CN112575037B - 一种嵌合人hla-dp基因组区域的人源化转基因小鼠模型的构建方法 - Google Patents
一种嵌合人hla-dp基因组区域的人源化转基因小鼠模型的构建方法 Download PDFInfo
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Abstract
本发明属于生物技术领域,涉及一种表达人HLA‑DP基因组区域的转基因动物模型的构建方法。本发明的BAC克隆含有124kb的HLA‑DP基因组区域(DOA‑DPA1*0103‑DPB1*0401),经过DNA测序、脉冲场凝胶电泳鉴定,纯化后通过原核显微注射技术,将BAC‑DNA引入小鼠的受精卵,再移植到假孕的小鼠输卵管,获得阳性转基因动物。与野生型小鼠杂交获得子代,通过分子生物学、免疫学、组织学技术鉴定分析,最终获得5个整合不同人HLA‑DP基因组区域的转基因小鼠品系。利用本发明的方法构建的小鼠模型在血细胞、脾脏、肺脏等主要组织器官上均存在高水平HLA‑DPA1、HLA‑DPB1的表达,可用于T细胞表位和表位特异性T细胞的免疫致病和免疫保护作用研究。
Description
技术领域
本发明属于生物技术领域,涉及嵌合人HLA-DP基因组区域的人源化转基因小鼠模型的构建方法,尤其涉及一种人MHC(HLA-DPA1*0103-DPB1*0401)转基因小鼠模型的构建方法。
背景技术
现有技术公开了主要组织相容性复合物(MHC)是一组决定移植组织是否相容、与免疫应答密切相关、紧密连锁的基因群,在人类中MHC被称为人类白细胞抗原(HLA)基因复合物。研究公开了组成MHC的各种基因成簇的分布在人第6号染色体短臂,主要包含了HLA I类、HLA II类和HLA III类基因,其中的HLA II类基因主要包括了HLA-DP、HLA-DQ、HLA-DR等三类基因簇,相应编码了HLAII类分子HLA-DP、HLA-DQ、HLA-DR等;HLAII类分子仅表达在成熟B细胞和抗原递呈细胞,如巨噬细胞和树突状细胞,部分细胞在特殊分子诱导或者某些病理状态下可以表达II类分子;这些分子的主要功能是从细胞外将病毒抗原递呈给T细胞,特异性的抗原通过递呈后可以有效地刺激T细胞,协助刺激产特异性抗体的B细胞产生相应抗体,以杀死或抑制该特定抗原。
现有技术中已制备多种MHC I类和IT类人源化小鼠模型,包括MHCI类人源化小鼠,例如A1,A2,A3,A11,A24,B7,B27或Cw3等;MHCII类人源化小鼠,例如DR1,DR2,DR3,DR4,DQ2,DQ3,DQ6或DQ8等,该些MHC类分子人源化转基因小鼠已被广泛用于传染性疾病、自身免疫疾病的研究,例如强直性脊柱炎(AS)、类风湿性关节炎(RA)、牛皮癣、胰岛素依赖性糖尿病(IDDM)、重症肌无力(MG)、多发性硬化症(MS)等;此外,所述人源化转基因小鼠在疫苗的研发和测试、评价治疗药物的安全性和免疫原性方面也在发挥重要作用。
研究显示,HLA-DP区域位于II类区域的着丝点端,全长75kb,包括有2个功能基因,2个假基因;其中DPA1和DPBl是经典的HLAII类基因,分别编码HLA-DP抗原分子的α、β链,这两个基因在基因组上是头对头排列的,即两个基因的5’端相向而立,两个基因的转录起始位点之间约间隔2.5kb的序列,是它们共同的调控序列;由于DP分子在细胞表面表达水平较低,因此是II类分子中最后被发现,而且也是研究最少的经典座位。研究显示,在小鼠MHC中没有DP相应的抗原分子,这是人和小鼠MHC的一个显著差别;基于这些特征,业内认为,DP区域可能与其他II类抗原分子有不同的功能或者进化模式。
疾病相关性研究表明,DP分子与幼年类风湿性关节炎、铍中毒及遗传性过敏症相关。2009年,Kamatani等基于人类全基因组关联研究(genome wide association study,GWAS),发现日本人群和泰国人群中HLA-DPA1及HLA-DPB1基因中的11个单核苷酸多态性(single nucleotidepolymorphism,SNP)位点与慢性乙型肝炎显著关联;随后研究证实在中国、韩国以及沙特阿拉伯、高加索人群中HLA-DP基因簇上的11个单核苷酸多态性位点(SNP)与乙肝病毒感染相关。进一步研究表明,HLA-DPB1基因3’UTR多态性(rs9277534G)影响HLA-DPB1基因的表达水平,进而可能影响到人群自身免疫性肝炎易感性。
有研究显示,HLA-DP基因座上的两SNPs(rs2395309,rs9277535)在中国南北汉族人群中与HBV的感染和感染后清除均呈显著相关;在骨髓移植中,HLA-DPB1的匹配程度对无关供者骨髓移植的效果具有显著影响;但HLA-DP在免疫反应中的作用迄今尚未阐明。
本领域研究者认为,不同疾病与HLA关联的机制可能有所不同,对此,目前有拟态学说、受体学说、免疫应答基因学说、连锁不平衡学说及共同表位学说等;另一种可能的机制,是HLA基因的表达调控的参与;不同等位基因型在抗原递呈细胞(APC)表面表达强度不同,从而改变了免疫反应的信号强度,促进炎症反应生成;有些疾病是HLA基因的异常表达引起的,如:胰岛B细胞表达II类基因引起自身免疫状态失调;而抗原递呈细胞如果没有HLAII分子的表达,则引起一种叫做裸淋巴细胞综合症(BLS)的免疫缺陷症,说明HLA基因的表达调控是免疫应答中极为关键的环节;此外,MHC类分子的表达能够决定免疫系统发育过程中T细胞群的形成,因此其表达水平对T细胞的发育命运至关重要;HLA基因的不同表达就有可能由此对免疫应答进行调控。
研究显示,HLA基因的表达调控与其他重要功能基因一样呈现出复杂的调控格局,更由于其重要的免疫学功能而表现出调控方式的特殊性。一般认为,HLA分子的表达调控主要在转录水平,受到了多个转录因子和其启动子区的顺式元件相互作用的影响。有研究显示,除了一些近端调控元件W/S、X1、X2、Y盒等之外,上游远端的LCR(locus controlregion)也被发现可与RFX和CIITA结合,对HLAII分子基因转录进行调控;在小鼠的H2Ea基因,甚至发现了位于基因上游20kb以外的调控元件。有报道显示5’UTR和3’UTR在HLA-DRA转录后水平的基因表达调控中发挥着重要作用,控制着mRNA的稳定性和降解速率,决定翻译速率和利用效率,而且还能对特定mRNA的时空性表达起重要作用。目前关于DP基因的表达调控研究较少,有限的资料表明,HLA-DPB1分子的表达调控不同于其他II类分子,类成淋巴细胞系45.EM2正常表达HLA-DQ、HLA-DR分子以及HLA-DPA1分子,但是不表达HLA-DPB l(Coiras等2002)。
业内共识,转基因小鼠是研究MHC类分子基因表达及功能的重要研究工具。相对于DQ和DR基因座位点,DP基因座位点的人源化转基因小鼠模型较少,目前仅有报道三种DP转基因小鼠(HLA-DPB1*0401、HLA-DPB1*0201、HLA-DPB1*1701),用于人慢性铍疾病分子机理研究(Tarantino-Hutchison等2009,Mack等2014);HLA-DP基因存在较多的遗传连锁不平衡现象;2013年2月WHO IMGT/HLA database(http://www.ebi.ac.uk/imgt/hla/,Release3.11.0)公布的HLA-DPA1和HLA-DPB1等位基因分别为36个和159个;但是,HLA-DPA1*0103/DPB1*0401(DP401)和HLA-DPA1*0103/DPB1*0402(DP402)两个基因型分布在20-60%的全世界人群中,DP401和DP402共享相似的抗原决定簇(Castelli等2002,JUNBAO等2005,Sidney等2010);且研究显示,HLA-DP4可以呈递来自流感病毒、HIV病毒、肿瘤细胞的多种多肽到CD4细胞(Castelli等2002,JUNBAO等2005,Cohen等2006,Sidney等2010)。
目前报道的HLA-DP人源化转基因小鼠整合人DP基因组片段最大为32kb。基于现有技术的基础与现状,本申请的发明人拟建立外源转基因的表达水平接近或达到原有的生理水平的动物模型,方便从动物整体水平研究DP基因的功能,提供一种嵌合人HLA-DP基因组区域的人源化转基因小鼠模型的构建方法。
发明内容
本发明的目的在于,基于现有技术的基础与现状,为建立外源转基因的表达水平接近或达到原有的生理水平的动物模型,方便从动物整体水平研究DP基因的功能,提供了一种嵌合人HLA-DP基因组区域的人源化转基因小鼠模型的构建方法。
本发明提出了使用BAC克隆进行原核显微注射,获得整合大片段DP基因组区域的HLA-DP人源化转基因小鼠的一种方法。本发明提供了一种高表达人HLA-DPA1和HLA-DPB1的转基因动物模型的构建方法。
本发明是通过以下技术方案实现的:
(1)BAC克隆的鉴定:BAC克隆CH501-138A21(GenBank:AL645931.7),含有全长为124899bp的HLA-DPA1/DPB1基因组区域,包含DOA、HLA-DPA1*0103、HLA-DPB1*0401全长基因序列;
常规碱裂解方法快速抽提BAC DNA,使用特异性引物常规PCR扩增基因序列,测序鉴定BAC克隆;使用的鉴定引物序列如下:P1:5’-TGTTGCTCCTTCTTCTTCCCC-3’P2:5’-TGGAATAGAGGATGCCAGGAG--3’,扩增1067bp的HLA-DPA1片段;P3:5’-CTCCTCTTCCCATCCTG-3’P4:5’-TCATCCACTTTCCTCCC-3’,扩增1478bp的HLA-DOA片段;P5:5’-GAAGGAAGGAAGGAAGGAAGG-3’P6:5’-GAAGAAAGATGGGGTTTGGAC-3’,扩增1272bp的HLA-P6片段;P7:5’-GAGGATTAGATGAGAGTGGCG-3’P8:5’-ATGAATCCCCAACCCAAAGTC-3’,扩增445bp的HLA-DPB1 ex2片段;P9:5’-CAGGAGCCACAGGAGTAT-3’P10:5’-AGCATTAACAGCACATAGGT-3’扩增588bp的Hotspot DPA1片段;P11:5’-CCATTCTCCATCTTCTCCTT-3’,P12:5’-CCTCCTCTGCTGTCCTAA-3’,扩增701bp的HLA-DPA2片段;
(2)脉冲场凝胶电泳鉴定BAC克隆完整性,
常规碱裂解方法快速抽提BAC DNA,使用限制性内切酶MluI和XhoI分别单酶切BAC克隆。Mlu I酶切产生2个酶切片段,9071bp和115828bp,XhoI酶切产生5个酶切片段,9834bp、24619bp、55093bp、8588bp和26765bp;脉冲场凝胶电泳参数:0.5%×TBE,14℃,1.0%低熔点凝胶,6v/cm,15h,120℃,Switch.Time 10s-80s,linear;
(3)纯化BAC克隆,
使用Nucleobond column(BAC 100kit)方法提纯显微注射用BAC-DNA,使用TE溶解BAC-DNA,4℃保存,使用前进行脉冲场凝胶电泳,确定BAC-DNA的完整性;注射前缓冲液(注射缓冲液:5mM Tris-HCl,pH 7.4;0.2mM EDTA,100mM NaCl)加入Spermidine和Spermine,以便能保存完整的BAC-DNA;
(4)使用原核显微注射的方法将步骤(3)中环状的BAC克隆引入小鼠的受精卵;
(5)将(4)中的受精卵移植到假孕的小鼠的输卵管;
(6)产生的小鼠基因组中整合有DOA、HLA-DPA1、HLA-DPB1基因序列;
(7)对步骤(6)获得的转基因动物使用常规PCR法进行鉴定,鉴定引物如上P1-P8;
(8)获得的阳性转基因小鼠同野生型小鼠杂交育种,获得F2代转基因小鼠;
(9)小鼠为野生型小鼠C57BL/6;
(10)使用Real-time PCR鉴定外源整合基因的拷贝数,以及DPA1和DPB1在主要组织中的基因表达水平。使用抗体进行免疫荧光染色,共聚焦显微镜观察DPA1和DPB1在脾脏上的表达分布。
在本发明建立的HLA-DP人源化转基因小鼠模型中,HLA-DP基因组DNA序列来自BAC克隆CH501-138A21,含有完整的DOA-DPA1基因以及DPB1基因3’端20kb。
本发明中通过原核显微注射的方法将完整的BAC克隆导入到野生型小鼠受精卵的原核中,待小鼠出生后用PCR鉴定人HLA-DP基因组片段是否整合入其基因组,从而获得转基因动物。
本发明的另一个目的是提供高水平表达人HLA-DPA1*0103/DPB1*0401(DP401)的转基因小鼠模型在筛选治疗传染性疾病和肿瘤的多肽疫苗中的应用,转基因小鼠模型中外源基因高表达是因整合了HLA-DP基因组区域所致。
本发明的转基因小鼠模型不仅可用于体内水平研究HLA-DP基因的表达调控等生物学功能,同时可用在评价和/或筛选能引起人HLA-DP4限制性免疫反应的物质。
本发明的有益之处是:HLA-DP4基因型分布在20-60%的世界人群中,HLA基因的不同表达对免疫应答反应进行调控,HLA-DPA1/DPB1基因表达调控方式存在差异,HLA-DP基因表达调控元件尚不清楚,将较完整HLA-DP基因组区域导入野生型小鼠体内,可获得接近HLA-DP生理水平表达的人源化转基因小鼠模型,因此筛选整合不同HLA-DP基因组区域的转基因小鼠,可在体内水平研究HLA-DP基因的表达调控机制;本发明提供的模型可有效筛选能引起人HLA-DP4限制性免疫反应的物质。
附图说明
图1,BAC克隆CH501-138A21序列构成及鉴定引物的位置。
图2脉冲场凝胶电泳鉴定MHCII类分子相关BAC克隆,CH501-138A21含有HLA-DP基因座全长,其中,2-7#泳道分别为CH501-254F23(147kb)、CH502-22I16(187kb)、CH17-440H14(207kb)、CH501-138A21(124kb)、CH502.213L12(160kb)和CH501-254F23(147kb)BAC质粒;8-9#泳道为Xho I酶切CH501-254F23产生4个片段,10#泳道为Sal I酶切CH501-254F23;11-12#泳道分别为Mlu I和Sac II酶切CH502-22I16;13-14#泳道分别为Xho I和Not I酶切CH17-440H14;15-16#泳道分别为Mlu I和Xho I酶切CH501-138A21;17-18#泳道分别为Xho I和SacII酶切CH502-213L12;19-21#泳道为空白组;1#泳道为Lambda PFGMarker。
图3,HLA-DP人源化转基因小鼠PCR鉴定图。
图4,HLA-DP鼠转基因基因拷贝数检测:
图5,实时定量PCR检测DPA1基因在HLA-DP人源化转基因小鼠血细胞中表达。
图6实时定量PCR检测DPA1基因在人源化转基因小鼠脾脏中表达。
图7实时定量PCR检测DPA1基因在人源化转基因小鼠肺脏中表达。
图8实时定量PCR检测DPB1基因在人源化转基因小鼠血细胞中表达。
图9实时定量PCR检测DPB1基因在人源化转基因小鼠脾脏中表达。
图10实时定量PCR检测DPB1基因在人源化转基因小鼠肺脏中表达。
图11RT-PCR检测HLA-DPA1/DPB1/DPA1-V2V3在入源化转基因小鼠DP3-3/3-9/5-2/5-4/8-3/在肺脏中表达。
图12RT-PCR检测HLA-DPA1/DPB1/DPA1-V2V3在人源化转基因小鼠DP3-3/3-9/5-2/5-4/8-3/在脾脏中表达。
图13RT-PCR检测HLA-DPA1/DPB1/DPA1-V2V3在人源化转基因小鼠DP3-3/3-9/5-2/5-4/8-3/在血细胞中表达
图14免疫组织化学染色检测人HLA-DPA1基因在人源化转基因小鼠DP3-3品系肾脏中组织学分布,其中,
A,HLA-DPA1抗体在野生型小鼠肾小球及肾小管无交叉反应;B,HLA-DPA1抗体在野生型小鼠肾小管无交叉反应;C,DP人源化转基因小鼠肾小球不存在外源HLA-DPA1转基因表达,肾小管表达广泛;D,DP人源化转基因小鼠中外源HLA-DPA1在远端肾小管及集合管的不同区段存在表达。
具体实施方式
下面结合实施例对本发明作进一步的说明。
下述实施例中所涉及的仪器、试剂、材料等,若无特别说明,均为现有技术中已有的常规仪器、试剂、材料等,可通过正规商业途径获得。下述实施例中所涉及的实验方法,检测方法等,若无特别说明,均为现有技术中已有的常规实验方法,检测方法等。
本发明结合附图和实施例作进一步的说明。
实施例1 BAC克隆的鉴定及纯化
将筛选到的含有目的基因的BAC克隆CH501-138A21的DH10B穿刺菌通过划线接种到含有氯霉素(12.5ug/ml)的固体LB培养基上,37℃培养16小时。挑单菌落接种到5ml含有氯霉素(12.5ug/ml)的液体LB培养基中振荡培养过夜。次日,按照常规碱裂解方法提取BACDNA,溶于TE中备用。
使用特异性引物常规PCR扩增基因序列,测序鉴定BAC克隆。使用的鉴定引物序列如下:P1:5’-TGTTGCTCCTTCTTCTTCCCC-3’P2:5’-TGGAATAGAGGATGCCAGGAG--3’,扩增1067bp的HLA-DPA1片段。P3:5’-CTCCTCTTCCCATCCTG-3’P4:5’-TCATCCACTTTCCTCCC-3’,扩增1478bp的HLA-DOA片段。P5:5’-GAAGGAAGGAAGGAAGGAAGG-3’P6:5’-GAAGAAAGATGGGGTTTGGAC-3’,扩增1272bp的HLA-P6片段。P7:5’-GAGGATTAGATGAGAGTGGCG-3’P8:5’-ATGAATCCCCAACCCAAAGTC-3’,扩增445bp的HLA-DPB1 ex2片段。P9:5’-CAGGAGCCACAGGAGTAT-3’P10:5’-AGCATTAACAGCACATAGGT-3’扩增588bp的Hotspot DPA1片段P11:5’-CCATTCTCCATCTTCTCCTT-3’,P12:5’-CCTCCTCTGCTGTCCTAA-3’,扩增701bp的HLA-DPA2片段。
脉冲场凝胶电泳(CHEF-DR 2 SYSTEM,BIO-RAD)
常规碱裂解方法快速抽提BAC DNA,使用限制性内切酶MluI和XhoI分别单酶切BAC克隆。Mlu I酶切产生2个酶切片段,9071bp和115828bp。XhoI酶切产生5个酶切片段,9834bp、24619bp、55093bp、8588bp和26765bp。脉冲场凝胶电泳参数:0.5%×TBE,14℃,1.0%低熔点凝胶,6v/cm,15h,120℃,Switch.Time 10s-80s,linear。脉冲电泳结束,凝胶EB中染色,拍照。
实施例2转基因小鼠的制备和鉴定
使用质粒抽提试剂盒XXX抽提BAC克隆CH501-138A21,TE溶解,脉冲场凝胶电泳确定抽提BAC DNA完整性后,使用显微注射液(XXX)调整终浓度至2ng/ul,原核显微注射,将注射后的卵细胞移植到假孕小鼠的输卵管,小鼠妊娠后约20天后分娩。
PCR法鉴定转基因首建鼠(F0):剪取15天大的小鼠尾巴,抽提基因组DNA,使用引物(P7:5’-GAGGATTAGATGAGAGTGGCG-3’P8:5’-ATGAATCCCCAACCCAAAGTC-3’)和引物(P1:5’-TGTTGCTCCTTCTTCTTCCCC-3’P2:5’-TGGAATAGAGGATGCCAGGAG--3’)进行初筛,PCR扩增DPB1和DPA1基因,阳性小鼠可获得约445bp的HLA-DPB1 ex2片段和1067bp的HLA-DPA1片段,对初筛获得阳性小鼠,继续使用引物(P3:5’-CTCCTCTTCCCATCCTG-3’P4:5’-TCATCCACTTTCCTCCC-3’)PCR扩增DOA基因,阳性小鼠可获得约1478bp的HLA-DOA片段。
进一步研究转基因小鼠中的外源转基因的传代情况,将转基因首建鼠(F0)与野生型C57BL/6小鼠杂交产生第一代转基因小鼠(F1),F1与野生型C57BL/6小鼠杂交产生第二代转基因小鼠(F2)。为确定外源转基因整合片段的大小,对每个阳性F1代鼠均使用下列引物PCR扩增鉴定,引物为:P1:5’-TGTTGCTCCTTCTTCTTCCCC-3’P2:5’-TGGAATAGAGGATGCCAGGAG--3’,扩增1067bp的HLA-DPA1片段。P3:5’-CTCCTCTTCCCATCCTG-3’P4:5’-TCATCCACTTTCCTCCC-3’,扩增1478bp的HLA-DOA片段。P5:5’-GAAGGAAGGAAGGAAGGAAGG-3’P6:5’-GAAGAAAGATGGGGTTTGGAC-3’,扩增1272bp的HLA-P6片段。P7:5’-GAGGATTAGATGAGAGTGGCG-3’P8:5’-ATGAATCCCCAACCCAAAGTC-3’,扩增445bp的HLA-DPB1 ex2片段。P9:5’-CAGGAGCCACAGGAGTAT-3’P10:5’-AGCATTAACAGCACATAGGT-3’扩增588bp的Hotspot DPA1片段P11:5’-CCATTCTCCATCTTCTCCTT-3’,P12:5’-CCTCCTCTGCTGTCCTAA-3’,扩增701bp的HLA-DPA2片段。对于获得的F2代转基因小鼠,鉴定方法与鉴定F1代转基因鼠的方法相同。
图3显示了HLA-DP转基因阳性小鼠的鉴定结果,转基因阳性出现不同的特异性条带,而野生型或阴性小鼠无特异性扩增,以BAC克隆DNA作为阳性对照,共获得三只转基因阳性首建鼠,这些小鼠经过杂交繁育建系,建立了含有不同HLA-DP基因组片段的5个转基因小鼠系;
进一步研究转基因小鼠中的外源转基因的整合情况,使用定量PCR方法对建立的F2转基因小鼠品系外源转基因的拷贝数进行绝对定量;抽提鼠尾DNA,定量PCR引物为:P135’-GGTGTTGCTCCTTCTTCTTCC-3’P145’-AACTCCTCCAGATGCCAGAC-3’,扩增289bp的HLA-DPA1基因片段;P155’-GAAGGAAGGAAGGAAGGAAGGA-3’P16 5’-CATTCAGGAACCATCGGACTTG-3’,扩增2179bp的HLA-DPB1基因片段,PCR反应条件为:95℃预变性5min,95℃10s,60℃40s,40个循环。
实施例3 RT-PCR和定量PCR法检测外源转基因在小鼠主要组织中的表达
按试剂盒说明书,使用Easypure@RNA kit(Trans,Beijing)抽提正常和转基因小鼠血细胞、脾脏、肺脏组织中的总RNA,DNAaseI消化后,逆转录(Yeasen,shanghai)产生第一条链cDNA,使用对血细胞、脾脏、肺脏组织cDNA进行real-time PCR反应,内参GAPDH,引物为:P17 5’-GTACAGACGCATAGACCAACAG-3’P18 5’-GAACTTGTCAATGTGGCAGATG-3’,扩增300bp的HLA-DPA1 cDNA片段;P19 5’-GGAACAGCCAGAAGGACATC-3’P20 5’-CAGGAACCATCGGACTTGAAT-3’,扩增217bp的HLA-DPB1 cDNA片段,P21 5’-GCCTCAGTTCCTCATCAC-3’P225’-CCTAAGTCCTCTTCTGTTCA-3’,扩增886bp的RT-PCR-DPA1cDNA片段;P235’-GTAATGGAGACTGGACCTTC-3’P245’-GACTTCAGAGCAACTTCTTG-3’,扩增386bp的RT-PCR-DPB1 cDNA片段;;P255’-GTAGATGTATCTCTCCAGGAAG-3’P26 5’-GAAACACGGTCACCTCAG-3’,扩增462bp的RT-PCR-DPA1-V2V3 cDNA片段;PCR反应条件为:95℃预变性5min,95℃10s,60℃40s,40个循环。
实施例4转基因在小鼠脾脏中的表达
组织的固定及包埋:取材后的组织立刻投于组织固定液中,4℃固定不超过24h。固定组织PBS冲洗数遍后,在4度冰箱中依次放置于10%-15%蔗糖溶液2小时,20%蔗糖溶液2小时,最后在30%蔗糖溶液中过夜直至组织块完全沉淀于溶液底部;在组织包埋盒中加入OCT,在液氮熏蒸速冻组织;
切片及免疫荧光染色:使用Leica组织切片机(Lecia CM1950)切片固定的组织块,8um的组织切片,切片在PBS漂洗3次,每次5min;0.3%TritonX-100破膜处理15min;PBS漂洗3次,每次5min;5%山羊血清室温下封闭15min;加入一抗HLA-DPA1(1∶400)、HLA-DPB1(1∶400),4℃12-24h;取出回温,PBS漂洗3次,每次5min;加入连有荧光素的相应的二抗,室温黑暗中孵育2h;PBS漂洗3次,每次5min;DAPI染色;PBS漂洗3次,每次5min;甘油封片,激光共聚焦显微镜下观察并拍照;
免疫组织化学染色:使用Leica组织切片机(Lecia CM1950)切片固定的组织块,8um的组织切片,切片在PBS漂洗3次,每次5min;0.3%Tri tonX-100破膜处理15min;3%过氧化氢溶液室温下孵育15分钟;PBS漂洗3次,每次5min。每张切片加1滴或50ul的非免疫性动物血清封闭,室温下孵育2-4hours,甩去血清,每张切片加1滴或50ul的第一抗体(HLA-DPA1或者HLA-DPB1),室温下孵育60分钟或4℃过夜,其中,参阅每种抗体的说明书;PBS漂洗3次,每次5min,每张切片加1滴或50ul生物素标记的第二抗体,室温下孵育45分钟,PBS漂洗3次,每次5min,每张切片加1滴或50ul链霉菌抗生物素,室温下孵育45分钟,PBS漂洗3次,每次5min,每张切片加2滴或100ul新鲜配制的DAB溶液,显微镜下观察3-10分钟,阳性显色为棕色,自来水冲洗,苏木素复染,0.1%HCL分化,PBS冲洗返蓝,二甲苯透明,树胶封片。
序列表
<110> 上海市公共卫生临床中心
<120> 一种嵌合人HLA-DP基因组区域的人源化转基因小鼠模型的构建方法
<130> 20190929
<160> 12
<170> SIPOSequenceListing 1.0
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tgttgctcct tcttcttccc c 21
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tggaatagag gatgccagga g 21
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ctcctcttcc catcctg 17
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tcatccactt tcctccc 17
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gaaggaagga aggaaggaag g 21
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<211> 21
<212> DNA
<213> P6
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gaagaaagat ggggtttgga c 21
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<212> DNA
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gaggattaga tgagagtggc g 21
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<212> DNA
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atgaatcccc aacccaaagt c 21
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caggagccac aggagtat 18
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<212> DNA
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agcattaaca gcacataggt 20
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ccattctcca tcttctcctt 20
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cctcctctgc tgtcctaa 18
Claims (4)
1.一种嵌合人HLA-DP基因组区域的人源化转基因小鼠模型的构建方法,其特征在于,其包括步骤:
(1) BAC克隆的鉴定:
BAC克隆CH501-138A21 GenBank: AL645931.7,含有全长为124899bp的HLA-DPA1/DPB1基因组区域,包含DOA、HLA-DPA1*0103、HLA-DPB1*0401全长基因序列;常规碱裂解方法快速抽提BAC DNA,使用特异性引物常规PCR扩增基因序列;
脉冲场凝胶电泳鉴定BAC克隆完整性;常规碱裂解方法快速抽提BAC DNA,使用限制性内切酶MluI和XhoI分别单酶切BAC克隆;脉冲场凝胶电泳参数:0.5%×TBE,14℃,1.0%低熔点凝胶,6v/cm,15h,120℃,Switch. Time 10s-80s,linear;
(2)BAC克隆纯化:
使用 核酸提取试剂盒NucleoBond® BAC 100 kit 的核酸提取柱Nucleobond column提纯显微注射用BAC-DNA,使用TE溶解BAC-DNA,4℃保存,使用前进行脉冲场凝胶电泳,确定BAC-DNA的完整性;注射前缓冲液加入Spermidine和 Spermin,以便能保存完整的BAC-DNA;
(3)使用原核显微注射的方法将步骤(2)中环状的BAC克隆引入小鼠的受精卵;
(4)将(3)中的受精卵移植到假孕的小鼠的输卵管;
(5)产生的小鼠基因组中整合有DOA、HLA-DPA1、HLA-DPB1基因序列;
(6)对步骤(5)获得的转基因动物使用常规PCR法进行鉴定,鉴定引物如上P1-P8;
(7)获得的阳性转基因小鼠同野生型小鼠杂交育种,获得F2代转基因小鼠;使用Real-time PCR鉴定外源整合基因的拷贝数,以及DPA1和DPB1在主要组织中的基因表达水平;使用抗体进行免疫荧光染色,共聚焦显微镜观察DPA1和DPB1在脾脏上的表达分布;
所述方法中测序鉴定BAC克隆,其中,使用的鉴定引物序列为:
P1:5’-TGTTGCTCCTTCTTCTTCCCC-3’ P2: 5’-TGGAATAGAGGATGCCAGGAG--3’,扩增1067bp的HLA-DPA1片段,P3:5’-CTCCTCTTCCCATCCTG-3’ P4:5’-TCATCCACTTTCCTCCC-3’,扩增1478bp的HLA-DOA片段,P5:5’-GAAGGAAGGAAGGAAGGAAGG-3’ P6:5’-GAAGAAAGATGGGGTTTGGAC-3’, 扩增1272bp的HLA-P6片段,P7:5’-GAGGATTAGATGAGAGTGGCG-3’ P8:5’-ATGAATCCCCAACCCAAAGTC-3’,扩增445bp的HLA-DPB1 ex2片段; P9 :5’-CAGGAGCCACAGGAGTAT-3’P10 :5’-AGCATTAACAGCACATAGGT-3’扩增588bp 的Hotspot DPA1片段;P11: 5’- CCATTCTCCATCTTCTCCTT-3’, P12: 5’-CCTCCTCTGCTGTCCTAA-3’,扩增701bp的HLA-DPA2片段;
所述方法中使用限制性内切酶MluI和XhoI分别单酶切BAC克隆;其中,MluⅠ酶切产生2个酶切片段,9071bp和 115828bp;XhoI酶切产生5个酶切片段,9834bp、24619bp、55093bp、8588bp和26765bp。
2.根据权利要求1所述的构建方法,其特征在于,所述注射前缓冲液加入Spermidine和Spermine中,注射缓冲液:5 mM Tris–HCl, pH 7.4; 0.2 mM EDTA, 100 mM NaCl 。
3.根据权利要求1所述的构建方法,其特征在于:所述的转基因小鼠后代品系通过下述方法鉴定:使用引物P1和P2检测转基因品系中DPA1的序列,使用引物P5和P6检测转基因品系中DPB1的序列,根据PCR检测结果判断转基因小鼠是否整合了外源基因。
4.权利要求1所述的构建方法建立的转基因小鼠模型在用于筛选治疗传染性疾病和肿瘤的多肽疫苗中的应用,所述的转基因小鼠模型高水平表达人HLA-DPA1*0103/DPB1*0401 。
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