CN111349655A - 一种免疫缺陷动物模型及其构建方法、用途 - Google Patents
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Abstract
本发明涉及动物基因工程领域,一种免疫缺陷动物模型及其构建方法、用途,将免疫缺陷动物的β2微球蛋白具有表达缺陷或功能性缺陷;免疫缺陷动物具有人源化HLA基因;通过使免疫缺陷动物的β2微球蛋白具有表达缺陷或功能性缺陷,使之不能与α链或称重链组装成有功能的MHC‑I,进而在一定程度上降低GVHD的发生,同时在免疫缺陷动物中引入人源化的HLA基因,以提高抗原递呈过程的人源化程度,进一步模拟人体抗原递呈和特异性免疫效应的过程,上述构建的免疫缺陷动物模型可有效降低GVHD的发生,并一定程度上重现人体抗原递呈和特异免疫反应,可以进行人细胞/组织/器官的异种移植模型构建及相关治疗方法的体内研究。
Description
技术领域
本发明涉及动物基因工程领域,具体涉及一种免疫缺陷动物模型及其构建方法、用途。
背景技术
通过运用动物模型,人们已经获得了大量关于细胞分化,个体发育,药物蹄选和病毒感染等研究成果。但是当运用动物模型来研究人类疾病时,发现单纯的动物模型存在诸多不足,分析其原因在于小鼠与人之间存在种属差异,导致他们之间无论在遗传背景,个体发育,还是在细胞功能,免疫识别上都存在着许多差别。为克服上述不足,人们通过对动物模型进行改进,构建了很多种类的免疫缺陷动物。目前,免疫缺陷程度最高的是第三代免疫缺陷小鼠NOD/SCID IL2rg-/-小鼠(日本的NOG,美国的NSG以及中国专利文献中201310229629.9中公开的NSI小鼠),该类小鼠缺失成熟的B、T、NK等淋巴细胞,进行原代异种细胞、组织、器官移植具有极高的效率,已成为人源化小鼠模型的金标准。
然而,申请人研究发现,将人的造血干细胞或是外周血移植到NOD/SCID IL2rg-/-小鼠中,造血干细胞来源的T细胞或是外周血来源的T细胞,会因为人和鼠之间种属不同,主要组织相容性复合物不相容,导致移植物抗宿主反应(graft-versus-host disease,GVHD)。受体小鼠会因为GVHD发生掉毛、体重下降、虚弱等,影响具体的实验结果,且小鼠抗原递呈细胞无法和异种移植细胞产生相关递呈反应,无法有效模拟人体抗原递呈过程和针对抗原的特异性免疫效应。因此,提供一种有效降低GVHD,并且针对抗原产生异种细胞的特异性免疫反应的免疫缺陷动物模型非常重要。
发明内容
因此,本发明要解决的技术问题在于一种免疫缺陷动物模型及其构建方法、用途,构建的免疫缺陷动物模型可有效降低GVHD的发生,并且针对抗原产生异种细胞的特异性免疫反应,可进行人的造血干细胞或是外周血移植等异种人细胞/组织/器官机器引起的免疫反应等相关实验的研究,使受体小鼠免受其异种T细胞GVHD的影响,也可产生抗原特异性的免疫细胞。
为此,本发明提供了如下技术方案:
一种免疫缺陷动物模型的构建方法,将免疫缺陷动物的β2微球蛋白具有表达缺陷或功能性缺陷;所述免疫缺陷动物具有人源化HLA基因。
进一步的,在所述构建方法中,所述人源化HLA(majorhistocompatibilitycomplex,human)基因包括HLA-A2.1(常见的HLA-A型等位基因)。
进一步的,在所述构建方法中,所述免疫缺陷动物为缺少B细胞、T细胞和/或NK细胞。
优选地,所述免疫缺陷动物为NOD基因背景。
优选地,所述免疫缺陷动物为重症联合免疫缺陷背景(SCID)。
优选地,所述免疫缺陷动物为IL2rg基因表达或者功能缺陷。
进一步的,在所述构建方法中,敲除所述免疫缺陷动物的β2m基因的全部或部分。
进一步的,在所述构建方法中,所述免疫缺陷动物为啮齿类动物,更进一步的为小鼠。
进一步的,在所述构建方法中,采用CRISPR/Cas9体系敲除,β2m基因的靶位点序列如SEQ ID NO.1所示。
进一步的,在所述构建方法中,基于所述靶位点序列设计引物组合成含粘性末端的线性化gRNA;优选的,所述引物组中的上游引物S Oligo如SEQ ID NO.2所示,下游引物ASOligo如SEQ ID NO.3所示。
进一步的,在所述构建方法中,采用CRISPR/Cas9体系敲除的步骤包括:
将含粘性末端的线性化gRNA和Cas9 mRNA注射入所述免疫缺陷动物的受精卵细胞质中并进行孵育,然后移植至雌性假孕免疫缺陷动物输卵管中发育,得到F0代,经基因型鉴定,随后通过杂交获得纯合子。
本发明提供了一种所述的构建方法构建得到的免疫缺陷动物模型。
本发明提供了一种所述的构建方法构建得到的免疫缺陷动物模型具有如下(1)-(4)中任意一个所示的用途;
(1)用于一种或者多种异种细胞、组织或器官的异种移植和体内功能研究;
(2)制备评价和/或筛选产品对人体引起的免疫效应的动物模型;
(3)制备和/或筛选引起人体免疫效应的产品;或
(4)用于免疫系统重建。
优选地,一种或者多种异种细胞、组织或器官中包含免疫细胞。
本发明技术方案,具有如下优点:
1.本发明提供的一种免疫缺陷动物模型的构建方法,将免疫缺陷动物的β2微球蛋白具有表达缺陷或功能性缺陷;所述免疫缺陷动物具有人源化HLA基因;申请人研究发现,将人的造血干细胞或是外周血移植到NOD/SCID IL2rg-/-小鼠中,造血干细胞来源的T细胞或是外周血来源的T细胞,会因为人和鼠之间种属不同,主要组织相容性复合物不相容,导致移植物抗宿主反应,而引起GVHD的主要原因是与CD8 T细胞结合并提成抗原肽的MHC-I类分子不匹配;由于所有MHC-I类分子都包含有两条不相连的多肽链:一条为MHC编码的α链或称重链,人类约44X103,小鼠约为47X103;另一条为独立染色体基因编码的β链(β2-微球蛋白,β2m),人类和小鼠均为12X103。因此,本发明使免疫缺陷动物自身的β2微球蛋白具有表达缺陷或功能性缺陷,通过部分或全部敲除β2m,使之不能与α链或称重链组装成有功能的MHC-I,进而在一定程度上降低GVHD的发生,同时在免疫缺陷动物中引入人源化的HLA基因,以提高人源化程度,并一定程度上重现人体抗原递呈和特异免疫反应,可以进行人细胞、组织或器官的异种移植模型构建及相关治疗方法的体内研究,使受体小鼠免受其T细胞GVHD的影响,还可以产生抗原递呈及其特异性的免疫效应。
2.本发明提供的一种免疫缺陷动物模型的构建方法,采用CRISPR/Cas9体系敲除,β2m基因的靶位点序列如SEQ ID NO.1所示,依据上述靶位点序列设计的Cas9敲除体系可以部分敲除小鼠β2m基因的2号外显子,实现将免疫缺陷动物的β2微球蛋白具有表达缺陷或功能性缺陷。
3.本发明提供的一种所述的构建方法构建得到的免疫缺陷动物模型,由于缺失鼠自身的β2微球蛋白,降低GVHD反应,同时具有的人源化的HLA-A2.1可表达人类HLA I类重链和轻链,提高了人源化程度,进一步模拟人体抗原递呈和特异性免疫效应的过程,因而可以用于人用于一种或者多种异种细胞、组织或器官的异种移植和体内功能研究;优选地,一种或者多种异种细胞、组织或器官中包含免疫细胞,同时可用于制备评价和/或筛选产品对人体引起的免疫保护水平的动物模型、制备和/或筛选引起人体免疫反应的产品或用于免疫系统重建。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,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的附图。
图1是本发明实施例1中β2m敲除位点选择示意图;
图2是本发明实施例1中体外扩增gDNA基因片段电泳图;
图3是本发明实施例1中回收纯化体外转录的Cas9 mRNA的电泳图;
图4是本发明实施例2中嵌合或杂合NSIB(F0代)的PCR扩增后测序结果图:
图5是本发明实施例2中NSIB小鼠纯合子与对照NSI小鼠的PCR扩增后测序比对结果图;
图6是本发明实施例3中NSIB小鼠纯合子与F1代杂合仔鼠流式图;
图7是本发明实施例3中NSIB HLA纯合子IL2rg-/-基因型鉴定结果图;
图8是本发明实施例3中NSIB HLA纯合子SCID基因测序结果;
图9是本发明实施例3中NSIB HLA纯合子β2m基因测序结果;
图10是本发明实施例3中NSIB HLA纯合子中HLA-A2.1基因表达量检测结果;
图11是本发明实施例4中NSI、NSIB和NSIB HLA小鼠体内检测到的CD8阳性T细胞占外周血白细胞的比例;
图12是本发明实施例5中NSIB HLA和NSIB荷瘤小鼠在皮下移植肿瘤细胞系35天后称量小鼠皮下形成的肿瘤组织块的重量。
具体实施方式
提供下述实施例是为了更好地进一步理解本发明,并不局限于所述最佳实施方式,不对本发明的内容和保护范围构成限制,任何人在本发明的启示下或是将本发明与其他现有技术的特征进行组合而得出的任何与本发明相同或相近似的产品,均落在本发明的保护范围之内。
实施例中未注明具体实验步骤或条件者,按照本领域内的文献所描述的常规实验步骤的操作或条件即可进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规试剂产品。
pMD18-T Simple(即pT7载体,带氨苄抗性)购自TaKaRa公司;
mirVanaTMmiRNA Isolation Kit试剂盒购自Ambion公司;
pcDNA3.3-hCas9(Cas9载体,Addgene NO.MLM3613);
mMESSAGE mMACHINE T7 Kit购自Invitrogen公司;
第三代免疫缺陷(NSI)小鼠(NOD/SCID IL2rg-/-)为按照中国专利文献ZL201310229629.9中公开的方法构建得到;
NODTg(HLA-A/H2-D/B2M)小鼠是从Jackson实验室购入(参见网址为https://www.jax.org/strain/006604),所述NOD Tg(HLA-A/H2-D/B2M)小鼠ID:006604,基因型为Tg(HLA-A/H2-D/B2M)1Dvs,该转基因编码一个人β2-微球蛋白(β2m)共价连接到人类HLA-A2.1基因的MHC类I、α1和α2结合域,以及小鼠H2-Db的α3、胞内和跨膜域;
小鼠胚胎培养基:HEPES(4-羟乙基哌嗪乙磺酸)20mmol/L,pH 7.4~7.8,非必需氨基酸总含量为0.1mmol/L,必需氨基酸总含量为0.1-0.6mmol/L;非必需氨基酸为包括甘氨酸、丙氨酸、脯氨酸、酪氨酸、丝氨酸、半胱氨酸、天冬酰胺、谷氨酰胺、天冬氨酸和谷氨酸;必需氨基酸包括赖氨酸、色氨酸、苯丙氨酸、甲硫氨酸、苏氨酸、异亮氨酸、亮氨酸和缬氨酸;
小鼠调整胚胎培养基:HEPES(4-羟乙基哌嗪乙磺酸)20mmol/L,pH7.4~7.8,丙酮酸浓度0.35mmol/L,谷氨酰胺浓度1mmol/L,葡萄糖浓度调至0.1mmol/L;
HLA-A2(APC)购自biolegend公司
H2Kb/H2Db(PE)购自biolegend公司
下述实施例中的基因测序如SCID基因测序、β2m基因测序、嵌合或杂合NSIB(F0代)(NOD-scid IL2rg-/-β2m-/-)免疫缺陷小鼠测序、NSIB小鼠纯合子与对照小鼠测序均由广州艾基生物技术有限公司完成。
实施例1 Cas9敲除体系的构建
1、靶点选择
利用ZiFiT Targeter Version网站设计遵循Cas9敲除靶点的GGN(17-18)NGG(N为任意碱基)的序列要求选择Cas9敲除的靶位点,并通过Ensembl/NCBI网站的“Blast”检索功能确定靶位点是基因组中的单一位点;
经选择,在β2m基因的2号外显子设计靶点(如图1所示,字体加粗序列是靶点序列,加下划线序列是PAM序列),设计得到的靶位点序列如SEQ ID NO.1所示。
靶点确认,依据打靶细胞的基因组设计扩增靶位点的高特异性引物,PCR扩增获得含靶位点片段;在靶位点中选择扩增片段唯一的限制性内切酶进行酶切电泳鉴定;酶切鉴定正确后,将PCR扩增产物送去测序鉴定;通过酶切、测序鉴定,确认打靶鉴定引物的特异性和酶切、测序鉴定的可行性;
2、构建打靶载体pT7-gDNA
(1)含粘性末端的、线性化guide DNA序列的制备
基因合成分别与靶位点序列相同和互补的两条引物,即多聚核苷酸单链(Oligo),分别为S Oligo和AS Oligo,分别如SEQ ID NO.2和SEQ ID NO.3所示;
按照下表1配制反应组分,然后加入1.5mL EP管中,置于沸水中加热10分钟后,冷却至室温,瞬间离心,获得编码guide RNA(gRNA)的含粘性末端的、线性化guide DNA序列(即L-gDNA),备用;
表1反应组分
(2)线性化空载体L-pT7的制备
通过限制性内切酶BbsI剪切空载体pMD18-T Simple(即pT7载体,带氨苄抗性),获得含粘性末端的线性化空载体pMD18-T Simple,即L-pT7;
(3)载体连接
通过DNA连接酶(如Takara公司Solution I),将步骤(2)获得的L-pT7和步骤(1)获得的L-gDNA连接成完整载体pT7-gDNA,转化、涂板、挑单克隆、摇菌、提取质粒DNA、酶切鉴定、质粒测序,筛选测序正确质粒备用;
3、gRNA体外转录
(1)扩增gDNA基因片段
以步骤2中测序正确的pT7-gDNA质粒(1-30ng)为模版,以T7-S和Tracr-Rev为引物,分别如SEQ ID NO.4和SEQ ID NO.5所示,扩增gDNA基因片段,使用的是TOYOBO高保真酶(KOD OneTM PCR Master Mix-Blue-Code No.KMM-201)进行PCR扩增;
将上述获得的产物电泳,电泳结果如图2所示,胶回收、溶解于30μL无(RNA)酶水中备用;
(2)gDNA体外转录成gRNA
通过体外转录试剂盒Kit(Ambion公司)将步骤(1)PCR扩增后的gDNA基因片段体外转录成gRNA,利用mirVanaTMmiRNA Isolation Kit试剂盒(Ambion公司)从转录体系中回收纯化体外转录的gRNA,用20μL无酶水溶解,-80℃保存备用;
4、Cas9体外转录
(1)线性化Cas9载体
按照下表2体系配置酶切反应液,37℃反应6h,用PmeI限制性内切酶剪切pcDNA3.3-hCas9(Cas9载体,Addgene NO.MLM3613),使载体线性化,电泳、跑胶、回收,用20μL无酶水溶解备用;
表2酶切反应体系
(2)体外转录带帽的Cas9 mRNA
按照下表3配制反应体系,于37℃反应4h,随后加入1μL DNase I(脱氧核糖核酸酶I),37℃反应15分钟,用mMESSAGE mMACHINE T7 Kit体外转录带帽的Cas9 mRNA,电泳、跑胶、回收带帽Cas9 mRNA;
表3反应体系
(3)加poly A
按照下表4配制反应体系,于37℃反应45分钟,在带帽Cas9 mRNA上加上poly A,获得稳定而且翻译效率也更高的RNA;
表4反应体系
(4)回收纯化体外转录的Cas9 mRNA
利用mirVanaTMmiRNA Isolation Kit试剂盒(Ambion公司)从转录体系中回收纯化体外转录的Cas9 mRNA,回收纯化体外转录的Cas9 mRNA的电泳图如图3所示,用10-20μL无酶水溶解,-80℃保存备用。
实施例2 NSIB小鼠的构建和验证
1、NSI小鼠超排
取若干NSI小鼠(NOD-scid IL2rg-/-),公鼠周龄:10-11周,雌鼠周龄:8周。母鼠于第一天13:00注射孕马血清促性腺激素(PMSG)7.5IU/只,第三天13:00注射人绒毛膜促性腺激素(HCG)7.5IU/只,第三天17:00每只雌鼠与2只雄鼠合笼,第四天8:00-9:00检查雌鼠阴道栓,从见栓雌鼠子宫获取NSI小鼠受精卵;
提供精子的NSI雄鼠和雌鼠的生产注意事项:NSI母代鼠与父代鼠(提供精子的NSI雄鼠和雌鼠的母代和父代)合笼见栓后,将父代鼠分出,然后放入拥有至少一次以上生产经验的ICR母鼠,以保证NSI母代鼠所产仔哺乳充足;
(2)取实施例1中获得的gRNA和Cas9 mRNA,将gRNA稀释至浓度为20ng/μL,Cas9mRNA稀释至浓度为20ng/μL,等体积混匀,将混合液原核注射入NSI小鼠受精卵胞质后,用小鼠胚胎培养基培养24小时后,将培养基替换为小鼠调整胚胎培养基继续培养7h后,移植入0.5dpc(二细胞期胚胎)假孕NSI母鼠输卵管壶腹部,每只小鼠移植受精卵数量为15个;
代孕NSI鼠饲养注意事项:生产代孕母鼠的NSI母代鼠与父代鼠合笼见栓后,将父代鼠分出,随后放入拥有至少一次以上生产经验的ICR母鼠,代孕母鼠出生后7天,将同窝雄性仔取出,以保证代孕母鼠的哺乳期营养供给充足;
(3)移植受精卵后的母鼠与生产过一次以上的ICR母鼠同笼(1只代孕NSI母鼠与1只ICR母鼠);
代孕母鼠产仔后,通过基因型鉴定(PCR扩增和测序)小鼠基因型,获得嵌合或杂合NSIB(F0代)(NOD-scid IL2rg-/-β2m-/-)免疫缺陷小鼠(PCR扩增后测序,如图4所示),通过进一步与NSI小鼠杂交获得NSIB小鼠纯合子,将NSIB小鼠纯合子与正常对照NSI小鼠进行PCR扩增测序比对,测序结果如图5所示(图中B11代表NSIB小鼠纯合子,WT代表正常对照NSI小鼠),获得β2m基因2号外显子缺失4bp碱基的NSIB小鼠纯合子。
实施例3 NSIB HLA小鼠的构建和鉴定
1、NSIB小鼠与NOD Tg(HLA-A/H2-D/B2M)小鼠杂交将实施例2中获得的NSIB小鼠纯合子与从Jackson实验室购入的NOD Tg(HLA-A/H2-D/B2M)小鼠杂交(HLA-A/H2-D/B2M为本发明专利所述的人源化HLA-A2.1基因,为人鼠嵌合基因),获得的F1代杂合仔鼠,待所述F1代杂合仔鼠生长到4周龄后用毛细血管眼眶静脉丛取血4滴到加入了200μL抗凝剂的EP管中,加入1mL的PBS(pH7.4)清洗,300g离心5分钟,弃上清,加入1mL红细胞裂解液(RCF)轻轻吹打混匀,裂解5分钟后离心,弃红色上清,再加500μL红细胞裂解液进行第二次裂解,离心,弃红色上清,每管细胞加入100μL的PBS制成细胞悬液,加入0.2μL的HLA-A2(APC)(100X)和0.2μL的H2Kb/H2Db(PE)(100X)抗体混匀,置于4℃环境中避光孵育30分钟,加入500μL PBS重悬细胞,离心弃上清,加入120μL的PBS(pH7.4)重悬细胞,NSIB小鼠纯合子作对照,用流式细胞仪(ACEA NovoCyte流式细胞仪)进行检测分析,结果如图6所示(图中,NIB代表NSIB小鼠纯合子,F1代表F1代杂合仔一号母鼠,F2代表F1代杂合仔二号母鼠、M1代表F1代杂合仔一号公鼠、M2代表F1代杂合仔二号公鼠、M3代表F1代杂合仔三号公鼠),经检测分析F1代杂合仔鼠为NSIB HLA杂合子小鼠。
2、NSIB HLA小鼠纯合子的构建和鉴定
将步骤1中获得的F1代的NSIB HLA杂合子小鼠进行杂交,获得F2代小鼠,然后通过基因型鉴定(PCR扩增和测序、qPCR)和流式分析,挑选IL2rg、SCID、鼠的缺失β2m基因纯合度高以及HLA-A2.1高表达的F2代小鼠与F1代NSIB HLA杂合子小鼠杂交,子代挑选基因型IL2rg、SCID、鼠的缺失β2m基因纯合度高以及HLA-A2.1高表达的仔鼠继续杂交四代后获得NSIB HLA纯合子免疫缺陷小鼠5只,代号分别是F1、F2、F3、M1和M2,F1代表一号母鼠,F2代表二号母鼠、M1代表一号公鼠、M2代表二号公鼠。
IL2rg-/-基因型鉴定:
以C57BL/6小鼠为WT小鼠,以上述获得的NSIB HLA纯合子免疫缺陷小鼠F1、F2、F3、M1和M2为实验小鼠,进行IL2rg-/-基因型鉴定,通过PCR鉴定,鉴定结果如图7所示。
SCID基因测序:
将上述获得的NSIB HLA纯合子免疫缺陷小鼠F1、F2、F3、M1和M2进行SCID基因测序,测序结果如图8所示,图中F1、F2、M1和M2的结果相同;
鼠β2m基因测序:
将上述获得的NSIB HLA纯合子免疫缺陷小鼠F1、F2、F3、M1和M2进行β2m基因测序,测序结果如图9所示,图中F1、F2、F3、M1和M2的结果相同;
人源化HLA-A2.1基因表达量
将上述获得的NSIB HLA纯合子免疫缺陷小鼠F1、F2、F3、M1和M2进行HLA-A2.1基因表达量的检测,通过RT-qPCR检测,检测结果如图10所示,图中heterozygote代表NSIB与NODTg(HLA-A/H2-D/B2M)小鼠杂交一代。
综上,本实施例获得的NSIB HLA小鼠纯合子的基因型为NOD/SCID IL2rg-/-β2m-/-Tg(HLA-A/H2-D/B2M)。
实施例4 NSIB HLA和NSIB小鼠可有效减低异种移植的GVHD反应
将人HLA-A2.1型外周血(市售)与生理盐水(0.85-0.9%)以1:2体积比混合获得外周血稀释液备用,在50ml离心管中加入15mL人外周血淋巴细胞分离液(STEMCELL淋巴细胞分离液Lymphoprep),并沿着管壁缓慢加入外周血稀释液30mL(分离液(Ficol):稀释液=1:2),18℃,300g离心30分钟,取出离心管,用巴氏吸管轻轻吸取第二层白细胞移入另一个试管,加入生理盐水充分洗涤,300g离心5分钟后,去除上清,加入T细胞培养液(培养基RPMI-1640+10%FBS+1%PS双抗),重悬获得PBMC溶液备用。
利用德国美天旎细胞分选磁珠--CD8+T Cell Isolation Kit试剂盒对PBMC进行磁珠分选:对PBMC溶液进行细胞计数,300g离心10分钟,去除上清,加入缓冲液重悬;再次洗涤,300g离心10分钟,去除上清,每107个细胞加入40ul缓冲液重悬PBMC;加入10ul CD8抗体,充分混匀,4℃孵育10分钟后,加入30ul缓冲液和20ul CD8磁珠Cocktail,4℃孵育15分钟;加入2mL缓冲液洗涤细胞,300g离心10分钟,去除上清,加入500ul缓冲液重悬,加入磁珠分选仪分选,获得CD8阳性T细胞溶液,300g离心5分钟,用T细胞培养液重悬备用。
分别在NSIB HLA、NSIB和NSI小鼠(每个品系设置5个重复组小鼠)体内通过静脉注射的方式移植入人HLA-A2.1型外周血CD8阳性T细胞,每只小鼠注射细胞数5×106个,并于细胞移植后28天检测小鼠外周血中CD8阳性T细胞占外周血白细胞的的比例,检测结果如图11所示,发现NSI小鼠外周血中的T细胞含量显著高于NSIB HLA和NSIB,且经观察发现NSI小鼠开始出现脱毛、弓背的病理症状,表明NSI小鼠体内发生了移植物排斥宿主反应GVHD,而NSIB HLA和NSIB小鼠则不表现出明显的脱毛和弓背等GVHD病理症状,表明NSIB HLA和NSIB小鼠β2m基因的缺失,小鼠MHC I基因表达异常,导致人体CD8阳性免疫细胞无法识别并杀伤排斥小鼠细胞,一定程度上缓解了GVHD。
实施例5 NSIB HLA小鼠可有效重现异种免疫细胞的抗原特异性免疫效应
分别在NSIB HLA、NSIB小鼠(每个品系设置4个重复组小鼠)体内通过皮下注射的方式移植入前列腺癌细胞系DU145(市售),每只小鼠注射细胞数5×105个,肿瘤细胞系移植7天后,通过静脉注射的方式移植入HLA-A2.1型外周血CD8 T细胞(实施例4中获得的CD8阳性T细胞溶液),每只小鼠注射细胞数5×106个,并于肿瘤移植35天后,分离小鼠皮下肿瘤,称量并记录肿瘤的重量,结果如图12所示,发现NSIB HLA小鼠皮下种植的肿瘤组织块显著小于NSIB小鼠皮下肿瘤组织块,说明由于NSIB HLA小鼠转基因表达了人源化的HLA-A2.1基因,所以小鼠的抗原递呈细胞可以将肿瘤抗原递呈给HLA-A2.1型外周血免疫细胞,并形成肿瘤抗原特异性的免疫细胞。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
SEQUENCE LISTING
<110> 湖南昭泰生物医药有限公司
<120> 一种免疫缺陷动物模型及其构建方法、用途
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Claims (10)
1.一种免疫缺陷动物模型的构建方法,其特征在于,将免疫缺陷动物的β2微球蛋白具有表达缺陷或功能性缺陷;所述免疫缺陷动物具有人源化HLA基因。
2.根据权利要求1所述的构建方法,其特征在于,所述人源化HLA基因包括HLA-A2.1。
3.根据权利要求2所述的构建方法,其特征在于,所述免疫缺陷动物为缺少B细胞、T细胞和/或NK细胞;
优选地,所述免疫缺陷动物为NOD基因背景;
优选地,所述免疫缺陷动物为重症联合免疫缺陷背景;
优选地,所述免疫缺陷动物为IL2rg基因表达或者功能缺陷。
4.根据权利要求1-3任一项所述的构建方法,其特征在于,敲除所述免疫缺陷动物的β2m基因的全部或部分。
5.根据权利要求4所述的构建方法,其特征在于,敲除β2m基因的2号外显子的全部或部分。
6.根据权利要求4或5所述的构建方法,其特征在于,采用CRISPR/Cas9体系敲除,β2m基因的靶位点序列如SEQ ID NO.1所示。
7.根据权利要求6所述的构建方法,其特征在于,基于所述靶位点序列设计引物组合成含粘性末端的线性化gRNA;优选的,所述引物组中的上游引物S Oligo如SEQ ID NO.2所示,下游引物AS Oligo如SEQ ID NO.3所示。
8.根据权利要求7所述的构建方法,其特征在于,采用CRISPR/Cas9体系敲除的步骤包括:
将含粘性末端的线性化gRNA和Cas9 mRNA注射入所述免疫缺陷动物的受精卵细胞质中并进行孵育,然后移植至雌性假孕免疫缺陷动物输卵管中发育,得到F0代,经基因型鉴定,随后通过杂交获得纯合子。
9.一种如权利要求1-8任一项所述的构建方法构建得到的免疫缺陷动物模型。
10.一种如权利要求1-8任一项所述的构建方法构建得到的免疫缺陷动物模型具有如下(1)-(4)中任意一个所述的用途;
(1)用于一种或者多种异种细胞、组织或器官的异种移植和体内功能研究;优选地,一种或者多种异种细胞、组织或器官中包含免疫细胞;
(2)制备评价和/或筛选产品对人体引起的免疫效应的动物模型;
(3)制备评估和/或筛选引起人体免疫效应的产品;或
(4)用于免疫系统重建。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104611371A (zh) * | 2015-02-10 | 2015-05-13 | 中国人民解放军军事医学科学院微生物流行病研究所 | 人mhc转基因小鼠模型的构建方法与应用 |
CN105578876A (zh) * | 2013-10-18 | 2016-05-11 | 大鹏药品工业株式会社 | 表达hlai类的非人动物 |
CN108531509A (zh) * | 2018-04-23 | 2018-09-14 | 中国人民解放军军事科学院军事医学研究院 | 人mhc转基因小鼠模型a2/dr15的构建方法与应用 |
CN110055223A (zh) * | 2018-01-19 | 2019-07-26 | 北京百奥赛图基因生物技术有限公司 | 一种B2m基因改造的免疫缺陷动物的制备方法及其应用 |
-
2020
- 2020-02-19 CN CN202010102723.8A patent/CN111349655A/zh active Pending
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105578876A (zh) * | 2013-10-18 | 2016-05-11 | 大鹏药品工业株式会社 | 表达hlai类的非人动物 |
CN104611371A (zh) * | 2015-02-10 | 2015-05-13 | 中国人民解放军军事医学科学院微生物流行病研究所 | 人mhc转基因小鼠模型的构建方法与应用 |
CN110055223A (zh) * | 2018-01-19 | 2019-07-26 | 北京百奥赛图基因生物技术有限公司 | 一种B2m基因改造的免疫缺陷动物的制备方法及其应用 |
CN108531509A (zh) * | 2018-04-23 | 2018-09-14 | 中国人民解放军军事科学院军事医学研究院 | 人mhc转基因小鼠模型a2/dr15的构建方法与应用 |
Non-Patent Citations (1)
Title |
---|
LEONARD D SHULTZ等: "Generation of functional human T-cell subsets with HLA-restricted immune responses in HLA class I expressing NOD/SCID/IL2r gamma(null) humanized mice", 《PROC NATL ACAD SCI U S A》 * |
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