CN104904661A - 一种人源化小鼠 - Google Patents
一种人源化小鼠 Download PDFInfo
- Publication number
- CN104904661A CN104904661A CN201510310055.7A CN201510310055A CN104904661A CN 104904661 A CN104904661 A CN 104904661A CN 201510310055 A CN201510310055 A CN 201510310055A CN 104904661 A CN104904661 A CN 104904661A
- Authority
- CN
- China
- Prior art keywords
- mouse
- born
- same parents
- people
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000011577 humanized mouse model Methods 0.000 title abstract 5
- 101000868279 Homo sapiens Leukocyte surface antigen CD47 Proteins 0.000 claims abstract description 67
- 102100032913 Leukocyte surface antigen CD47 Human genes 0.000 claims abstract description 64
- 210000004027 cell Anatomy 0.000 claims abstract description 32
- 210000001539 phagocyte Anatomy 0.000 claims abstract description 10
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 8
- 101150084532 CD47 gene Proteins 0.000 claims abstract description 7
- 230000004927 fusion Effects 0.000 claims abstract description 7
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 69
- 241000699670 Mus sp. Species 0.000 claims description 9
- 239000012528 membrane Substances 0.000 claims description 5
- 125000000539 amino acid group Chemical group 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 230000008447 perception Effects 0.000 claims description 4
- 101100477532 Mus musculus Sirpa gene Proteins 0.000 claims description 3
- 230000006870 function Effects 0.000 abstract description 8
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 238000001727 in vivo Methods 0.000 abstract description 3
- 102000044459 human CD47 Human genes 0.000 abstract 2
- 230000003834 intracellular effect Effects 0.000 abstract 2
- 239000002609 medium Substances 0.000 description 12
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 230000014509 gene expression Effects 0.000 description 8
- 210000001744 T-lymphocyte Anatomy 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 101150036449 SIRPA gene Proteins 0.000 description 6
- 108010046722 Thrombospondin 1 Proteins 0.000 description 6
- 102000007614 Thrombospondin 1 Human genes 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 5
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 5
- 241000700605 Viruses Species 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- 108090000978 Interleukin-4 Proteins 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 2
- 241000222122 Candida albicans Species 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 101000863873 Homo sapiens Tyrosine-protein phosphatase non-receptor type substrate 1 Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100029948 Tyrosine-protein phosphatase non-receptor type substrate 1 Human genes 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 229940095731 candida albicans Drugs 0.000 description 2
- 230000003915 cell function Effects 0.000 description 2
- 230000009849 deactivation Effects 0.000 description 2
- 210000004443 dendritic cell Anatomy 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 229940041022 streptomycins Drugs 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 230000009385 viral infection Effects 0.000 description 2
- 108700031361 Brachyury Proteins 0.000 description 1
- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 101100193633 Danio rerio rag2 gene Proteins 0.000 description 1
- 101150066002 GFP gene Proteins 0.000 description 1
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 1
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108020004684 Internal Ribosome Entry Sites Proteins 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 101100059528 Mus musculus Cd47 gene Proteins 0.000 description 1
- 101100335081 Mus musculus Flt3 gene Proteins 0.000 description 1
- 101100193635 Mus musculus Rag2 gene Proteins 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 1
- 108020005091 Replication Origin Proteins 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108020000999 Viral RNA Proteins 0.000 description 1
- 108091093126 WHP Posttrascriptional Response Element Proteins 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000011316 allogeneic transplantation Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 238000009739 binding Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000007321 biological mechanism Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 210000002798 bone marrow cell Anatomy 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 108040003610 interleukin-12 receptor activity proteins Proteins 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000005259 peripheral blood Anatomy 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 230000001960 triggered effect Effects 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开一种人源化小鼠。该人源化小鼠其人源细胞上表达小鼠-人融合CD47基因如SEQ ID NO.1所示,该基因的胞外部分为小鼠CD47序列,而穿膜部分和胞内部分为人CD47序列;或者胞外部分和穿膜部分为小鼠CD47氨基酸序列,胞内部分为人CD47氨基酸序列。本发明转入该小鼠-人融合CD47基因的人源细胞,在不被小鼠吞噬细胞吞噬的同时,能使小鼠体内环境下胞外信息传递到人源细胞并被人源细胞所感知并作出应有反应,使人源化小鼠中的人源细胞正常生长发育并发挥功能。
Description
技术领域
本发明属于基因工程领域,涉及一种人源化小鼠,尤其是一种在人源细胞表达人鼠融合CD47基因进而促进人源细胞在小鼠体内正常生长发育并发挥功能的人源化小鼠。
背景技术
人类免疫学、免疫病理学等的研究发现转化为临床疗法时常受到不能直接在人体进行体内实验研究的限制。人类疾病的小鼠模型虽然能提供很多有用信息,但是由于人类生理与动物生理有显著的差别,利用动物模型得到的实验结果有时不能适用到人体上,而非人类的灵长类动物研究能够填平物种亲缘关系太远而存在的差别沟壑,但是其使用受限于其高昂的费用、缺乏近交系和伦理问题。在转化医学时代,复杂的生物学机制需要体内实验分析,因此,这就需要一种新的动物模型,该模型要能支持体内人体组织和细胞的研究。人源化小鼠刚好符合这个要求,该模型的使用能获得对很多人类感染和炎症疾病的深入研究。
1988年世上第一个成功地用重症联合免疫缺陷(SCID)小鼠建立了异基因移植小鼠模型【Mosier DE,et al.Nature 1988;335:256–9.】。目前制作免疫系统人源化小鼠是通过在免疫缺陷小鼠移植人体造血干细胞。常见的受体鼠有NOD-scid IL2rγ-/-小鼠、BALB/c背景的Rag2-/-IL2rγ-/-小鼠和C57BL/6背景的Rag2-/-IL2rγ-/-CD47-/-小鼠【Lavender KJ,et al.Blood.2013,122(25):4013-4020】。通过敲除IL2rγ、Rag2基因或Prkdc基因自然突变能导致免疫功能障碍,使B、T和NK细胞缺失,从而能进行异种移植【Shultz LD,et al.Nat Rev Immunol.2007;7:118–130;Ito M,et al.Blood 2002;100:3175–3182;Shultz LD,et al.J Immunol.2005;174:6477–6489】。
NOD遗传背景的小鼠相对缺乏吞噬细胞活性【Legrand N,et al.J Immunol 2006;176:2053–2058;Shultz LD,et al.J Immunol 1995;154:180–191】,而BALB/c和C57BL/6小鼠吞噬细胞活性较强,阻碍了人源化T、NK细胞的发育。吞噬细胞的活性能通过CD47和SIRPα分子间的相互识别并结合而被抑制【Barclay AN.2009;Curr OpinImmunol 21:47–52】。SIRPα表达在巨噬细胞和树突状细胞上,该分子能识别并结合CD47分子,而CD47分子广泛表达于几乎所有的细胞上【Kharitonenkov A,et al.Nature 1997;386:181–186;Seiffert M,et al.Blood 1999;94:3633–3643;Lindberg FP,etal.J Cell Biol 1993;123:485–496】。不表达CD47的造血细胞会很快被血液中的巨噬细胞和树突状细胞所清除【Oldenborg PA,et al.Science 2000;288:2051–2054;Blazar BR,et al.J Exp Med 2001;194:541–550】,因此CD47是一个识别自身细胞的必要标记物。由于人源细胞表达的CD47不同于小鼠CD47,小鼠SIRPα分子不能识别人CD47分子,因此人源细胞容易被小鼠吞噬细胞吞噬,从而使建立人源化小鼠难于成功。基于此,近来针对SIRPα和CD47分子进行了基因修饰来阻止小鼠细胞吞噬人源细胞,比如在人源造血干细胞转入小鼠CD47分子【Legrand N,et al.Proc Natl Acad Sci USA.2011;108(32):13224–13229】,在小鼠转入人SIRPα基因【StrowigT,et al.Proc Natl Acad Sci USA.2011;108(32):13218–13223】,敲除小鼠CD47基因【Lavender KJ,et al.Blood.2013,122(25):4013-4020】等,取得了较好的效果。
尽管在人源细胞表达小鼠CD47能阻止吞噬细胞吞噬人源细胞,但是由于CD47分子本身也是一个信号分子,它不仅能与SIRPα反应,还能与多种其他分子进行反应(如thrombospondin,integrins,其他的SIRP家族成员SIRPγ等)【van Beek EM,et al.J Immunol 2005;175:7781–7787;Sarfati M.et al.Current Drug Targets,2008;9:842-850】,因此,CD47除了作为SIRPα配体而起到识别自我组织的作用外,它还通过与其他配体结合而介导其他许多生理活动,比如影响T细胞功能等(CD47与thrombospondin-1(TSP-1)或可溶性CD47单克隆抗体结合能抑制T细胞功能【Avice MN,et al.J.Immunol.2000;165(8):4624-31;Avice MN,et al.J.Immunol.2001;167(5):2459-68;Li Z,et al.J.Immunol.2001;166(4):2427-36;Waclavicek M,et al.J.Immunol.1997;159(11):5345-54】)、影响细胞自我更新(CD47与配体TSP-1结合后通过抑制c-myc等的表达而影响细胞自我更新等【Kaur S,et al.Sci Rep.2013;3:1673】)、以及影响炎性反应(CD47与CD47单克隆抗体或L-SIRP-alpha结合抑制IL-12R的表达并下调活化的T细胞对IL-12的反应【Latour S,et al.J Immunol.2001;167(5):2547-54.】)。因此CD47不仅仅作为一个与它反应的受体的配体而存在,它本身也是一个信号分子或受体,起到传递胞外信号进细胞的作用。
在现有的制作人源化小鼠技术中,为了不使小鼠巨噬细胞和DC细胞吞噬人源细胞,只是把小鼠CD47基因表达于人源细胞上,该表达于人源细胞上的小鼠CD47分子只是起到让小鼠吞噬细胞识别人源细胞为自身细胞而不吞噬人源细胞的作用,但是由于小鼠CD47分子与人CD47分子存在差异,很多胞外信号并不能通过小鼠CD47分子被人源细胞感知并触发应有的生理反应,而本身就表达在人源细胞上的人CD47分子对小鼠CD47的配体或受体又不能识别,从而使得人源细胞在小鼠体内缺乏很多需要通过CD47传递的胞外信号的刺激,细胞外的信息不能畅达至细胞内,从而影响到人源细胞在小鼠体内生长发育的正常进行与功能的正常发挥。这一点已经被我们实验所证实,比如在表达了小鼠CD47的人源T细胞,小鼠TSP-1或可溶性CD47单克隆抗体不能抑制该人源T细胞的功能,而对于小鼠T细胞则其功能能被小鼠TSP-1或可溶性CD47单克隆抗体抑制,说明在人源细胞表达的小鼠CD47不能把胞外信息顺利传递到人源细胞内部。
发明内容
本发明的目的是为了克服现有制备人源化小鼠技术的在人源细胞表达的小鼠CD47不能顺畅地把胞外信号进传递入胞内的缺点,本发明通过基因改建提供一种人源化小鼠,该小鼠不仅能使小鼠吞噬细胞识别人源细胞为自身细胞而不再吞噬人源细胞,而且能使人源细胞在小鼠体内获得正常的胞外信息,促进其在小鼠体内进行正常的生长发育并发挥功能,从而提高人源化小鼠的应用价值。
本发明解决其技术问题所采用的技术方案是:
本发明的人源化小鼠,其人源细胞上表达小鼠-人融合CD47基因,该基因的胞外部分为小鼠CD47序列,而穿膜部分和胞内部分为人CD47序列;或者胞外部分和穿膜部分为小鼠CD47氨基酸序列,胞内部分为人CD47氨基酸序列。
本发明的人源化小鼠,人源细胞上表达的融合CD47分子,其胞外部分能被小鼠SIRPα、TSP-1及其他小鼠CD47的配体或受体所识别,胞内部分能很好地把胞外所受到的信号让人源细胞感知并作出应有的反应,达到既能使小鼠吞噬细胞识别人源细胞为自身细胞而不再吞噬,又能使人源细胞在小鼠体内环境下获得正常的胞外信息的目的。
本发明的有益效果是转入该小鼠-人融合CD47基因的人源细胞,在不被小鼠吞噬细胞吞噬的同时,能使小鼠体内环境下胞外信息传递到人源细胞并被人源细胞所感知并作出应有反应,使人源化小鼠中的人源细胞正常生长发育并发挥功能。
附图说明
图1是本发明融合CD47的载体质粒图,其中各元件如下:
元件 | 功能 |
Pcmv/5’LTR | 启动转录整个病毒RNA |
CAG | 启动转录下游基因 |
Fused CD47 | 小鼠-人融合基因 |
IRES | 内部核糖体进入位点,启动下游GFP基因翻译 |
copGFP | 绿色荧光蛋白基因,筛选基因 |
WPRE | 提高病毒转录本的稳定性 |
3’ΔLTR | 终止转录 |
Ori | Col E1中复制起始点 |
AMPr | 氨苄青霉素抗性基因 |
图2是经免疫接种灭活的白色念珠菌3天后,对照人源化小鼠(人源细胞表达小鼠CD47)和融合CD47人源化小鼠(人源细胞表达小鼠-人融合CD47)中人源CD4和白介素4双阳性细胞白介素表达强度的比较图。
具体实施方式
下面结合附图和实施例对本发明进一步说明(下面序列ID 1是本发明融合CD47序列)。
1、人-鼠CD47融合基因的构建
首先,小鼠CD47(如GENBANK accession number:AB012693)的第1至第142氨基酸残基与人CD47(如GENBANK accession number:BT006907)的第145至305氨基酸残基融合在一起,相对应的核苷酸序列为序列ID 1。根据该设计好的序列,在5’端ATG前加上三个保护核苷酸和XbaI限制性内切酶识别序列以及Kozak序列GCCACC,在终止密码子后加上BglII限制性内切酶识别序列和三个保护核苷酸。该序列DNA委托生物技术公司合成。
以上合成好的DNA经BglII和XbaI酶切,分离片段后克隆入慢病毒表达载体多克隆位点相应的酶切点间。图1为融合CD47载体质粒图。
2、慢病毒的制备
将293T细胞培养在6孔细胞培养板中的改良杜氏伊格尔培养基(Dulbecco's Modified Eagle's Medium,DMEM)完全培养基(在DMEM完全培养基中添加10%胎牛血清、100单位/ml青霉素、100μg/ml链霉素、2mM L-谷氨酰胺等)中(37℃,5﹪CO2),待细胞90﹪汇合时,用含5﹪FBS、不含抗生素的DMEM新鲜培养基替换掉老培养基,每孔加1.5ml培养基,放入37℃,5﹪CO培养箱备用。取两个1.5ml离心管,每管加入预先37℃加热的OPTI-MEM培养基250μl,然后在其中一管中加入经纯化的2.5μg上述构建好的慢病毒CD47融合蛋白表达载体质粒(或作为对照的慢病毒小鼠CD47表达载体质粒)以及VSV-G质粒和包装质粒,混匀,常温5分钟;同时在另一管中加入Lipofectamine 2000脂质体5μl,混匀,常温5分钟。把两管内容物加在一起混匀,常温放置20分钟,然后加到细胞培养孔中,轻轻摇晃均匀,然后置于37℃,5﹪CO2培养,6小时后用新鲜完全DMEM培养基(在DMEM培养基中添加了10%胎牛血清、100单位/ml青霉素、100μg/ml链霉素、2mM L-谷氨酰胺等)置换掉老培养基,再培养约30个小时后收集上清液,500g离心5分钟,取上清,-80度保存备用。
3、人CD34造血干细胞分离、病毒感染及移植
从产妇获取新鲜脐带血,按照CD34磁珠分离试剂盒(STEM CELL公司)产品使用说明进行CD34阳性细胞分离。分离后的CD34阳性细胞培养于添加有100纳克/毫升人干细胞因子(rhSCF)、100纳克/毫升Flt3配体(Flt3-L)、50纳克/毫升促血小板生成素(Tpo)的完全培养基【含有15%胎牛血清、2毫摩尔/升谷氨酰胺、0.1毫摩尔/升非必需氨基酸、1%青霉素/链霉素(100x,Gibco)的高糖改良杜氏伊格尔培养基(Dulbecco's Modified Eagle's Medium,DMEM)】,48小时后用制备好的慢病毒上清液进行病毒感染,感染方法为离心感染法,即在细胞中加好含有6微克每毫升polybrene的病毒上清液,然后在离心机上900g离心45分钟,连续两天进行两次离心感染。第二次感染后换上新鲜含有细胞因子的完全培养基,培养24小时后进行GFP阳性细胞流式细胞仪筛选,筛选出的GFP阳性细胞进行移植。移植前免疫缺陷的受体小鼠(BALB/c Rag2-/-IL2rγ-/-小鼠)经不同剂量137Cs源放射线照射或不照射,从尾静脉注入感染好的CD34阳性骨髓细胞。
4、移植后免疫应答
移植后6周测定小鼠外周血人源细胞比例,人源细胞表达小鼠CD47的人源化小鼠(对照人源化小鼠)和人源细胞表达小鼠-人融合CD47的人源化小鼠(融合CD47人源化小鼠)中人源细胞比例分别为62.9±23.6%和67.4±20.2%,两者无显著差别。
挑选人源细胞达到80%以上的人源化小鼠,接种5×105灭活的白色念珠菌来免疫小鼠。接种后3天,从小鼠取出脾脏细胞,用红细胞裂解液去除红细胞,用1%胎牛血清的PBS洗两遍,然后用FITC标记的抗CD4抗体和APC标记的抗人CD45抗体对脾脏白细胞冰上孵育30分钟,然后用含1%胎牛血清的PBS洗两遍,然后在固定-透膜剂(Cytofix/Cytoperm)中固定和透膜15分钟,用透膜冲洗液冲洗两遍,然后用PE标记的抗白介素4(anti-IL-4)抗体在冰上标记30分钟,细胞用含1%胎牛血清的PBS洗两遍,重悬于400μl的PBS中,上流式细胞仪进行检测。结果见图2,对照人源化小鼠的人源CD4和白介素4双阳性细胞表达的白介素4显著小于融合CD47人源化小鼠的人源CD4和白介素4双阳性细胞,提示后者有更好的体液免疫应答。
本发明上述融合CD47DNA序列,如SEQ ID NO.1所示,具体是:
ATGTGGCCCTTGGCGGCGGCGCTGTTGCTGGGCTCCTGCTGCTGCGGTTCAGCTCAACTACTGTTTAGTAACGTCAACTCCATAGAGTTCACTTCATGCAATGAAACTGTGGTCATCCCTTGCATCGTCCGTAATGTGGAGGCGCAAAGCACCGAAGAAATGTTTGTGAAGTGGAAGTTGAACAAATCGTATATTTTCATCTATGATGGAAATAAAAATAGCACTACTACAGATCAAAACTTTACCAGTGCAAAAATCTCAGTCTCAGACTTAATCAATGGCATTGCCTCTTTGAAAATGGATAAGCGCGATGCCATGGTGGGAAACTACACTTGCGAAGTGACAGAGTTATCCAGAGAAGGCAAAACAGTTATAGAGCTGAAAAACCGCACGGTTTCGTGGTTTTCTCCAAATGAAAAGATCCTCATTGTTATTTTCCCAATTTTTGCTATACTCCTGTTCTGGGGACAGTTTGGTATTAAAACACTTAAATATAGATCCGGTGGTATGGATGAGAAAACAATTGCTTTACTTGTTGCTGGACTAGTGATCACTGTCATTGTCATTGTTGGAGCCATTCTTTTCGTCCCAGGTGAATATTCATTAAAGAATGCTACTGGCCTTGGTTTAATTGTGACTTCTACAGGGATATTAATATTACTTCACTACTATGTGTTTAGTACAGCGATTGGATTAACCTCCTTCGTCATTGCCATATTGGTTATTCAGGTGATAGCCTATATCCTCGCTGTGGTTGGACTGAGTCTCTGTATTGCGGCGTGTATACCAATGCATGGCCCTCTTCTGATTTCAGGTTTGAGTATCTTAGCTCTAGCACAATTACTTGGACTAGTTTATATGAAATTTGTGGAATAACTGAAGTGAAGTGATGGACTCCGATTTGGAGAGTAG
上述实施例并非是对于本发明的限制,本发明并非仅限于上述实施例,只要符合本发明要求,均属于本发明的保护范围。
SEQUENCE LISTING
<110> 王彦刈
<120> 一种人源化小鼠
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 912
<212> DNA
<213> 人工合成
<400> 1
atgtggccct tggcggcggc gctgttgctg ggctcctgct gctgcggttc agctcaacta 60
ctgtttagta acgtcaactc catagagttc acttcatgca atgaaactgt ggtcatccct 120
tgcatcgtcc gtaatgtgga ggcgcaaagc accgaagaaa tgtttgtgaa gtggaagttg 180
aacaaatcgt atattttcat ctatgatgga aataaaaata gcactactac agatcaaaac 240
tttaccagtg caaaaatctc agtctcagac ttaatcaatg gcattgcctc tttgaaaatg 300
gataagcgcg atgccatggt gggaaactac acttgcgaag tgacagagtt atccagagaa 360
ggcaaaacag ttatagagct gaaaaaccgc acggtttcgt ggttttctcc aaatgaaaag 420
atcctcattg ttattttccc aatttttgct atactcctgt tctggggaca gtttggtatt 480
aaaacactta aatatagatc cggtggtatg gatgagaaaa caattgcttt acttgttgct 540
ggactagtga tcactgtcat tgtcattgtt ggagccattc ttttcgtccc aggtgaatat 600
tcattaaaga atgctactgg ccttggttta attgtgactt ctacagggat attaatatta 660
cttcactact atgtgtttag tacagcgatt ggattaacct ccttcgtcat tgccatattg 720
gttattcagg tgatagccta tatcctcgct gtggttggac tgagtctctg tattgcggcg 780
tgtataccaa tgcatggccc tcttctgatt tcaggtttga gtatcttagc tctagcacaa 840
ttacttggac tagtttatat gaaatttgtg gaataactga agtgaagtga tggactccga 900
tttggagagt ag 912
Claims (4)
1.一种人源化小鼠,其特征在于其人源细胞上表达小鼠-人融合CD47基因。
2.如权利要求1所述的一种人源化小鼠,其特征在于其人源细胞上表达的小鼠-人融合CD47分子,胞外部分为小鼠CD47氨基酸序列,穿膜部分和胞内部分为人CD47氨基酸序列;或者胞外部分和穿膜部分为小鼠CD47氨基酸序列,胞内部分为人CD47氨基酸序列。
3.如权利要求1所述的一种人源化小鼠,其特征在于其人源细胞上表达的小鼠-人融合CD47基因如SEQ ID NO.1所示,为小鼠CD47的第1至第142氨基酸残基与人CD47的第145至305氨基酸残基融合在一起。
4.如权利要求1所述的一种人源化小鼠,其特征在于人源化小鼠,人源细胞上表达的融合CD47分子,其胞外部分能被小鼠SIRPα、TSP-1及其他小鼠CD47的配体或受体所识别,胞内部分能很好地把胞外所受到的信号让人源细胞感知并作出应有的反应,达到既能使小鼠吞噬细胞识别人源细胞为自身细胞而不再吞噬,又能使人源细胞在小鼠体内环境下获得正常的胞外信息的目的。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510310055.7A CN104904661B (zh) | 2015-06-05 | 2015-06-05 | 一种人源化小鼠 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510310055.7A CN104904661B (zh) | 2015-06-05 | 2015-06-05 | 一种人源化小鼠 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104904661A true CN104904661A (zh) | 2015-09-16 |
CN104904661B CN104904661B (zh) | 2018-07-24 |
Family
ID=54074459
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510310055.7A Expired - Fee Related CN104904661B (zh) | 2015-06-05 | 2015-06-05 | 一种人源化小鼠 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104904661B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2018177440A1 (en) * | 2017-03-31 | 2018-10-04 | Beijing Biocytogen Co., Ltd | Genetically modified non-human animal with human or chimeric cd47 |
US10973212B2 (en) | 2017-03-31 | 2021-04-13 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric SIRPa |
US11071290B2 (en) | 2016-08-31 | 2021-07-27 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric CTLA-4 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007033221A2 (en) * | 2005-09-13 | 2007-03-22 | The General Hospital Corporation | Methods and compositions for inhibition of immune responses |
WO2014022853A1 (en) * | 2012-08-03 | 2014-02-06 | Sanford Applied Biosciences, L.L.C. | Complex chromosome engineering for production of human antibodies in transgenic animals |
CN103665165A (zh) * | 2013-08-28 | 2014-03-26 | 江苏匡亚生物医药科技有限公司 | 一种靶向人CD47-SIRPα信号通路的双特异性抗体及其制备方法和用途 |
CN103804495A (zh) * | 2012-11-07 | 2014-05-21 | 深圳大学 | 抗肿瘤基因工程二价类抗体及其制备方法及抗肿瘤基因工程药物 |
US20150089679A1 (en) * | 2013-09-23 | 2015-03-26 | Regeneron Pharmaceuticals, Inc. | Non-human animals having a humanized signal-regulatory protein gene |
-
2015
- 2015-06-05 CN CN201510310055.7A patent/CN104904661B/zh not_active Expired - Fee Related
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007033221A2 (en) * | 2005-09-13 | 2007-03-22 | The General Hospital Corporation | Methods and compositions for inhibition of immune responses |
WO2014022853A1 (en) * | 2012-08-03 | 2014-02-06 | Sanford Applied Biosciences, L.L.C. | Complex chromosome engineering for production of human antibodies in transgenic animals |
CN103804495A (zh) * | 2012-11-07 | 2014-05-21 | 深圳大学 | 抗肿瘤基因工程二价类抗体及其制备方法及抗肿瘤基因工程药物 |
CN103665165A (zh) * | 2013-08-28 | 2014-03-26 | 江苏匡亚生物医药科技有限公司 | 一种靶向人CD47-SIRPα信号通路的双特异性抗体及其制备方法和用途 |
US20150089679A1 (en) * | 2013-09-23 | 2015-03-26 | Regeneron Pharmaceuticals, Inc. | Non-human animals having a humanized signal-regulatory protein gene |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11071290B2 (en) | 2016-08-31 | 2021-07-27 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric CTLA-4 |
US11096383B2 (en) | 2016-08-31 | 2021-08-24 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Method of using a genetically modified mouse that expresses a humanized CTLA-4 gene |
WO2018177440A1 (en) * | 2017-03-31 | 2018-10-04 | Beijing Biocytogen Co., Ltd | Genetically modified non-human animal with human or chimeric cd47 |
US10918095B2 (en) | 2017-03-31 | 2021-02-16 | Beijing Biocytogen Co., Ltd | Genetically modified mice expressing humanized CD47 |
US10973212B2 (en) | 2017-03-31 | 2021-04-13 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified non-human animal with human or chimeric SIRPa |
US11723348B2 (en) | 2017-03-31 | 2023-08-15 | Biocytogen Pharmaceuticals (Beijing) Co., Ltd. | Genetically modified mice expressing humanized CD47 |
Also Published As
Publication number | Publication date |
---|---|
CN104904661B (zh) | 2018-07-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7068498B2 (ja) | 二重shRNAを用いて強化された免疫細胞及びそれを含む組成物 | |
ES2781073T3 (es) | Receptores de antígenos quiméricos del promotor MND | |
ES2806126T3 (es) | Receptores de antígenos quiméricos (CAR) dirigidos de forma selectiva a complejos proteicos | |
CN109715808A (zh) | 用于选择性蛋白质表达的组合物和方法 | |
UA125962C2 (uk) | Біспецифічна антигензв'язуюча молекула до ox40 та фібробласт-активуючого білка (fap) | |
CN109862912A (zh) | 携带双特异性T细胞衔接器(BiTE)的腺病毒 | |
US20180094280A1 (en) | Vector formulations | |
CN108137669A (zh) | 抗ror1嵌合抗原受体 | |
CN109097402B (zh) | 一种重组载体CAR-CD244-antiCD19的制备方法 | |
KR102617818B1 (ko) | 인간 t 세포 수용체 알파 불변 영역 유전자에 대한 특이성을 갖는 최적화된 조작된 뉴클레아제 | |
JP2022031784A (ja) | Bcmaを標的とするキメラ抗原受容体およびその製造方法と使用 | |
JP2021501604A (ja) | 操作された細胞の治療用組成物を生成するためのプロセス | |
CN105408473A (zh) | 工程化嵌合抗原受体(car)t细胞的人应用 | |
AU2014339926A1 (en) | Polyclonal gamma delta T cells for immunotherapy | |
CN111269941B (zh) | 一种基于双色荧光系统的活化car-t细胞的示踪及定量方法 | |
EP3412775A1 (en) | Mesenchymal stem cell expressing trail and cd, and use thereof | |
CA3201621A1 (en) | Genetically engineered cells and uses thereof | |
WO2010027062A1 (ja) | B細胞由来iPS細胞およびその用途 | |
CN104904661A (zh) | 一种人源化小鼠 | |
Weidner et al. | Genetic in vivo engineering of human T lymphocytes in mouse models | |
CN106255749A (zh) | 用于重组表达感兴趣多肽的新型脊椎动物细胞和方法 | |
CN114921496B (zh) | 一种具有nk细胞及adcc能力的人源化免疫系统动物模型的构建方法及其应用 | |
CN110373428A (zh) | 一种促进细胞移植和基因表达的转基因载体系统及其应用 | |
WO2022225981A2 (en) | Chimeric costimulatory receptors, chemokine receptors, and the use of same in cellular immunotherapies | |
CN111378624A (zh) | 一种靶向性抗肿瘤t细胞及其制备方法和应用 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
TA01 | Transfer of patent application right |
Effective date of registration: 20180222 Address after: Yuhang District, Hangzhou City, Zhejiang Province, 310000 West No. 1500 Building No. 2 room 716 Applicant after: Hangzhou positive Biotechnology Co.,Ltd. Address before: Room 704, unit 704, No. 13, Changning street, Xiacheng City, Zhejiang, Zhejiang Applicant before: Wang Yanyi |
|
TA01 | Transfer of patent application right | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180724 |