CN116814689B - 特异性表达hMRGPRX2的转基因小鼠的构建方法及其应用 - Google Patents
特异性表达hMRGPRX2的转基因小鼠的构建方法及其应用 Download PDFInfo
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Abstract
本发明提供一种特异性表达hMRGPRX2的转基因小鼠的构建方法及其应用。本发明通过将人的MRGPRX2替换小鼠的同源基因Mrgprb2表达在小鼠肥大细胞中，成功构建出一种人源化小鼠，为研究MRGPRX2基因功能，筛选治疗肥大细胞上hMRGPRX2介导的药源性假性过敏以及神经感觉炎性反应相关疾病的药物提供了动物模型。
Description
技术领域
本发明涉及生物技术领域，具体地说，涉及一种特异性表达hMRGPRX2的转基因小鼠的构建方法及其应用。
背景技术
药物引起的过敏反应占所有药物副作用的15％，且其中很大一部分是非IgE介导的，被称为假性过敏反应，主要表现为给药部位的红肿、皮疹，、瘙痒以及全身性过敏反应，体温，血压和心率的变化等，严重影响生活质量。但目前仍然缺乏缓解或治疗药源性假性过敏反应的有效药物。最近研究表明，药源性假性过敏反应的发生与肥大细胞上高表达的受体MrgprX2有密切关系，它的激活可引发非IgE途径的肥大细胞脱颗粒。在野生小鼠上发现一些阳离子分子和神经肽类可以引发类过敏反应症状，但在Mrgprb2敲除转基因小鼠上却不能引发相同的过敏发应，用IgE介导的底物作用在Mrgprb2敲除的转基因小鼠上，依旧可以引发过敏反应，因此说明Mrgprb2介导了非IgE介导的肥大细胞脱颗粒反应。小鼠Mrgprb2与人MRGPRX2具有高度同源性，并且在体外转染Mrgprb2与MRGPRX2的HEK细胞上已经验证Mrgprb2与MRGPRX2均可以被一些阳离子药物和神经肽类激活。因此，hMRGPRX2是治疗假性药源性过敏反应的药物靶点，hMRGPRX2的拮抗剂将是治疗这类过敏反应的备选药物。
另外，神经炎性领域的很多疾病(特异性皮炎，荨麻疹等)也都与肥大细胞相关，并且近期研究证明感觉神经元分泌的一些物质(SP)可以直接激活肥大细胞上高表达的hMRGPRX2,进而介导肥大细胞释放前炎性因子和趋化因子，促进免疫细胞的迁移，因此，hMRGPRX2将是该疾病领域中具有潜力的靶点。
目前开发治疗假性药源性过敏以及神经免疫炎性疾病的技术瓶颈主要在于，缺乏精准的可以完整反应药理机制的动物模型，导致动物实验中应用有效的药物在转向临床用于人类时往往无效。这种药物疗效的差异会引起昂贵的临床前和临床研究失败。因此，特异性靶向人源蛋白/受体开发治疗疾病的药物将是今后药物开发的趋势，亟需建立合适的动物模型用于在体研究hMRGPRX2的功能，筛选和验证hMRGPRX2拮抗剂对药源性过敏反应以及感觉神经炎性反应的抑制作用，还可以筛选目前引起过敏反应副作用的上市药品。
发明内容
本发明的目的是提供一种特异性表达hMRGPRX2(人源MRGPRX2)的转基因小鼠的构建方法及其应用。
本发明的技术原理：MRGPRX2可以介导肥大细胞脱颗粒相关的药源性过敏与神经炎性反应；小鼠的Mrgprb2与人MRGPRX2同源且均高表达在肥大细胞表面；通过人MRGPRX2替换小鼠Mrgprb2激活小鼠肥大细胞可以引起脱颗粒。本发明利用CRISPR-Cas9基因敲入技术，构建特异性表达hMRGPRX2的转基因小鼠模型。此模型可以用于研究hMRGPRX2的功能，筛选并验证hMRGPRX2的拮抗剂，开发用于治疗药源性假性过敏和神经炎性反应相关疾病的药物。
为了实现本发明目的，第一方面，本发明提供一种特异性表达hMRGPRX2的转基因小鼠的构建方法，所述方法包括：构建CRISPR-Cas9系统介导的小鼠Mrgprb2基因敲除且定点整合外源基因①的转基因小鼠，命名为hMRGPRX2-Cre。
所述外源基因①是由hMRGPRX2基因(编码区)和重组酶基因之间通过P2A序列(P2A自切割肽段序列)连接而成，且所述hMRGPRX2基因的3’端修饰有3×HA标签序列；其中，所述hMRGPRX2基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示，hMRGPRX2基因的CDS序列如SEQ ID NO:2所示，mRNA对应的cDNA序列如SEQ ID NO:3所示。
进一步地，所述方法包括：根据小鼠Mrgprb2基因序列，构建基于CRISPR-Cas9系统的sgRNA和Cas9表达载体，分别体外转录得到sgRNA和Cas9 mRNA，并根据sgRNA作用位点构建含有外源基因①且可以整合至宿主基因组中的供体质粒，然后将体外转录得到的sgRNA和Cas9 mRNA以及供体质粒共同转入野生型小鼠的受精卵中，然后将克隆胚胎通过非手术法移入小鼠子宫内进行妊娠，获得转基因小鼠。
本发明的CRISPR-Cas9系统靶向Mrgprb2阳性的小鼠细胞。
进一步地，所述sgRNA靶向小鼠Mrgprb2基因外显子2。
优选地，sgRNA作用位点的核苷酸序列为5′-AGGTGCTTAGATTCTTGATTAGG-3′和5′-TTAGGCACCGAAGGTTCAGGCGG-3′(SEQ ID NO:7-8)。
前述的方法，所述重组酶为Cre重组酶。
进一步地，所述外源基因①为hMRGPRX2-3×HA-P2A-iCre-WPRE-pA，其核苷酸序列如SEQ ID NO:4所示。
进一步地，所述供体质粒含有顺次连接的小鼠Mrgprb2基因5’同源臂片段、外源基因①和小鼠Mrgprb2基因3’同源臂片段。
优选地，所述小鼠Mrgprb2基因5’、3’同源臂片段的核苷酸序列分别如SEQ ID NO:5和6所示。
优选地，所述小鼠为C57BL/6J。
第二方面，本发明提供按照所述方法构建的转基因小鼠的以下任一应用：
(1)用作构建治疗或缓解人药源性假性过敏以及神经感觉炎性反应相关疾病的动物模型；
(2)用于筛选治疗或缓解人药源性假性过敏以及神经感觉炎性反应相关疾病的药物；
(3)用于检测引起人药源性假性过敏瘙痒症的物质；
(4)用于筛选hMRGPRX2受体的激动剂或抑制剂；
(5)用于hMRGPRX2基因功能的研究。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明通过将人的MRGPRX2替换小鼠的同源基因Mrgprb2表达在小鼠肥大细胞中，成功构建出一种人源化小鼠，即将hMRGPRX2特异性表达在小鼠Mrgprb2阳性的细胞中。提取小鼠皮肤组织中RNA进行RT-PCR和体外细胞钙流测试证明了hMRGPRX2功能性地表达在小鼠肥大细胞中。在体足底皮内注射hMRGPRX2激动剂C48/80证明了hMRGPRX2可以介导肥大细胞脱颗粒。该人源化小鼠可用于筛选治疗肥大细胞上hMRGPRX2介导的药源性假性过敏以及神经感觉炎性反应相关疾病的药物，为后续开展临床实验提供理论基础和实验依据。本发明为研究hMRGPRX2的功能和筛选治疗肥大细胞上hMRGPRX2介导的药源性假性过敏以及神经感觉炎性反应相关疾病的药物提供了模式动物。
附图说明
图1为本发明较佳实施例中hMRGPRX2-Cre转基因小鼠的基因型鉴定结果以及RT-PCR结果；其中，a：hMRGPRX2-Cre转基因小鼠基因信息模式图；b：hMRGPRX2-Cre转基因小鼠的基因型鉴定结果，c：对hMRGPRX2-Cre转基因小鼠的皮肤组织提取RNA，进行RT-PCR。
图2为本发明较佳实施例中hMRGPRX2-Cre转基因小鼠的细胞水平验证结果。从hMRGPRX2-Cre转基因小鼠腹壁提取的肥大细胞，用hMRGPRX2受体激动剂C48/80处理细胞后钙成像结果代表性曲线。
图3为本发明较佳实施例中用hMRGPRX2受体激动剂C48/80可以引起hMRGPRX2-Cre转基因小鼠肥大细胞脱颗粒；其中，a：hMRGPRX2-Cre转基因小鼠足底伊文思蓝外渗情况；b：hMRGPRX2-Cre转基因小鼠中引起的后爪皮下组织伊文思蓝含量的统计结果。
具体实施方式
本发明提供一种特异性表达hMRGPRX2受体的转基因小鼠，该小鼠模型可以用于研究hMRGPRX2的功能，筛选并验证hMRGPRX2的拮抗剂，开发用于治疗肥大细胞上hMRGPRX2介导的药源性假性过敏以及神经感觉炎性反应相关疾病的药物。
本发明的理论基础是基于人体内的MRGPRX2，其高表达在人体肥大细胞中，且容易被多种外源性物质和内源性神经肽类激活，这一特性对于反应化合物致敏特性和引发炎性紧密相关。本发明成功构建得到在肥大细胞中成功表达hMRGPRX4的人源化小鼠。用人源MRGPRX2替换小鼠Mrgprb2得到转基因小鼠hMRGPRX2-Cre，其中hMRGPRX2的表达是依赖于小鼠内源Mrgprb2的启动子。
本发明通过将人源MRGPRX2高表达在小鼠肥大细胞中，构建了一种人源化小鼠，为研究MRGPRX2基因的功能，筛选治疗肥大细胞上hMRGPRX2介导的药源性假性过敏以及神经感觉炎性反应相关疾病的药物提供了动物模型。
本发明采用如下技术方案：
本发明提供一种特异性表达hMRGPRX2的转基因小鼠的构建方法，所述方法包括：构建CRISPR-Cas9系统介导的小鼠Mrgprb2基因敲除且定点整合外源基因①的转基因小鼠品系hMRGPRX2-Cre；其中，所述外源基因①是hMRGPRX2基因编码序列(3’端带有3×HA标签)和重组酶基因(3’端带有WPRE-pA元件)之间通过P2A自切割肽段序列(5′-GCTACTAACTTCAGCCTGCTGAAGCAGGCTGGAGACGTGGAGGAGAACCC TGGACCT-3′)连接而成，且所述CRISPR-Cas9系统靶向Mrgprb2阳性的小鼠细胞。
前述的方法，所述重组酶为Cre重组酶。
进一步地，所述方法包括：根据小鼠Mrgprb2基因序列，构建基于CRISPR-Cas9系统的sgRNA和Cas9表达载体，分别体外转录得到sgRNA和Cas9 mRNA，并根据sgRNA作用位点构建含有外源基因①的且可以整合至宿主基因组中的供体质粒，然后将体外转录得到的Cas9mRNA、sgRNA和供体质粒共同转入野生型大鼠的受精卵中，然后将克隆胚胎通过非手术法移入小鼠子宫内进行妊娠，获得转基因小鼠。
所述小鼠为C57BL/6J小鼠。
本发明还提供按照上述方法构建的转基因小鼠的以下任一应用：
(1)用作构建治疗或缓解人药源性假性过敏以及神经感觉炎性反应相关疾病的动物模型；
(2)用于筛选治疗或缓解人药源性假性过敏以及神经感觉炎性反应相关疾病的药物；
(3)用于检测引起人药源性假性过敏瘙痒症的物质；
(4)用于筛选hMRGPRX2受体的激动剂或抑制剂；
(5)用于hMRGPRX2基因功能的研究。
本发明利用CRISPR-Cas9技术构建的小鼠转基因品系，命名为hMRGPRX2-Cre，本发明用包含hMRGPRX4和Cre重组酶的基因序列取代小鼠的Mrgprb2基因，其中hMRGPRX4和Cre重组酶之间用P2A自切割肽段序列连接，使其在翻译的过程中断裂成两个功能完整的蛋白。此外，本发明在hMRGPRX2基因的3’端插入3×HA标签序列，可以在后期通过抗体染色确定受体的表达。在该品系中，hMRGPRX2将利用Mrgprb2的启动子进行转录和翻译表达在Mrgprb2阳性的小鼠细胞中；由于Cre重组酶的表达，此品系与报告品系交配，可以将多种工具基因表达在Mrgprb2阳性的小鼠细胞中，用于Mrgprb2阳性的小鼠细胞的标记和操作。
本发明中涉及的术语：
肥大细胞：来源于骨髓的多功能祖细胞，由骨髓刚进入外周血液循环系统的肥大细胞仍处于未成熟状态，只有当它们的前体细胞迁移到最终定居的地方才能完成分化和/或成熟。作为一种重要的免疫细胞，它在人和动物体内分布广泛，其胞浆颗粒内含有多种生物活性物质，如生物胺、肿瘤坏死因子、白三烯、前列腺素、血小板活化因子等，肥大细胞通过释放这些生物活性物质发挥其功能。
MRGPRX2：mas相关G蛋白偶联受体成员X2(MRGPRX2)是灵长类特有的G蛋白偶联受体。MRGPRX2主要分布在肥大细胞(mast cell)中，它可以感知包括聚阳离子化合物和多肽等多种分泌素(secretagogues)引发肥大细胞脱颗粒反应，进而引起假过敏反应(pseudoallergic reactions)。虽然瘙痒可以引发抓挠行为，对机体起到防御保护作用，但受体过度激活会导致过敏、炎症等多种疾病的发生。
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。
以下实施例中使用的C57BL/6J小鼠按照常规饲养方法进行。载体pCS、TV-4G购自百奥赛图(北京)医药科技股份有限公司。
实施例1hMRGPRX2-Cre小鼠的构建
在小鼠Mrgprb2基因中，用hMRGPRX2-3xHA-P2A-iCre-WPRE-pA(SEQ ID NO:4)替换Mrgprb2基因外显子2(exon2)上第1位氨基酸到终止子对应的核苷酸序列。sgRNA设计在小鼠Mrgprb2基因exon2替换区。基于sgRNA的设计原则，在靶位点区域设计多条候选sgRNA，根据sgRNA活性检测结果并结合sgRNA的位置和特异性，最终选择2个合适sgRNA用于受精卵注射。将sgRNA(5′-AGGTGCTTAGATTCTTGATTAGG-3′和5′-TTAGGCACCGAAGGTTCAGGCGG-3′，综合sgRNA特异性、活性及sgRNA切割位点与重组位点的距离，最终优选sgRNA1和sgRNA10进行打靶)和Cas9基因连入带T7启动子的pCS-4G质粒载体(百奥赛图(北京)医药科技股份有限公司)上并进行体外转录，得到进行显微注射的gRNA和Cas9mRNA。
根据靶基因Mrgprb2序列，分别设计并合成5’、3’同源臂片段(SEQ ID NO:5和6)，然后将5’同源臂片段、目的基因(hMRGPRX4-3×HA-P2A-iCre-WPRE-pA)片段和3’同源臂片段依次连接到TV-4G载体上，即为供体质粒。
将Cas9 mRNA、sgRNA和供体质粒按一定比例混合，显微注射到小鼠受精卵中，注射后至出生得到F0小鼠。由于胚胎早期卵裂速度很快，因此得到的F0大鼠为嵌合体。故以F0小鼠鼠尾进行鉴定得到的F0基因型仅供参考，不能代表其一定为可遗传的基因突变型，可遗传的基因型需待F1大鼠鼠尾检测后确定。
1、hMRGPRX2-Cre转基因小鼠的基因型鉴定及hMRGPRX2-Cre转基因小鼠皮肤组织的RT-PCR结果
1)采用一步裂解法提取小鼠组织(脚趾)的基因组：含有0.1mg/ml的蛋白酶K的一步裂解液。
2)将200ul上述裂解液加到组织中，56℃孵育10小时；然后80℃孵育10分钟。
3)按照正常PCR程序扩增目的片段用于鉴定小鼠基因型。
4)用于鉴定hMRGPRX4-Cre等位基因的引物为(5′-3′)：
A-WT-F：TACCAAGTGCATTTAAATTAATGCTCC
A-WT-R：AGGGATGTTGATGTCAATTGAGTAGAAG
利用上述引物，扩增得到的WT等位基因片段大小为533bp。
A-WT-F：TACCAAGTGCATTTAAATTAATGCTCC
A-Mut-R：ATGAAAAGGATCAGGAAGACCGGGA
利用上述引物，扩增得到的hMRGPRX4-Cre等位基因片段大小为325bp。
5)应用RNA提取试剂盒对hMRGPRX2-Cre转基因小鼠皮肤进行RNA提取，然后应用反转录试剂盒，将其反转录成cDNA.
P-hMRGPRX2-1：GGGAACAGAAAGTACAACAG
P-hMRGPRX2-2：CTACACCAGACTGCTTCTCG
利用上述引物，扩增的hMRGPRX4cDNA的目的片段大小为970bp。hMRGPRX2-Cre转基因小鼠的基因型鉴定结果见图1。如图所示，利用A-WT-F/A-WT-R引物，野生型小鼠等位基因的条带大小为533bp；利用A-WT-F/A-Mut-R引物，对应的hMRGPRX2-Cre等位基因条带大小为325bp；可见编号为1E9X191-0103的小鼠为杂合子。如图所示，利用P-hMRGPRX2-1/P-hMRGPRX2-2引物扩增hMRGPRX2cDNA的目的条带大小为970bp。
2、hMRGPRX2-Cre转基因小鼠的细胞水平验证
1)利用钙成像溶液将激动剂C48/80配制成适当浓度的溶液；
2)参照文献(The Use of Human and Mouse Mast Cell and Basophil Culturesto Assess Type 2Inflammation)中的方法提取小鼠腹壁肥大细胞；
3)用钙成像溶液稀释Calcium 6到指定浓度；将稀释好的Calcium 6加入到细胞中，50ul/孔，37℃培养箱静置2h；
4)用高通量实时荧光检测分析系统记录细胞内钙信号的变化。
hMRGPRX2-Cre转基因小鼠的细胞水平验证结果见图2。
实施例2hMRGPRX2-Cre转基因小鼠的伊文思蓝外渗实验
具体方法如下：
1)将激动剂C48/80配制成适当浓度的溶液；
2)小鼠尾静脉注射100ul 0.625％的伊文思蓝溶液；
3)伊文思蓝注射完成15min左右后小鼠左侧后足底皮内注射10ul的10mg/ml C48/80，右侧后足注射等量生理盐水作为对照；
4)15分钟后观察足底伊文思蓝渗漏情况；
5)安乐死动物，收集一定量的足底皮下组织，从中提取伊文思蓝，用分光光度仪在620nm波长下测试组织中伊文思蓝含量。
图3结果显示，激动剂C48/80可以引起hMRGPRX2-Cre转基因小鼠的肥大细胞脱颗粒。
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。
参考文献:
[1]McNeil,B.,Pundir,P.,Meeker,S.etal.Identification of a mast-cell-specific receptor crucial for pseudo-allergic drug reactions.Nature 519,237–241(2015).https://doi.org/10.1038/nature14022.
[2]Michele A Grimbaldeston.Mast cell-MrgprB2:sensing secretagogues ora means to overreact？Immunology and Cell Biology 93,221–223(2015)；doi:10.1038/icb.2015.10.
[3]Dustin P.Green,Nathachit Limjunyawong,Naina Gour,Priyanka Pundir,Xinzhong Dong.A Mast-Cell-Specific Receptor Mediates Neurogenic Inflammationand Pain.Neuron 101,412–420.DOI:https://doi.org/10.1016/j.neuron.2019.01.012.
[4]Wichayapha Manorak,Chizobam Idahosa,KshitijGupta.Etal.Upregulation of Mas-related G Protein coupled receptor X2 inasthmatic lung mast cells and its activation by the novel neuropeptidehemokinin-1.Respiratory Research 19:1(2018).DOI 10.1186/s12931-017-0698-3.
[5]Can Cao,Hye Jin Kang,Isha Singh,etal.STRUCTURE,FUNCTION ANDPHARMACOLOGY OF HUMAN ITCH GPCRs.Nature.600(7887):170–175(2021).doi:10.1038/s41586-021-04126-6.
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[7]Radu,M.,Chernoff,J.An in vivo Assay to Test Blood VesselPermeability.J.Vis.Exp.(73),e50062,doi:10.3791/50062(2013).
Claims (3)
1.特异性表达hMRGPRX2的转基因小鼠的构建方法，其特征在于，所述方法包括：构建CRISPR-Cas9系统介导的小鼠Mrgprb2基因敲除且定点整合外源基因①的转基因小鼠；
所述外源基因①是由hMRGPRX2基因和重组酶Cre基因之间通过P2A序列连接而成，且所述hMRGPRX2基因的3’端修饰有3×HA标签序列；其中，所述hMRGPRX2基因编码的蛋白质的氨基酸序列如SEQ ID NO:1所示；所述外源基因①的核苷酸序列如SEQ ID NO:4所示；
根据小鼠Mrgprb2基因序列，构建基于CRISPR-Cas9系统的sgRNA和Cas9表达载体，分别体外转录得到sgRNA和Cas9 mRNA，并根据sgRNA作用位点构建含有外源基因①且可以整合至宿主基因组中的供体质粒，然后将体外转录得到的sgRNA和Cas9 mRNA以及供体质粒共同转入野生型小鼠的受精卵中，然后将克隆胚胎通过非手术法移入小鼠子宫内进行妊娠，获得转基因小鼠；
所述CRISPR-Cas9系统靶向Mrgprb2阳性的小鼠细胞；
所述sgRNA靶向小鼠Mrgprb2基因外显子2，sgRNA作用位点的核苷酸序列为5′-AGGTGCTTAGATTCTTGATTAGG-3′和5′-TTAGGCACCGAAGGTTCAGGCGG-3′；
所述供体质粒含有顺次连接的小鼠Mrgprb2基因5’同源臂片段、外源基因①和小鼠Mrgprb2基因3’同源臂片段；
所述小鼠Mrgprb2基因5’、3’同源臂片段的核苷酸序列分别如SEQ ID NO:5和6所示。
2.根据权利要求1所述的方法，其特征在于，所述小鼠为C57BL/6J。
3.按照权利要求1或2所述方法构建的转基因小鼠的以下任一应用：
(1)用作构建治疗或缓解人药源性假性过敏以及神经感觉炎性反应相关疾病的动物模型；
(2)用于筛选治疗或缓解人药源性假性过敏以及神经感觉炎性反应相关疾病的药物；
(3)用于检测引起人药源性假性过敏瘙痒症的物质；
(4)用于筛选hMRGPRX2受体的激动剂或抑制剂；
(5)用于hMRGPRX2基因功能的研究。
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