CN113528571B - hACE2人源化转基因猪的构建方法及应用 - Google Patents
hACE2人源化转基因猪的构建方法及应用 Download PDFInfo
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Abstract
本发明公开了hACE2人源化转基因猪的构建方法及应用。采用CRISPR‑Cas9基因编辑技术,用hACE2cDNA替换猪自身的ACE2基因,避免了人源ACE2表达的同时猪内源ACE2也表达,由此造成猪模型无法很好地再现人类疾病特征的问题。本发明构建的hACE2人源化猪模型是能够模拟人类COVID‑19临床症状和治疗反应的理想大动物猪模型,可用于SARS‑CoV‑2感染和发病机制的研究,特别适合于由SARS‑CoV‑2感染所致的多器官病症、重症、慢性疾病以及后遗症的研究,并可供诊断技术、抗病毒疗法和疫苗的开发与测试,候选药物疗效和安全性的评价,该模型有助于解决当前缺乏有效新冠肺炎动物模型的困境。
Description
技术领域
本发明属于基因工程技术领域,具体地说,涉及一种hACE2人源化转基因猪的构建方法及应用。
背景技术
新型冠状病毒(又称严重急性呼吸系统综合征冠状病毒2,SARS-CoV-2)是2019年以来新发现的一种病毒,可导致人类病毒性肺炎/肺部感染。研究SARS-CoV-2感染和发病的机制,开发和测试针对该病毒的诊断技术、抗病毒疗法和疫苗,模拟其临床症状和治疗反应,评价候选药物的疗效和安全性,确定与发病、严重程度和病程相关的生物标志物,都需要大量与人类临床发病机制类似的动物模型。
新冠肺炎患者表现出从无症状到严重肺炎甚至死亡的广泛疾病症状(Wu andMcGoogan,2020),仅依靠单个动物模型不能模拟和反映人类临床所表现出的不同疾病症状,可能具有误导性。对疫苗和抗病毒药物有效性的研究传统上是使用严重疾病模型,可能无法模拟大多数患者的常见病理,并可能限制对感染动力学和传播等的理解。
能够重现COVID-19临床和病理特征的动物模型至关重要,目前已证实多种动物能够感染SARS-CoV-2并被用于该疾病的研究和候选治疗策略的测试,但感染后只表现为轻微症状或无疾病感染。研究表明,仓鼠适合用于研究人类的轻度SARS-CoV-2感染和宿主对病毒的防御反应,但其显著缺陷是感染SARS-CoV-2后第14天,肺部病理恢复正常(Chan etal.,2020;Sia et al.,2020)。猫适合作为无症状至中度SARS-CoV-2感染的动物模型,据Shi等人的报道,病毒虽然可以在猫的上呼吸道有效复制,但是没有出现体重减轻或呼吸窘迫等严重的临床症状(et al.,2020;Shen et al.,2020;Shi et al.,2020)。雪貂被认为是最接近人类的SARS-CoV-2感染和传播模型,包括人工养殖的雪貂在内,均能观察到对病毒十分易感,但症状轻微,且检测到的肺部病毒滴度较低,仅能模拟中度疾病(Hobbs and Reid,2020;Kim et al.,2020;Richard et al.,2020)。非人灵长类动物适合作为研究SARS-CoV-2复制的模型,食蟹猴通过鼻内和气管内联合给药感染SARS-CoV-2后,病毒可在上呼吸道和下呼吸道传播,但临床症状较轻,仅能模拟轻度COVID-19临床症状(Gao et al.,2020;Lu et al.,2020;Rockx et al.,2020;Woolsey et al.,2020)。同样,接种病毒后,恒河猴出现了体重减轻和肺炎,但无发热等临床症状(Munster et al.,2020;Shan et al.,2020)。此外,非人灵长类动物模型的繁殖速度较慢、饲喂养殖成本高、实验操作困难和伦理原因等问题也进一步限制了它们的广泛应用(Takayama,2020;Abdel-Moneim and Abdelwhab,2020;Pandey et al.,2020)。
此外,由于大多数COVID-19重症病例的特点是具有潜在的慢性疾病,因此需要包含这些已存在疾病的动物模型来重现COVID-19重症病例。除了导致因急性呼吸窘迫综合征(ARDS)导致的急性肺损伤(ALI)之外,部分COVID-19患者还可能出现器官损害,包括急性肾、心、肝功能障碍(Chen et al.,2020;Wang et al.,2020;Yang et al.,2020)。高血压、心血管疾病和糖尿病等共患病(Wang et al.,2020)。为了全面了解和治疗COVID-19疾病,需要开发一种适合的对SARS-CoV-2病毒易感并伴有人类常见共患病的动物模型。
综上,由于SARS-CoV-2感染和致病机制研究、治疗策略和疫苗开发的急迫性,模拟人类COVID-19的大动物模型需求量巨大,因此亟需开发一种能够准确再现COVID-19的动物模型。
发明内容
本发明的目的是提供一种hACE2人源化转基因猪的构建方法及应用。
本发明构思如下:猪模型与人类医学密切相关,在器官大小、生理结构及营养代谢等各方面接近于人类,可应用于人类疾病模型制备、人类异种器官移植等领域。
猪模型会因饮食引起的肥胖而发展出代谢综合征和心肺疾病。在患有代谢综合征的猪(如肥胖的Ossabaw小型猪)鼻内接种PRCV(猪呼吸道冠状病毒),会出现急性呼吸窘迫综合征(ARDS)、多器官衰竭、弥漫性肺泡损伤以及血液炎症反应和肺纤维化等症状,类似于患有严重COVID-19的代谢综合征患者(Heegaard et al.,2020)。因此猪模型可以加速COVID-19高危人群基础疾病机制、生物标志物和治疗方法的研究。研究表明,猪源ACE2可以结合SARS-CoV-2病毒表面的spike蛋白,然而通过鼻腔给猪接种大剂量的SARS-CoV-2后,在鼻腔或直肠拭子中并未检测到病毒,表明病毒在猪体内没有发生复制,猪对SARS-CoV-2不易感(Schlottau et al.,2020;Shi et al.,2020)。发明人利用基因编辑技术,通过构建靶向猪ACE2基因的打靶载体,以及携带hACE2基因的同源修复载体,然后将两种载体共同导入猪胎儿成纤维细胞系中,将获得的转基因细胞系作为核移植供体细胞用于代孕母猪,待其生产后,即为hACE2人源化转基因猪。该猪模型能够模拟人类COVID-19临床症状和治疗反应的大动物猪模型,可用于SARS-CoV-2感染和发病机制的研究,并可供诊断技术、抗病毒疗法和疫苗的开发与测试,候选药物疗效和安全性的评价。
为了实现本发明目的,第一方面,本发明提供一种靶向编辑猪血管紧张素转化酶ACE2基因的CRISPR-Cas9系统,包括猪血管紧张素转化酶ACE2基因打靶载体和携带人血管紧张素转化酶ACE2基因的同源修复载体。
所述打靶载体是基于CRISPR-Cas9技术设计的靶向猪血管紧张素转化酶ACE2基因的打靶载体,其包含靶向猪血管紧张素转化酶ACE2基因的sgRNA序列以及编码Cas9核酸酶的核酸序列。
所述同源修复载体包含如下Donor DNA:人血管紧张素转化酶ACE2基因的上游同源臂-人血管紧张素转化酶ACE2基因的cDNA序列-polyA序列-人血管紧张素转化酶ACE2基因的下游同源臂。
本发明中,猪血管紧张素转化酶ACE2基因在NCBI上的参考序列编号为100144303。
人血管紧张素转化酶ACE2基因在NCBI上的参考序列编号为59272。
Cas9核酸酶在NCBI上的参考序列编号为57852564。
优选地,所述打靶载体中sgRNA作用位点位于猪血管紧张素转化酶ACE2基因的1号外显子起始密码子ATG附近。
更优选地,sgRNA作用位点的DNA序列为5′-CCACTGAGGAACTGGCCAAGACA-3′。
优选地,人血管紧张素转化酶ACE2基因的上游同源臂的序列如SEQ ID NO:1所示,人血管紧张素转化酶ACE2基因的下游同源臂的序列如SEQ ID NO:2所示。
更优选地,Donor DNA的序列如SEQ ID NO:3所示。
打靶载体的出发载体可以是pX459。
同源修复载体的出发载体可以是pUC19或pUC57。
第二方面,本发明提供所述CRISPR-Cas9系统在制备hACE2人源化转基因细胞系中的应用。
第三方面,本发明提供一种hACE2人源化转基因细胞系,将所述CRISPR-Cas9系统导入猪胎儿成纤维细胞系中(导入方法优选电穿孔),获得的中靶阳性细胞克隆,即为hACE2人源化转基因细胞系。
第四方面,本发明提供一种猪克隆胚胎的制备方法,以所述hACE2人源化转基因细胞系作为核移植供体细胞,离体的猪卵母细胞为核移植受体细胞,通过核移植技术获得猪克隆胚胎。
第五方面,本发明提供一种hACE2人源化转基因猪的构建方法,将按照上述方法制备的猪克隆胚胎通过非手术法移入母猪子宫内进行妊娠,获得hACE2人源化转基因猪。
本发明中,猪的品种可以是巴马香猪,也可以是大白/长白等大型猪品种。
第六方面,本发明提供按照上述方法制备得到的hACE2人源化转基因猪在新型冠状病毒SARS-CoV-2感染和致病机理研究、药物和疫苗研发中的应用。
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hACE2人源化转基因猪的构建方法是采用CRISPR-Cas9基因编辑技术,用人血管紧张素转化酶ACE2基因的cDNA序列替换猪自身的血管紧张素转换酶ACE2基因的表达,从而避免了人源ACE2表达的同时猪内源ACE2也同样表达,由此可能造成猪模型无法很好地再现人类疾病特征的问题。人源ACE2 cDNA受到猪ACE2基因启动子的调控,使其可以在猪的特定组织器官中表达,且hACE2的表达水平接近于猪内源ACE2的表达水平。
(二)本发明利用CRISPR/Cas9基因编辑技术实现猪中hACE2的敲入,相比于传统的同源重组介导的基因敲入,CRISPR/Cas9技术具有sgRNA设计简单,基因敲入效率高等优势。
(三)为了保证同源重组介导的外源人ACE2基因片段的插入效率,选择效率最高且安全的sgRNA进行基因编辑操作。
(四)本发明构建的hACE2人源化猪模型是能够模拟人类COVID-19临床症状和治疗反应的理想大动物猪模型,可用于SARS-CoV-2感染和发病机制的研究,特别适合于由SARS-CoV-2感染所致的潜在慢性疾病的研究,并可供诊断技术、抗病毒疗法和疫苗的开发与测试,候选药物疗效和安全性的评价,该模型有助于解决当前缺乏有效的新冠肺炎动物模型的困境。
(五)本发明构建的hACE2人源化猪模型也适用于SARS等同样通过人类细胞表面受体蛋白ACE2介导入侵细胞的病毒,该动物模型可以用于致病机理研究或作为药筛模型。
(六)本发明提供的ACE2人源化猪模型,是利用体细胞核移植的方法制备的,可在短时间内获得同一细胞来源且遗传背景相同的个体,保证了猪内源ACE2的完全替换,克服了在小鼠中常见的使用受精卵原核注射方法易产生嵌合体的缺点。
附图说明
图1为本发明较佳实施例中为猪ACE2基因位点打靶并插入人源ACE2的策略。
图2为本发明较佳实施例中人源ACE2插入阳性的猪胎儿成纤维单克隆细胞系的鉴定情况。PCR引物为人源ACE2定点插入的跨同源臂检测引物(Right Arm,RA)及人源ACE2定点插入的跨同源臂检测引物(Left Arm,LA)。IBRS+为同源修复模板即阳性对照;BM-wt为野生型巴马猪基因组;neg为水即阴性对照;红色标记为人源ACE2插入阳性的猪胎儿成纤维单克隆细胞系编号。
图3为本发明较佳实施例中出生后1日龄及3日龄的ACE2人源化仔猪。
图4为本发明较佳实施例中3头ACE2人源化仔猪各组织DNA水平hACE2插入PCR检测。PCR引物为人源ACE2定点插入的跨同源臂检测引物(hACE2 knockin RA F2+R2)及针对人源ACE2的特异性引物(hACE2 F1+R2)。IBRS2 donor为同源修复模板即阳性对照;BM-wt为野生型巴马猪基因组;水为阴性对照;BM3-25为用于核移植的人源ACE2插入阳性的猪胎儿成纤维单克隆细胞系编号。
图5为本发明较佳实施例中ACE2人源化仔猪各组织转录水平hACE2表达的定量PCR检测。提取hACE2敲入猪及同窝野生型对照猪不同组织的总RNA并逆转录,以GAPDH为内参基因。数据以平均数±标准差表示,**p≤0.01,***p≤0.001,n=3。KI,敲入。
图6为本发明较佳实施例中ACE2人源化仔猪各组织hACE2蛋白表达水平的WesternBlot检测。提取hACE2敲入猪及同窝野生型对照猪不同组织的总蛋白,以GAPDH为内参基因。kDa,千道尔顿;WT,同窝野生型对照猪。
图7为本发明较佳实施例中利用免疫荧光分析人源ACE2蛋白(绿色)在hACE2敲入猪的肺、肾、小肠和脑中的定位。细胞核经DAPI染色(蓝色)。随机选择有代表性的图像。比例尺为50μm。KI,敲入;WT,同窝野生型对照猪。
图8为本发明较佳实施例中SARS-CoV-2感染后hACE2敲入猪及同窝野生型对照猪肺、肾上皮细胞的细胞病变效应。比例尺为50μm。KI,敲入;WT,同窝野生型对照猪。
图9为本发明较佳实施例中SARS-CoV-2感染后,hACE2敲入猪及同窝野生型对照猪肺、肾上皮细胞中病毒核蛋白表达水平的Western Blot检测。以GAPDH为内参基因。kDa,千道尔顿;KI,敲入;WT,同窝野生型对照猪;L,肺上皮细胞;K,肾上皮细胞。
图10为本发明较佳实施例中SARS-CoV-2感染后,hACE2敲入猪及同窝野生型对照猪肺、肾上皮细胞中病毒核蛋白表达的免疫荧光分析。病毒核蛋白经染色为红色,细胞核经DAPI染色(蓝色)。随机选择有代表性的图像。比例尺为25μm。KI,敲入;WT,同窝野生型对照猪。
具体实施方式
本发明提供的一种ACE2人源化的COVID-19敏感猪模型,即hACE2人源化转基因猪。本发明采用如下技术方案:
(1)利用CRISPR/Cas9系统介导外源基因靶向敲入,实现了猪ACE2基因位点的人源ACE2插入;
(2)分别在转录水平和蛋白水平对敲入猪模型中人源ACE2的表达情况进行检测;
(3)确定SARS-CoV-2在ACE2人源化猪模型中的感染和复制效率。
步骤(1)中所述外源hACE2基因靶向敲入通过以下方法实现:
①设计靶向猪ACE2基因第一外显子起始密码子ATG附近的sgRNAs并优化效率;
②利用Cas9核酸酶针对sgRNA靶位点进行切割,引入含有人源ACE2的cDNA及polyA终止子的同源定向修复载体,从而实现人源ACE2序列替换猪ACE2基因的目的;
③制备人源ACE2插入阳性的猪胎儿成纤维单克隆细胞系;
④制备人源ACE2插入阳性的敲入猪模型。
步骤①所述sgRNA的效率优化通过以下方法实现:合成并组装多个sgRNAs,利用sgRNAs和Cas9核酸酶表达质粒电转IBRS2细胞(猪肾细胞)。利用Sanger测序鉴定靶位点处序列碱基的插入或删除情况,并评估这些sgRNAs的靶向效率。选择效率最高的sgRNA进行后续实验。
步骤②所述同源定向修复载体包含人源ACE2的cDNA序列及polyA终止子序列,并取ATG上游长度约1kb左右的序列作为其同源重组左臂,取第一外显子末端下游长度约1kb左右的序列作为其同源重组右臂。本发明提供的ACE2人源化猪模型中,人源ACE2cDNA受到猪ACE2基因启动子的调控,使其可以在猪的特定组织器官中表达。
步骤③利用步骤①和②所述CRISPR/Cas9打靶插入载体系统对从巴马小型猪(巴马香猪)胚胎中分离获得的猪胎儿成纤维细胞进行电穿孔处理,获得单克隆细胞系后,利用针对外源插入hACE2序列及跨同源臂的PCR和Sanger测序鉴定其基因型。
步骤④将步骤③所述检测人源ACE2插入阳性的单克隆细胞系作为体细胞核移植的供体细胞用于代孕母猪,待其生产后,从仔猪的各组织中提取基因组DNA,通过针对外源插入hACE2序列及跨同源臂的PCR对人源ACE2的插入情况进行鉴定。
步骤(2)中所述敲入猪模型中人源ACE2的表达情况通过以下方法检测:
⑤利用定量PCR方法检测了敲入猪模型各组织中人源ACE2的转录水平;
⑥利用Western Blot检测敲入猪不同器官中的人源ACE2蛋白的表达情况;
⑦利用免疫荧光分析检测敲入猪不同器官中的人源ACE2蛋白的表达情况。
步骤⑤所述定量PCR检测方法以GAPDH为内参基因,检测材料为敲入猪的肝、肺、肾、小肠及大脑等组织的cDNA,定量PCR引物为针对人源ACE2 cDNA的特异性检测引物。
步骤⑤中所述针对人源ACE2 cDNA的特异性定量PCR引物通过以下方法筛选:针对人源ACE2基因序列跨内含子设计多对引物,分别以人cDNA和猪cDNA为PCR底物,选择可在人cDNA中扩增出特异性片段而在猪cDNA中无扩增片段的引物作为特异性检测引物。
步骤⑥所述Western Blot检测方法以GAPDH为内参基因,检测材料为敲入猪的肺、肾、小肠及大脑等组织的总蛋白,检测抗体为针对人源ACE2的特异性抗体。
步骤⑦所述免疫荧光分析检测方法的材料为敲入猪的肺、肾、小肠及大脑等组织切片,检测抗体为针对人源ACE2的特异性抗体。
步骤(3)所述SARS-CoV-2在ACE2人源化猪模型中的感染和复制效率通过以下方法确定:
⑧分离并培养ACE2人源化猪模型及同窝野生型对照猪的原代肺、肾上皮细胞;
⑨观察SARS-CoV-2感染后,原代上皮细胞的细胞病变效应;
⑩通过Western Blot及免疫荧光分析检测SARS-CoV-2感染后原代上皮细胞中病毒核蛋白的表达情况。
步骤⑧所述原代上皮细胞均从出生后一天的ACE2人源化猪模型及同窝野生型对照仔猪肺、肾组织中分离并培养。
步骤⑨所述SARS-CoV-2感染前应将原代上皮细胞汇合度调整为一致。
步骤⑨所述SARS-CoV-2感染剂量(MOI)与感染时间需根据所使用的毒株进行灵活调整。
步骤⑩所述Western Blot检测方法以GAPDH为内参基因,检测材料为ACE2人源化猪模型及同窝野生型对照猪原代肺、肾上皮细胞经SARS-CoV-2感染后的总蛋白,检测抗体为针对SARS-CoV-2病毒核蛋白的特异性抗体。
步骤⑩所述免疫荧光分析检测方法的材料为SARS-CoV-2感染后的ACE2人源化猪模型及同窝野生型对照猪的原代肺、肾上皮细胞,检测抗体为针对SARS-CoV-2病毒核蛋白的特异性抗体。
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1人源化ACE2猪模型的构建方法
本发明中人源化ACE2猪模型制备的技术流程主要包括以下三个方面:
第一,猪ACE2打靶及人源ACE2插入策略(图1)
在猪ACE2基因的第一外显子ATG附近设计sgRNA,利用Cas9核酸酶针对靶点进行切割,然后引入含有hACE2 cDNA-polyA的同源修复载体,取hACE2基因起始密码子ATG上游1kb左右大小片段作为其同源重组左臂(left arm,LA,SEQ ID NO:1),取第一外显子末端下游1kb左右大小片段作为其同源重组的右臂(Right arm,RA,SEQ ID NO:2)。从而实现hACE2cDNA替换猪ACE2基因的目的,此人源ACE2 cDNA受到猪ACE2启动子的控制,可以在特定组织器官中的表达。
具体地,CRISPR/Cas9打靶插入载体系统包括猪内源ACE2基因打靶载体和携带hACE2基因的同源修复载体;
所述打靶载体是基于CRISPR-Cas9技术设计的靶向猪内源ACE2基因的打靶载体,其包含靶向猪内源ACE2基因的sgRNA序列以及编码Cas9核酸酶的核酸序列。所述打靶载体的出发载体为pX459(购自Addgene)。
所述同源修复载体(同源定向修复载体)包含如下Donor DNA:hACE2上游同源臂(SEQ ID NO:1)-hACE2 cDNA序列-polyA序列-hACE2下游同源臂(SEQ ID NO:2)。所述同源修复载体的出发载体为pUC57(购自Addgene)。
Donor DNA的序列如SEQ ID NO:3所示。
第二,人源ACE2插入阳性的猪胎儿成纤维单克隆细胞系制备及检测。将CRISPR/Cas9打靶插入载体系统(打靶载体与同源修复载体的摩尔比为1:1)对从巴马小型猪胚胎中分离获得的猪胎儿成纤维细胞进行电穿孔处理,获得单克隆细胞系后,利用跨同源臂的引物进行PCR鉴定其基因型。
具体步骤如下:
1.猪胎儿成纤维细胞转染,具体操作为:
质粒:sgRNA及Cas9核酸酶表达载体pX459,带有嘌呤霉素抗药筛选标记;同源定向修复载体,包含同源臂与hACE2 cDNA及polyA,经限制性内切酶SpeI和NotI(New EnglandBiolabs)酶切线性化后可用于转染。
细胞:巴马小型猪胎儿成纤维细胞,细胞量为1×106/反应。
电转化程序:2B-Nucleofector Device(Lonza)程序A-024。
药物筛选:嘌呤霉素(1μg/mL)筛选2天。
2.单克隆细胞系中hACE2插入DNA水平检测(图2)
获得单克隆细胞系后,提取基因组进行PCR鉴定,引物为人源ACE2定点插入的跨同源臂检测引物(Right Arm,RA)及人源ACE2定点插入的跨同源臂检测引物(Left Arm,LA)。经统计,单克隆细胞系中人源ACE2的敲入效率约为3.53%(3/85)。
第三,人源ACE2插入阳性猪模型的制备及检测(图3)
将检测人源ACE2插入阳性的单克隆细胞系作为体细胞核移植的供体细胞用于代孕母猪,待其生产后,对仔猪的各组织中hACE2的插入和表达情况分别从DNA水平、转录水平及蛋白水平进行鉴定。
具体方法如下:
1.将检测人源ACE2插入阳性的单克隆细胞系(BM3-25)作为体细胞核移植的供体细胞用于3头代孕母猪,共获得10头仔猪,从仔猪的各组织中提取基因组DNA,PCR鉴定引物为人源ACE2定点插入的跨同源臂检测引物(hACE2 knockin RA F2+R2)及针对人源ACE2的特异性引物(hACE2 F1+R2)。经检测,仔猪各组织中均可检测到hACE2的插入,即鉴定为人源ACE2敲入阳性猪(图4)。
hACE2 knockin RA F2:5'-TCCAGGATTCCAAAACACTGATG-3'
hACE2 knockin RA R2:5'-ATCTCCTGGATTTCTCTGAGG-3'
hACE2 F1:5'-TGTCCAAAACATGAATAATGCTG-3'
hACE2 R2:5'-TGGTTTAATCTCTTCAAAGGTATGT-3'
2.利用定量PCR方法检测敲入猪模型各组织中人源ACE2的转录水平(图5)。使用RaPure总RNA试剂盒(Magen,R4011-03)提取hACE2敲入仔猪和同窝野生型对照猪各组织的总RNA,取2μg RNA逆转录成cDNA进行定量PCR,以GAPDH为内参基因,测定hACE2的相对mRNA水平。hACE2的特异性引物为hACE2-F3(5'-GTTTTGAATAGCGCCCAACC-3')和hACE2-R3(5'-TCTTGGCCTGTTCCTCAATG-3');GAPDH的特异性引物为GAPDH-F(5'-ACCCAGAAGACTGTGGATGGC-3')和GAPDH-R(5'-AGCCAGAGGCAAAGTGATAGATA-3')。结果表明,敲入猪的肾、肝、小肠、肺等组织中人源ACE2的mRNA水平显著高于同窝野生型对照猪。
3.利用Western Blot检测敲入猪不同器官中的人源ACE2蛋白的表达情况(图6)。用RIPA裂解缓冲液(Beyotime,Shanghai,China;P0013B)提取hACE2敲入仔猪和同窝野生型对照猪的各组织的总蛋白。取10μg蛋白在上样缓冲液中99℃变性10分钟,用浓度为6%的SDS/PAGE胶分离,再转移至聚氟乙烯膜上。抗ACE2一抗(Abcam;ab108252),工作浓度为1:1000稀释,抗GAPDH一抗(碧云天;AF1186),工作浓度为1:100稀释。
4.利用免疫荧光分析检测敲入猪不同器官中的人源ACE2蛋白的表达情况(图7)。将hACE2敲入仔猪和同窝野生型对照猪的各组织在4℃冰箱中用4%多聚甲醛固定过夜。在10%和30%的蔗糖溶液中连续脱水后,用OCT包埋并在-80℃冷冻。用冰冻切片机制备12μm组织切片。冷冻切片利用5%牛血清白蛋白封闭,抗ACE2一抗(Abcam;ab108252)的工作浓度为1:100稀释,使用含有DAPI的中性树胶染色并封片。图像均为激光共聚焦显微镜拍摄。Western Blot和免疫荧光分析结果显示,hACE2在敲入猪各器官中均高表达。
实施例2人源化ACE2猪模型对SARS-CoV-2易感性的检测
本发明中人源化ACE2猪模型对SARS-CoV-2易感性的检测主要包括以下三个方面:
第一,分离并培养ACE2人源化猪模型及同窝野生型对照猪的原代肺、肾上皮细胞。
具体操作为:收集hACE2敲入仔猪和同窝野生型对照猪的肾和肺组织,用PBS洗涤后用200U/mL I型胶原酶在37℃下消化15分钟。原代上皮细胞培养基为向DMEM基础培养基中添加15%胎牛血清、1%青霉素和链霉素和10ng/μL上皮生长因子,原代上皮细胞在37℃,5%CO2条件下培养。
第二,观察SARS-CoV-2感染后,原代上皮细胞的细胞病变效应(图8)。
具体操作为:攻毒实验均在生物安全三级实验室中进行,当ACE2人源化猪模型及同窝野生型对照猪原代肺、肾上皮细胞的汇合度达到80%时,接种毒株为SARS-CoV-2(hCoV-19/China/CAS-B001R/2020),感染倍数(MOI)为0.01,在接种72小时后,观察细胞病变效应情况,并收集细胞和培养基上清进行Western Blot和免疫荧光分析。结果表明,与同窝野生型对照组猪相比,hACE2敲入猪的原代上皮细胞感染SARS-CoV-2后表现出显著的细胞病变效应。
第三,通过Western Blot(图9)及免疫荧光分析(图10)检测SARS-CoV-2感染后原代上皮细胞中病毒核蛋白的表达情况。
具体方法为:
1.用RIPA裂解缓冲液(Beyotime,Shanghai,China;P0013B)提取经SARS-CoV-2感染后的hACE2敲入仔猪和同窝野生型对照猪原代肺、肾上皮细胞总蛋白。取10μg蛋白在上样缓冲液中99℃变性10分钟,用浓度为6%的SDS/PAGE胶分离,再转移至聚氟乙烯膜上。SARS-CoV-2(2019-nCoV)核衣壳一抗(义翘神州;40143-R019),工作浓度为1:1000稀释。
2.利用经SARS-CoV-2感染后hACE2敲入仔猪和同窝野生型对照猪的原代肺、肾上皮细胞制作爬片,SARS-CoV-2(2019-nCoV)核衣壳一抗(义翘神州;40143-R019),工作浓度为1:100稀释,利用DAPI进行细胞核染色后封片。图像均为激光共聚焦显微镜拍摄。WesternBlot和免疫荧光分析结果显示,hACE2基因敲入仔猪的原代上皮细胞对SARS-CoV-2的感染敏感。
实施例3 sgRNA的优化实验
本发明在猪ACE2基因位点第一外显子起始密码子ATG附近设计了9条sgRNA,并在IBRS2细胞中测试了这些sgRNA的基因打靶效率,各sgRNA序列及针对猪ACE2基因的打靶效率如表1所示:
表1
从表1可以看出,在IBRS2细胞中,针对猪ACE2位点的sgRNA3基因打靶效率最高,因此选择将其作为后续CRISPR/Cas9介导外源人ACE2基因插入系统中使用的靶位点。
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。
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序列表
<110> 北京复昇生物科技有限公司
<120> hACE2人源化转基因猪的构建方法及应用
<130> KHP211117240.9
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 997
<212> DNA
<213> 人(Homo sapiens)
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acaccctggg aatgaggaca ctggtccggc tcccgagagt caaaccactg acgtctcttt 120
gaattctacg ccatgatcca tggctctgga tgaccagaac ctgacaaggg cacgctctga 180
atttcaggcc atggggaggc attctgagtt ccttgtgacc ttggctgagt tctgagcagg 240
taagtgcaag ggatagacgc acacagtgaa atcagccaca gagccagggt ttaacacggg 300
catctgagaa atggaaaatg aaccttttta cgtttttcca ggttaagtca ggagagttaa 360
cttttgcagt ggaaggtagt cttataatta aaaaaaaaat ggccgtggca attaaaattc 420
atcaaaaaat gatctccata aggatgaggg caaagttgtt tattcaggaa gggaaaagat 480
tacccaagta gagagtttct ttgaatatga gtttgaaatg aaaaggaaag agggctaact 540
attaacccaa ctgtctgtga aacgtataag tctcaacctt acccccatga tccctaattc 600
tagagttcgt tgctcactgg aaaatcgcga caccttcagt gtatctttaa cagattttaa 660
ggaacatatt aaccaaatgt acaagttttg atttggccat aaagttcgag gaaagctatg 720
gttctctagg attaatgaat aacatttgtt tatttgattt actttaagaa atcattctaa 780
aatctgttta catatctgtc ctctccggga tgaattttat gttggttcag cagattgttt 840
actgttttat cttcttcttc cttttttttt ttttttttcc gtcttccctg ctcagtgccc 900
aacccaagtt caaaggctga tgagagagga aaactcatga ggaggtttta ctctagggaa 960
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<211> 1000
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gaaaatgaaa agggaaatca aagaaatgct ctgagcagtg agattggatg tctgcctccc 120
tatttctgat tctgagtccc agatggctaa acaattgacc tttgggttca tttgggaaat 180
tgttacaaaa taattcgctg gtctcagtcc agattcttgg aaaccagtga acaggcttta 240
gaaagttgcc accataattc tcatcatagt caggctttca aagcatttca tggaaatgcc 300
ataagttatt gtgttaataa tttccccaag cccacaatgt caacaggaat gtctaaagac 360
gctctacaaa tgatgtatta actgaaaaat gcaactggag ctttagcgaa agagctagct 420
agtaattaag atcgcatggg tcgggtgagg ttggccctgg gaagttcatt gtcatggaac 480
ttcaccgtca tggagctccg agggacttcc agggggtacg gagtccacag agccctccaa 540
ggaaaaccgg aattaaacca gtgctgacaa atctcagagt gctctgggaa agcaagatat 600
tttgtttgac aagtgcaagc tttcaggaaa tttcttcctt cgcagatccc aaagcagtgc 660
tggaaacgaa gcagcattat ttaagcaaag ttttttggca tttgttccag caatgcattc 720
cttcaaaaag tgtagttcag aagcttattt tttcaaatag gaggttgcat gtttaaagta 780
aattattgtt attcactgga cagatcaaat ttgagagctg ctgcctcatt tgagcacaat 840
taggagattt tttttttttt ctttttaggg ctgcacctgc ggcatatgga ggttcccagg 900
ctagaggtct aattggagct acagctgcca acctacacca cagctcatgg caatgccgga 960
tccttaaccc actgagcgag gccagggatc aaacctgaaa 1000
<210> 3
<211> 2697
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cctagggcca ccatgtcaag ctcttcctgg ctccttctca gccttgttgc tgtaactgct 60
gctcagtcca ccattgagga acaggccaag acatttttgg acaagtttaa ccacgaagcc 120
gaagacctgt tctatcaaag ttcacttgct tcttggaatt ataacaccaa tattactgaa 180
gagaatgtcc aaaacatgaa taatgctggg gacaaatggt ctgccttttt aaaggaacag 240
tccacacttg cccaaatgta tccactacaa gaaattcaga atctcacagt caagcttcag 300
ctgcaggctc ttcagcaaaa tgggtcttca gtgctctcag aagacaagag caaacggttg 360
aacacaattc taaatacaat gagcaccatc tacagtactg gaaaagtttg taacccagat 420
aatccacaag aatgcttatt acttgaacca ggtttgaatg aaataatggc aaacagttta 480
gactacaatg agaggctctg ggcttgggaa agctggagat ctgaggtcgg caagcagctg 540
aggccattat atgaagagta tgtggtcttg aaaaatgaga tggcaagagc aaatcattat 600
gaggactatg gggattattg gagaggagac tatgaagtaa atggggtaga tggctatgac 660
tacagccgcg gccagttgat tgaagatgtg gaacatacct ttgaagagat taaaccatta 720
tatgaacatc ttcatgccta tgtgagggca aagttgatga atgcctatcc ttcctatatc 780
agtccaattg gatgcctccc tgctcatttg cttggtgata tgtggggtag attttggaca 840
aatctgtact ctttgacagt tccctttgga cagaaaccaa acatagatgt tactgatgca 900
atggtggacc aggcctggga tgcacagaga atattcaagg aggccgagaa gttctttgta 960
tctgttggtc ttcctaatat gactcaagga ttctgggaaa attccatgct aacggaccca 1020
ggaaatgttc agaaagcagt ctgccatccc acagcttggg acctggggaa gggcgacttc 1080
aggatcctta tgtgcacaaa ggtgacaatg gacgacttcc tgacagctca tcatgagatg 1140
gggcatatcc agtatgatat ggcatatgct gcacaacctt ttctgctaag aaatggagct 1200
aatgaaggat tccatgaagc tgttggggaa atcatgtcac tttctgcagc cacacctaag 1260
catttaaaat ccattggtct tctgtcaccc gattttcaag aagacaatga aacagaaata 1320
aacttcctgc tcaaacaagc actcacgatt gttgggactc tgccatttac ttacatgtta 1380
gagaagtgga ggtggatggt ctttaaaggg gaaattccca aagaccagtg gatgaaaaag 1440
tggtgggaga tgaagcgaga gatagttggg gtggtggaac ctgtgcccca tgatgaaaca 1500
tactgtgacc ccgcatctct gttccatgtt tctaatgatt actcattcat tcgatattac 1560
acaaggaccc tttaccaatt ccagtttcaa gaagcacttt gtcaagcagc taaacatgaa 1620
ggccctctgc acaaatgtga catctcaaac tctacagaag ctggacagaa actgttcaat 1680
atgctgaggc ttggaaaatc agaaccctgg accctagcat tggaaaatgt tgtaggagca 1740
aagaacatga atgtaaggcc actgctcaac tactttgagc ccttatttac ctggctgaaa 1800
gaccagaaca agaattcttt tgtgggatgg agtaccgact ggagtccata tgcagaccaa 1860
agcatcaaag tgaggataag cctaaaatca gctcttggag ataaagcata tgaatggaac 1920
gacaatgaaa tgtacctgtt ccgatcatct gttgcatatg ctatgaggca gtacttttta 1980
aaagtaaaaa atcagatgat tctttttggg gaggaggatg tgcgagtggc taatttgaaa 2040
ccaagaatct cctttaattt ctttgtcact gcacctaaaa atgtgtctga tatcattcct 2100
agaactgaag ttgaaaaggc catcaggatg tcccggagcc gtatcaatga tgctttccgt 2160
ctgaatgaca acagcctaga gtttctgggg atacagccaa cacttggacc tcctaaccag 2220
ccccctgttt ccatatggct gattgttttt ggagttgtga tgggagtgat agtggttggc 2280
attgtcatcc tgatcttcac tgggatcaga gatcggaaga agaaaaataa agcaagaagt 2340
ggagaaaatc cttatgcctc catcgatatt agcaaaggag aaaataatcc aggattccaa 2400
aacactgatg atgttcagac ctccttttag accggtgaat tctaactaga gctcgctgat 2460
cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 2520
ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 2580
cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 2640
gggaggattg ggaagagaat agcaggcatg ctggggactt aagctcgagg gcgcgcc 2697
Claims (3)
1.猪克隆胚胎的制备方法,其特征在于,以hACE2人源化转基因细胞系作为核移植供体细胞,离体的猪卵母细胞为核移植受体细胞,通过核移植技术获得猪克隆胚胎;
所述hACE2人源化转基因细胞系是将靶向编辑猪血管紧张素转化酶ACE2基因的CRISPR-Cas9系统导入猪胎儿成纤维细胞系中,获得的中靶阳性细胞克隆,即为hACE2人源化转基因细胞系;
所述靶向编辑猪血管紧张素转化酶ACE2基因的CRISPR-Cas9系统包括猪血管紧张素转化酶ACE2基因打靶载体和携带人血管紧张素转化酶ACE2基因的同源修复载体;
所述打靶载体是基于CRISPR-Cas9技术设计的靶向猪血管紧张素转化酶ACE2基因的打靶载体,其包含靶向猪血管紧张素转化酶ACE2基因的sgRNA序列以及编码Cas9核酸酶的核酸序列;
所述同源修复载体包含如下Donor DNA:SEQ ID NO:1所示的血管紧张素转化酶ACE2基因的上游同源臂-人血管紧张素转化酶ACE2基因的cDNA序列-polyA序列-SEQ ID NO:2所示的血管紧张素转化酶ACE2基因的下游同源臂;
其中,猪血管紧张素转化酶ACE2基因在NCBI上的参考序列编号为100144303;人血管紧张素转化酶ACE2基因在NCBI上的参考序列编号为59272;
sgRNA作用位点的DNA序列为5′-CCACTGAGGAACTGGCCAAGACA-3′;
Donor DNA的序列如SEQ ID NO:3所示。
2.hACE2人源化转基因猪的构建方法,其特征在于,将按照权利要求1所述方法制备的猪克隆胚胎通过非手术法移入母猪子宫内进行妊娠,获得hACE2人源化转基因猪。
3.按照权利要求2所述方法制备得到的hACE2人源化转基因猪在新型冠状病毒SARS-CoV-2感染和致病机理研究、药物和疫苗研发中的应用。
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