1ΔDB |
5 | pAP1-Luc+pcDNA-FosB |
6 | pAP1-Luc+pcDNA-FosB+pcDNA-Fosl1-NT |
7 | pAP1-Luc+pcDNA-FosB+pcDNA-Fosl1ΔDB |
8 | pAP1-Luc+pcDNA-Fosl2 |
9 | pAP1-Luc+pcDNA-Fosl2+pcDNA-Fosl1-NT |
10 | pAP1-Luc+pcDNA-Fosl2+pcDNA-Fosl1ΔDB |
②将上述每个实验组中的质粒DNA(各500ng,每组总的质粒DNA量为1.5μg,不足质粒DNA用pcDNA3.1补齐)用无菌水配置成10μL的注射液,然后用显微注射仪将2nL的注射液注射到热带爪蛙受精卵(1细胞期)的动物极,注射后的受精卵放置于0.1×MBS胚胎培养液(由5×MBS(配置方法:称取NaCl 25.9g、Hepes 11.9g、NaHCO3 1.0g、KCl 0.4g、MgSO4 0.5g、Ca(NO3)2 0.8g、CaCl2 0.2g,加入800mL去离子水并混匀,pH调至7.4,容量瓶定容至1000mL,高压灭菌后4℃保存备用)稀释而成)中,并在25℃的培养箱中孵育48小时。收集胚胎,利用萤火虫萤光素酶报告基因检测试剂盒(碧云天,RG005)及BioTek Synergy4多功能酶标仪(BioTek)检测荧光素酶的活性。结果表明,pcDNA-Fosl1-NT对pcDNA-Fos、pcDNA-FosB及pcDNA-Fosl2所诱导的荧光素酶的活性并无显著影响,而pcDNA-Fosl1ΔDB对pcDNA-Fos、pcDNA-FosB及pcDNA-Fosl2所诱导的荧光素酶的活性均有不同程度的抑制作用(图2)。上述结果说明,突变体Fosl1-NT的过表达能有效抑制野生型Fosl1蛋白的转录活性,并对Fos家族其他蛋白(Fos、FosB及Fosl2)的功能无显著影响。因此,突变体Fosl1-NT可用作特异性抑制野生型Fosl1蛋白功能的dominant-negative突变体(dnFosl1)。
实施例6:转基因载体pMlc2-dnFosl1-T2A-EGFP的构建
以实施例4和实施例5所筛选到的dnFosl1突变体(Fosl1-NT)所对应的编码DNA序列为基础,分别合成dnFosl1-T2A-EGFP融合DNA片段(上海捷瑞生物工程有限公司),并将合成的融合DNA片段分别克隆到pMlc2-NTR-T2A-Dendra2opt载体(已在我们前期申请的专利:202010650078.3中公开)的EcoRI与HpaI酶切位点之间,替换原有载体中的NTRopt-T2A-Dendra2opt片段,进而构建pMlc2-dnFosl1-T2A-EGFP转基因载体(图3)。
实施例7:转基因热带爪蛙品系的构建
将实施例6构建成功的pMlc2-dnFosl1-T2A-EGFP转基因载体分别与I-SceI归位内切酶共同注射(将500ng转基因载体与20U的I-SceI核酸酶(NEB)用无菌水配置成10μL的注射液,每个受精卵注射2nL)到热带爪蛙受精卵(1细胞期)的动物极,注射后的受精卵放置于0.1×MBS胚胎培养液(由5×MBS(配置方法:称取NaCl 25.9g、Hepes 11.9g、NaHCO3 1.0g、KCl 0.4g、MgSO4 0.5g、Ca(NO3)2 0.8g、CaCl2 0.2g,加入800mL去离子水并混匀,pH调至7.4,容量瓶定容至1000mL,高压灭菌后4℃保存备用)稀释而成)中,并在21℃的培养箱中孵育过夜,于第二天早上将培养箱温度调至25℃继续孵育胚胎发育至St45期,利用荧光体式显微镜筛选心脏特异性表达绿色荧光(EGFP)的F0胚胎。结果表明,在pMlc2-dnFosl1-T2A-EGFP转基因载体注射胚胎中筛选到了心脏能特异性表达EGFP报告基因的F0代蝌蚪(图4)。将F0代阳性蝌蚪饲养至成蛙,进而建立心肌细胞特异性失活Fosl1蛋白的转基因热带爪蛙品系Tg(Mlc2-dnFosl1-T2A-EGFP)。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110>暨南大学
<120> 一种热带爪蛙Fosl1蛋白突变体及其应用
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 123
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Fosl1-NT突变体的氨基酸序列
<400> 1
Met Tyr Arg Asp Phe Thr Gly Ala Phe Pro Gln Pro Asp Ser Gly Cys
1 5 10 15
Ser Ser His Gly Leu Arg Asn Ser Gly Ser Ser Pro Ile Leu Gly Thr
20 25 30
Ser Gly Met Gly His Ser Val Arg Leu Asn Pro Pro Pro Glu Asp Ala
35 40 45
Gln Gln Lys Tyr Pro Val His Thr Ser Ser Gly Gln Phe Ile Pro Thr
50 55 60
Leu Asn Ala Ile Thr Ser Ser Gln Asp Leu Asn Trp Met Ile Gln Pro
65 70 75 80
Ser Val Arg Pro Leu Asn Leu Pro Pro Tyr Gln Ser Pro Arg His Gly
85 90 95
Val Ile Arg Asn Met Gly Gly Val Leu Ser Met Gly Arg Arg Arg Asn
100 105 110
Gly Glu His Leu Ser Pro Glu Glu Glu Glu Arg
115 120
<210> 2
<211> 372
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Fosl1-NT突变体的核苷酸序列
<400> 2
atgtacagag acttcactgg agccttccct cagccagatt cggggtgcag cagccacggc 60
ctacggaact cgggatcttc cccaatttta ggaacttcag gaatgggaca cagcgtcaga 120
ctgaacccac cacccgagga cgcgcagcag aagtatccag ttcacacatc ctcgggtcaa 180
tttatcccaa cattgaatgc tatcacatca agtcaggacc ttaactggat gatccaacca 240
agtgtacgcc ccctgaattt accaccatac caaagccctc ggcatggggt aatccgtaac 300
atgggaggag tcttgagtat ggggcgaagg agaaatggag aacatctgtc accagaggaa 360
gaggagcgat ag 372
<210> 3
<211> 274
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Fosl1∆DB突变体的氨基酸序列
<400> 3
Met Tyr Arg Asp Phe Thr Gly Ala Phe Pro Gln Pro Asp Ser Gly Cys
1 5 10 15
Ser Ser His Gly Leu Arg Asn Ser Gly Ser Ser Pro Ile Leu Gly Thr
20 25 30
Ser Gly Met Gly His Ser Val Arg Leu Asn Pro Pro Pro Glu Asp Ala
35 40 45
Gln Gln Lys Tyr Pro Val His Thr Ser Ser Gly Gln Phe Ile Pro Thr
50 55 60
Leu Asn Ala Ile Thr Ser Ser Gln Asp Leu Asn Trp Met Ile Gln Pro
65 70 75 80
Ser Val Arg Pro Leu Asn Leu Pro Pro Tyr Gln Ser Pro Arg His Gly
85 90 95
Val Ile Arg Asn Met Gly Gly Val Leu Ser Met Gly Arg Arg Arg Asn
100 105 110
Gly Glu His Leu Ser Pro Glu Glu Glu Glu Arg Tyr Leu Gln Ala Glu
115 120 125
Thr Asp Lys Leu Glu Glu Glu Lys Ser Ser Leu Gln Lys Glu Ile Ala
130 135 140
Glu Leu Gln Lys Gln Lys Asp Lys Leu Glu Leu Ile Leu Glu Ala His
145 150 155 160
Gln Pro Ile Cys Lys Phe Pro Asp Ser His His Asn Met Gln Gln Val
165 170 175
Asp Ser Ser Arg Leu Val Lys Lys Glu Pro His Glu Glu Ser Pro Arg
180 185 190
Gly Pro Lys Val Asn Leu Pro Arg Ile Glu Leu Ser Asp Thr Ile Leu
195 200 205
Glu Pro Glu Ala Leu His Thr Pro Thr Leu Met Lys Thr Pro Ser Ile
210 215 220
Thr Pro Phe Thr Pro Asn Leu Ile Phe Thr Tyr Pro Gly Pro Gln Glu
225 230 235 240
Ser Cys Ser Thr Ala His Arg Arg Leu Ser Arg Ser Ser Ser Ser Gly
245 250 255
Ser Ser Gly Glu Gln Ser Pro Cys Ser Leu Ser Ser Asn Ser Leu Leu
260 265 270
Thr Leu
<210> 4
<211> 825
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Fosl1∆DB突变体的核苷酸序列
<400> 4
atgtacagag acttcactgg agccttccct cagccagatt cggggtgcag cagccacggc 60
ctacggaact cgggatcttc cccaatttta ggaacttcag gaatgggaca cagcgtcaga 120
ctgaacccac cacccgagga cgcgcagcag aagtatccag ttcacacatc ctcgggtcaa 180
tttatcccaa cattgaatgc tatcacatca agtcaggacc ttaactggat gatccaacca 240
agtgtacgcc ccctgaattt accaccatac caaagccctc ggcatggggt aatccgtaac 300
atgggaggag tcttgagtat ggggcgaagg agaaatggag aacatctgtc accagaggaa 360
gaggagcgat acctgcaggc agagacagac aaacttgaag aagagaagtc atccctccag 420
aaagaaattg ctgagctgca gaagcagaag gataagctgg aactcatcct tgaggctcac 480
cagcctatat gcaagtttcc tgactcccat cacaacatgc agcaagtgga ctcctccagg 540
ctggttaaga aggaaccaca tgaagagtca cccaggggac ctaaagtcaa ccttcccagg 600
atagagctga gcgacacaat cctagagcca gaggcccttc acaccccaac actcatgaag 660
acaccatcca ttactccttt tacgccaaat ttgatattca cttatcctgg tccacaagaa 720
tcatgttcta cagcgcaccg aaggctgagc agaagcagca gcagtggtag tagtggagag 780
caaagtccat gttctctaag ttctaacagc ctactgactc tttag 825
<210> 5
<211> 269
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Fosl1∆LZ突变体的氨基酸序列
<400> 5
Met Tyr Arg Asp Phe Thr Gly Ala Phe Pro Gln Pro Asp Ser Gly Cys
1 5 10 15
Ser Ser His Gly Leu Arg Asn Ser Gly Ser Ser Pro Ile Leu Gly Thr
20 25 30
Ser Gly Met Gly His Ser Val Arg Leu Asn Pro Pro Pro Glu Asp Ala
35 40 45
Gln Gln Lys Tyr Pro Val His Thr Ser Ser Gly Gln Phe Ile Pro Thr
50 55 60
Leu Asn Ala Ile Thr Ser Ser Gln Asp Leu Asn Trp Met Ile Gln Pro
65 70 75 80
Ser Val Arg Pro Leu Asn Leu Pro Pro Tyr Gln Ser Pro Arg His Gly
85 90 95
Val Ile Arg Asn Met Gly Gly Val Leu Ser Met Gly Arg Arg Arg Asn
100 105 110
Gly Glu His Leu Ser Pro Glu Glu Glu Glu Arg Arg Arg Val Arg Arg
115 120 125
Glu Arg Asn Lys Val Ala Ala Ala Lys Cys Arg Asn Arg Arg Lys Glu
130 135 140
Leu Thr Asp Tyr Glu Leu Ile Leu Glu Ala His Gln Pro Ile Cys Lys
145 150 155 160
Phe Pro Asp Ser His His Asn Met Gln Gln Val Asp Ser Ser Arg Leu
165 170 175
Val Lys Lys Glu Pro His Glu Glu Ser Pro Arg Gly Pro Lys Val Asn
180 185 190
Leu Pro Arg Ile Glu Leu Ser Asp Thr Ile Leu Glu Pro Glu Ala Leu
195 200 205
His Thr Pro Thr Leu Met Lys Thr Pro Ser Ile Thr Pro Phe Thr Pro
210 215 220
Asn Leu Ile Phe Thr Tyr Pro Gly Pro Gln Glu Ser Cys Ser Thr Ala
225 230 235 240
His Arg Arg Leu Ser Arg Ser Ser Ser Ser Gly Ser Ser Gly Glu Gln
245 250 255
Ser Pro Cys Ser Leu Ser Ser Asn Ser Leu Leu Thr Leu
260 265
<210> 6
<211> 810
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Fosl1∆LZ突变体的核苷酸序列
<400> 6
atgtacagag acttcactgg agccttccct cagccagatt cggggtgcag cagccacggc 60
ctacggaact cgggatcttc cccaatttta ggaacttcag gaatgggaca cagcgtcaga 120
ctgaacccac cacccgagga cgcgcagcag aagtatccag ttcacacatc ctcgggtcaa 180
tttatcccaa cattgaatgc tatcacatca agtcaggacc ttaactggat gatccaacca 240
agtgtacgcc ccctgaattt accaccatac caaagccctc ggcatggggt aatccgtaac 300
atgggaggag tcttgagtat ggggcgaagg agaaatggag aacatctgtc accagaggaa 360
gaggagcgaa gacgagtgag acgtgagcgg aacaaggtag cagcagcaaa atgccgtaat 420
cgtcgcaagg agctaacaga ttacgaactc atccttgagg ctcaccagcc tatatgcaag 480
tttcctgact cccatcacaa catgcagcaa gtggactcct ccaggctggt taagaaggaa 540
ccacatgaag agtcacccag gggacctaaa gtcaaccttc ccaggataga gctgagcgac 600
acaatcctag agccagaggc ccttcacacc ccaacactca tgaagacacc atccattact 660
ccttttacgc caaatttgat attcacttat cctggtccac aagaatcatg ttctacagcg 720
caccgaaggc tgagcagaag cagcagcagt ggtagtagtg gagagcaaag tccatgttct 780
ctaagttcta acagcctact gactctttag 810
<210> 7
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> FNT-Fw
<400> 7
ggggtaccgc caccatgtac agagacttca ctgg 34
<210> 8
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> FNT-Re
<400> 8
cggaattcct atcgctcctc ttcctctggt gaca 34
<210> 9
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> F∆DB-Re
<400> 9
gcctgcaggt atcgctcctc ttcctctggt gaca 34
<210> 10
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> F∆DB -Fw
<400> 10
aagaggagcg atacctgcag gcagagacag acaa 34
<210> 11
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> FCT-Re
<400> 11
cggaattcct aaagagtcag taggctgtta gaac 34
<210> 12
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> F∆LZ-Re
<400> 12
aggatgagtt cgtaatctgt tagctccttg cgac 34
<210> 13
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> F∆LZ -Fw
<400> 13
taacagatta cgaactcatc cttgaggctc acca 34
<210> 14
<211> 63
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> T2A
<400> 14
ggcagtggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc 60
cca 63
Claims (7)
1.一种热带爪蛙Fosl1蛋白突变体,其特征在于:其氨基酸序列如SEQ ID NO.1所示。
2.根据权利要求1所述的热带爪蛙Fosl1蛋白突变体,其特征在于:所述的Fosl1蛋白突变体的编码序列如SEQ ID NO.2所示。
3.权利要求1所述的热带爪蛙Fosl1蛋白突变体、其编码序列、含有其编码序列的重组表达载体或工程菌在构建Fosl1蛋白功能缺失型热带爪蛙模型中的应用。
4.根据权利要求3所述的应用,其特征在于:
所述的应用的具体操作为:将所述的Fosl1蛋白突变体的编码序列克隆到表达载体中,构建含有Fosl1蛋白突变体的转基因载体,并注射热带爪蛙受精卵的动物极,孵育,筛选成功表达Fosl1蛋白突变体的胚胎,培养至成蛙,获得Fosl1蛋白失活的热带爪蛙品系。
5.权利要求1所述的热带爪蛙Fosl1蛋白突变体、其编码序列、含有其编码序列的重组表达载体或工程菌在构建心肌细胞特异性失活Fosl1蛋白的热带爪蛙模型中的应用。
6.根据权利要求5所述的应用,其特征在于:
所述的应用的具体操作为:
S1、根据编码所述的Fosl1蛋白突变体的DNA序列,合成dnFosl1-T2A-EGFP融合DNA片段,并将合成的融合DNA片段克隆到pMlc2-NTR-T2A-Dendra2opt载体的EcoRI与HpaI酶切位点之间,替换原有载体中的NTRopt-T2A-Dendra2opt片段,进而构建pMlc2-dnFosl1-T2A-EGFP转基因载体;其中,所述的融合DNA片段中dnFosl1是Fosl1蛋白突变体,其核苷酸序列如SEQ ID NO.2所示;T2A是连接肽,其核苷酸序列如下所示:GGCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCTGGCCCA;
S2、将步骤S1所制备的pMlc2-dnFosl1-T2A-EGFP转基因载体与I-SceI归位内切酶共同注射热带爪蛙受精卵的动物极,孵育,筛选心脏特异性表达绿色荧光的F0胚胎,培养至成蛙,获得心肌细胞特异性失活Fosl1蛋白的热带爪蛙品系Tg(Mlc2-dnFosl1-T2A-EGFP)。
7.根据权利要求6所述的应用,其特征在于:
所述的共同注射方式为:500ng转基因载体和20U I-SceI归位内切酶用无菌水配置成10μL的注射液,每个受精卵注射2nL;
所述的共同注射用的热带爪蛙受精卵为1细胞期热带爪蛙受精卵;
所述的孵育为孵育胚胎至45期;
所述的筛选采用荧光体式显微镜实现;
所述的培养所用的培养液为MBS胚胎培养液,配制方法为称取NaCl 25.9 g、Hepes11.9 g、NaHCO3 1.0 g、KCl 0.4 g、MgSO4 0.5 g、Ca(NO3)2 0.8 g、CaCl2 0.2 g,加入800 mL去离子水并混匀,pH调至7.4,容量瓶定容至1000 mL,高压灭菌后4℃保存备用,使用前稀释50倍;
所述的培养的温度为注射后的12小时内为21℃,然后再调整为25℃。
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