CN108070033A - 一种3bnc-car分子的构建及其在杀灭hiv-1感染细胞中的应用 - Google Patents
一种3bnc-car分子的构建及其在杀灭hiv-1感染细胞中的应用 Download PDFInfo
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- CN108070033A CN108070033A CN201711322015.XA CN201711322015A CN108070033A CN 108070033 A CN108070033 A CN 108070033A CN 201711322015 A CN201711322015 A CN 201711322015A CN 108070033 A CN108070033 A CN 108070033A
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Classifications
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- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1036—Retroviridae, e.g. leukemia viruses
- C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
- C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/7051—T-cell receptor (TcR)-CD3 complex
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0636—T lymphocytes
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
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- C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
- C12N2740/15041—Use of virus, viral particle or viral elements as a vector
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及感染性疾病免疫治疗技术领域,具体涉及一种3BNC‑CAR分子的构建及其在杀灭HIV‑1感染细胞中的应用。本发明通过一种能够识别感染HIV‑1病毒细胞表面的gp120的单链抗体ScFv制成嵌合型抗原受体3BNC‑CAR,将3BNC‑CAR编码基因转入质粒载体,并将转入了3BNC‑CAR编码基因的慢病毒载体转导到CD8+T淋巴细胞,所得的CAR‑T淋巴细胞在体外实验和体内实验中均发现具有显著的抑制、杀灭HIV感染细胞的活性,能够作为活性成份制备清除HIV‑1感染细胞的药物,具有良好的应用前景。
Description
技术领域
本发明涉及感染性疾病免疫治疗技术领域,具体涉及一种3BNC-CAR分子的构建及其在杀灭HIV-1感染细胞中的应用。
背景技术
艾滋病是由人类免疫缺陷病毒1型(Human Immunodeficiency Virus 1,HIV-1)感染引起的一种重大威胁人类生命安全的传染病,目前世界上艾滋病毒感染者仍有3700万。我国艾滋病毒感染者逐年增加,其总患病人数已突破百万。目前尚无有效疫苗且现有药物不能彻底治愈。
高效抗逆转录病毒治疗(HAART)可降低HIV/AIDS的发病率和病死率,显著延长了患者寿命,甚至降低了HIV的传播。但也面临着很多挑战:1)患者必须终生服药,需付出昂贵的经济代价;2)严重的毒副作用;3)耐药毒株的出现;4)更为重要的是cART不能彻底清除病毒,主要是因为药物仅对复制中的病毒有效,而对HIV在感染早期建立的潜伏性病毒“储藏库”(reservoir)是无效的。一旦抗逆转录病毒治疗中断,病毒储藏库中的整合前病毒再度激活,几乎所有患者体内病毒血症会迅速反弹。
联合抗逆转录病毒疗法(cART)可以有效的抑制病毒复制,然而由于病毒整合在被感染细胞中形成一个稳定的潜伏感染储存库,被感染者一旦停cART治疗,病毒血症即在短时间内再次爆发,这构成了治愈HIV-1感染的主要障碍。
要找到治愈艾滋病的一个重大挑战,就是如何能够重新激活潜伏的HIV从而被机体的免疫系统自发识别和清除(shock and kill)。同时,研究发现,即使在接受cART治疗的感染者体内,也出现不可逆转的免疫损伤,特别是细胞毒性T细胞(CTL)的数量减少和功能缺陷,这表明通过抗逆转录病毒治疗重建的机体免疫系统将不能有效的清除那些被激活的细胞,需要通过联合增强机体HIV特异性免疫应答的方法来增强对HIV储藏库的清除效果。因此,对HIV潜伏性病毒“储藏库”的形成及激活策略和以重建机体免疫功能的探索都是迈向治愈HIV时代极有价值的研究方向。
由于嵌合抗原受体具有高亲和力和MHC非依赖性等优点,特别是由两个CD28及4-1BB、CD3ζ偶联形成的3代CAR可增强T细胞的杀瘤能力并延长了其在体内的存活时间,从而在白血病和淋巴瘤等肿瘤免疫治疗中取得了令人鼓舞的成效。
Roberts等人尝试用CAR-T细胞治疗HIV感染,他们选取CD4序列作为单链抗体用于结合感染细胞表面的gp120,虽然具有部分杀感染细胞功能且经过多年的努力,但最终以失败而告终。其主要原因有以下几个方面:1.使用逆转录病毒载体转导效率较低,为了获得足够的可回输CAR-T细胞,过度的体外扩增导致回输后细胞死亡和CAR分子的丢失。2.CAR分子设计本身存在缺陷,其中CD4结构域可能引起转导的CTLs被HIV感染或者病毒感染细胞通过下调CD4分子的表达而逃脱CAR-T细胞的杀伤。
发明内容
本发明为了克服现有技术的上述不足,提供了一种新的能识别HIV病毒感染细胞表面gp120的单链抗体ScFv以及由此单链抗体制成的嵌合型抗原受体(CAR),称为3BNC-CAR分子。
本发明的另一目的是提供一种3BNC-CAR载体基因修饰的CD8+T淋巴细胞。
本发明的再一个目的是提供3BNC-CAR分子修饰的CD8+T淋巴细胞在制备杀灭HIV感染细胞药物方面的用途。
为了实现上述目的,本发明是通过以下技术方案实现的:
提供了一种单链抗体scFv,该单链抗体序列作为整个CAR分子的胞外结合结构域能够识别HIV病毒感染细胞表面的gp120,其氨基酸序列如SEQ ID NO.2所示。
本发明也提供了编码上述的单链抗体ScFv的基因,编码基因的核苷酸序列如SEQID NO.1所示。
本发明还提供了一种杀灭HIV-1感染细胞的嵌合型抗原受体3BNC-CAR,该嵌合型抗原受体是通过N端到C端顺次拼接信号肽、权利要求1所述的单链抗体ScFv、CD8hinge、白细胞抗原分化群分子跨膜区CD28-TM及其细胞内结构域(ICD)、4-1BB和白细胞抗原分化群3的ζ链CD3得到的,所得到的嵌合型抗原受体的氨基酸序列如SEQ ID NO.4所示。
本发明同时提供了编码上述的嵌合型抗原受体3BNC-CAR的基因,编码基因的核苷酸序列如SEQ ID NO.3所示。
一种基因修饰的CD8+T淋巴细胞,它是通过使用上述的嵌合型抗原受体3BNC-CAR分子转导至CD8+T淋巴细胞中制备得到的。
进一步地,所述基因修饰的CD8+T细胞由以下方法制备得到:
(1)从外周血分离PBMC后再用磁珠阳选获得CD8+T细胞,经anti-CD3/28磁珠(细胞与磁珠比例为1:3)激活CD8+T细胞;
(2)细胞激活12小时后,加入重组有3BNC-CAR分子的慢病毒感染;
(3)从病毒感染后第三天开始,细胞计数并根据细胞状态和增殖情况补加培养基,细胞浓度调整至0.6x106/ml,并补充IL-2100U/Ml,进一步扩增细胞直到满足回输的细胞数。
本发明同时提供了一种上述3BNC-CAR基因修饰的CD8+T淋巴细胞的用途,所述3BNC-CAR基因修饰的CD8+T淋巴细胞是应用于制备清除HIV-1感染细胞的制剂。
本发明的有益效果:(1)本发明中克服早期设计的缺陷,利用能与病毒蛋白Gp120高度特异性结合的广谱中和抗体作为ScFv,能与98%的HIV-1的病毒株结合,增加该CAR-T细胞的广谱性;(2)本发明中使用含有SIN(Self-inactivating)结构的PTK质粒来生产慢病毒载体增加安全性的同时,改造CAR分子细胞内为双刺激分子以其提高3BNC-CAR-T细胞的扩增和存活特性,增加临床有效性和安全性。(3)本发明中的能表达嵌合型抗原受体的基因修饰CD8+T淋巴细胞在体外实验和体内实验中均发现具有显著的抑制、杀灭HIV病毒的活性,能够作为活性成份制备抗HIV感染药物;(4)本发明PG9-CAR制备的CD8+T细胞表达CAR分子的能力强,且明显高于N6-CAR制备的CD8+T细胞,因此为临床上降低药物副作用和药物成本提供了依据。
附图说明
图1为本发明构建的抗HIV感染的嵌合型抗原受体的结构示意图;
图2为本发明构建的PTK-EF1α-3BNC慢病毒载体结构示意图;
图3为本发明中3BNC-CAR在被转导的CD8+T淋巴细胞中的表达水平(A)及其刺激后功能性增殖能力(B)检测;
图4为本发明中3BNC-CAR转导的CD8+T淋巴细胞体外杀灭HIV感染细胞活性检测;
图5为共培养条件下本发明中3BNC-CAR转导的CD8+T淋巴细胞抑制病毒的活性检测;
图6为本发明中3BNC-CAR转导的CD8+T淋巴细胞杀灭人源化小鼠体内的HIV感染细胞活性检测。
具体实施方式
展示一下实例来具体说明本发明的某些实施例,且不应解释为限制本发明的范围。对本发明公开的内容可以同时从材料、方法和反应条件进行改进,所有这些改进,均应落入本发明的的精神和范围之内。
实施例1:
本发明提供了一种单链抗体ScFv,其氨基酸序列如SEQ ID NO.2所示,该单链抗体ScFv是通过串联针对感染HIV病毒细胞表面gp120的抗体轻链、重链可变区而得,其编码基因的核苷酸序列如SEQ ID NO.1所示。
以上述的单链抗体ScFv为来源可以构建一种治疗HIV感染的嵌合抗原受体3BNC-CAR分子,具体的拼接方法为:顺次拼接信号肽、能识别感染HIV病毒细胞表面的gp120单链抗体ScFv、CD8hinge、白细胞抗原分化群分子跨膜区CD28-TM+ICD、4-1BB和CD3(白细胞抗原分化群分子3)的ζ链,最后得到完整的能治疗HIV的嵌合型抗原受体(CAR)分子,其氨基酸序列见SEQ ID NO.4,其结构如图1所示,编码该嵌合型抗原受体3BNC-CAR分子的基因的核苷酸序列如SEQ ID NO.3所示。
以SEQ ID NO.4所示的嵌合型抗原受体3BNC-CAR分子详细介绍本发明CAR分子的结构设计。3BNC-CAR分子的序列氮端是CAR来源的ScFv序列,可特异性识别感染HIV病毒细胞表面的gp120;CAR分子的序列碳端是以三代CAR结构为基础,包括CD8hinge、CD28TM+ICD、4-1BB和CD3ζ胞内结构域串联组成,通过CD28分子的跨膜结构域将ScFv和胞内信号分子相连。在上述的结构下各个片段能发挥如下的功能:信号肽可以将CAR分泌到胞外,CD28TM+ICD将本发明的CAR锚钉在细胞膜上;ScFv特异性识别识别感染HIV病毒细胞表面的gp120;CD3ζ为胞内信号激活序列,在ScFv结合抗原后CD3ζ激活信号,启动淋巴细胞的杀伤活性。
实施例2:CAR分子重组构建PTK-EF-1α-3BNC质粒表达载体
按照SEQ ID NO.4所示序列合成3BNC-CAR分子,将全长3BNC-CAR分子的编码基因通过基于无缝重组克隆技术插入目的表达载体(见图2)。经过多次试验,首选的质粒载体为以PTK881载体为骨架,将CMV启动子替换成EF1α启动子后改造成的PTK-EF1α载体,核苷酸序列如SEQ ID NO.5所示,最终获得了插入CAR基因并能表达CAR的重组质粒PTK-EF1α-3BNC载体。
病毒包装步骤如下:
1)取两个均装有16ml DMEM培养液的离心管,向其中一管加入960μg PEI,另一管加入320μg预混的PTK881载体质粒,漩涡震荡,室温平衡10分钟。
2)取一个10ml的移液管将混有PEI的培养基吹起来,将混有质粒的培养基一滴一滴地加入PEI中,室温温育30分钟。
3)取一个T175瓶,向其中加入3ml胎牛血清,将和PEI混合的PTK-EF1α-3BNC载体质粒加入其中,然后将多层细胞培养瓶中的培养基倒入到T175瓶,上下左右颠倒与质粒混匀,最后将T175瓶中的培养基倒回到多层细胞培养瓶中。37℃,5%CO2培养箱培养3天,收获上清。收集的上清4000rpm(3000g),30min离心去除293T细胞碎片。
4)将慢病毒液上清用0.22μm滤膜过滤后,分装到250ml的离心瓶中,于4℃,30000g离心2.5小时,离心后将离心瓶小心转移至生物安全柜,用真空泵去上清,留沉淀,加入T细胞培养基500μl/离心瓶,用枪将沉淀吹散混匀,即得到含3BNC-CAR分子的慢病毒载体,立即使用或分装后于-80℃保存。
实施例3:制备能表达嵌合型抗原受体的CD8+T细胞
步骤1:分离患者PBMC细胞
(1)采集人外周血样本60-80ml,边采集边摇晃使得外周血与抗凝剂充分混合;
(2)将外周血转入50ml离心管中,用DPBS缓冲液按1:1稀释外周血,混匀。将稀释好的血样缓慢加入室温的15ml人淋巴细胞分离液的离心管中。方法如下:用10ml移液管吸取血样,伸至分离液液面上方0.5cm处,血样自然滑落铺至分离液面上,然后轻轻加入血样,注意不要冲破液面;
(3)配平离心30min,慢升慢降;
(4)离心完成后,离心管出现明显分层由下至上:红细胞层,粒细胞层,Ficoll层、单个核细胞层和血浆层。吸取血浆层至距白膜层5mm左右处弃之。小心吸取红细胞层以上的所有液体至离心管中,PBS稀释,与细胞悬液体积比应大于1:3,混匀。
(5)离心(1600r/min)5min,PBS重悬细胞混匀,取少量细胞计数。
(6)离心300g(1200r/min)5min,上清液送检无菌检测。
步骤2:分选CD8+T细胞
(1)步骤1中的PBMC细胞,用30ml生理盐水重悬后取样计数(取样完后,补加至50ml混匀,500g、10min、18℃、升快、降快离心,去上清),计数后按每107/80μLbuffer混匀(如果上清没有去干净,建议不用加buffer),加入按每107/20μL CD8Microbeads重悬,4-8℃孵育15min。
(2)孵育完成后,使用按每107个1~2ml buffer洗细胞,500g、10min离心。
(3)用500μLbuffer重悬多达108个细胞(若细胞数目比较多,buffer使用的也会多)。
(4)将美天旎专用LS柱,置于磁力架上,使用3ml buffer冲洗LS柱后,将细胞重悬液加入到LS柱里,使之流尽。用3ml buffer洗LS柱三次,每次需要流尽。将LS柱离开磁力架,使用5ml buffer,加入到LS柱中,用活塞将标记后的细胞冲洗出来(可以冲洗两次,确保标记的细胞均可以冲洗出来)。
(5)CD8+T细胞冲洗出来后,用生理盐水重悬至30ml,取样计数,500g、10min、18℃离心,得细胞沉淀,即可用于培养。
步骤3:CD8+T细胞激活
(1)将步骤2中CD8+T细胞,计数,按密度为2x106/ml加入培养瓶中,混匀放入CO2培养箱培养2小时。
(2)取出培养瓶,轻摇,使沉降于底部的悬浮细胞浮起,移液管吸取培养基转入离心管中,少许培养基洗培养瓶将悬浮细胞全部收集,混匀计数。
(3)根据细胞计数调整细胞浓度,按1.2x106/ml的浓度接种培养瓶中(100~120mlin a T150,50~60ml in a T75,15~29ml in a T25),加CD3/CD28磁珠,按细胞与磁珠比例为1:3(加之前磁珠用培养基洗涤3次,去除保存液),加入IL-2100U/mL,混匀放入CO2培养箱培养,收集细胞。
步骤4:CD8+T细胞被转导3BNC-CAR分子制备CAR-T细胞
加入磁珠12小时后,取适量步骤3中生长状态良好的CD8+T细胞悬液,放入离心管中300g离心5分钟。弃去上清液,以1x106/ml细胞的比例加入嵌合型抗原受体(CAR)病毒载体,同时加入终浓度4μg/ml的Polybrene,混匀。将细胞悬液在37℃条件下小体积孵育。孵育4小时后补加适量T细胞完全培养基进行培养。细胞培养的第3天,细胞计数并根据细胞状态和增殖情况补加培养基,细胞浓度调整至0.6x106/ml,并补充IL-2100U/mL。细胞培养的第5天,细胞混匀转离心管中,在磁力架上去除磁珠,细胞计数并补加培养基,并补充IL-2100U/mL,细胞密度调整至0.6x106/ml继续培养。并利用流式检测SCFV的表达,同时,取部分CD8+T淋巴细胞经goat anti-human Fab antibody抗体刺激并连续传代,以确定anti-gp120CAR转导的CD8+T淋巴细胞自扩增能力,结果如图3所示。
结果表明,Anti-gp120CAR病毒转导细胞培养到五天后,有70%的CD8+T细胞表达CAR分子,当抗体goat anti-human Fab antibody特异性地与CD8+T细胞表达CAR分子结合后,能有效和剂量依赖地激活细胞的增殖,随着传代次数增加,CAR分子阳性细胞种群比例也逐渐升高。本发明的3BNC-CAR制备的CD8+T细胞表达CAR分子的能力强,且明显高于N6-CAR制备的CD8+T细胞,因此为临床上降低药物副作用和药物成本提供了依据。
实施例4:CAR-T细胞体外杀灭HIV感染细胞活性的检测
为了进一步检测3BNC-CAR的功能,我们将3BNC-CAR-T细胞与两株HIV-1感染的细胞系H9-NL4-3和H9-NDK分别进行混合培养,在U形底的96孔板中进行细胞杀伤实验。首先利用Calcein-AM标记HIV感染细胞系H9和阴性对照细胞,取100μl(含靶细胞数量104)于96孔板中,取100μl梯度稀释的CAR-T细胞加入相应的96孔板中,确保效靶比范围为5:1到10:1,每孔终体积为200μl。室温200g离心30分钟,37℃孵育2-3小时。离心取上清测定荧光并计算出裂解百分数并用于判断3BNC-CAR-T细胞对HIV感染细胞的细胞毒性,实验结果如图4所示。
结果显示,从5:1到10:1效靶比区间范围内,3BNC-CAR-T细胞以剂量依赖的方式显著杀伤两个毒株HIV感染的靶细胞系,而对对照靶细胞没有明显的杀伤效果,且杀伤HIV感染靶细胞系的能力明显优于N6-CAR-T细胞,说明3BNC-CAR-T细胞杀伤靶细胞作用是HIV-gp120特异性的。
实施例5:共培养条件下CAR-T细胞体外抑制病毒复制活性的检测
为了进一步证明3BNC-CAR-T细胞在清除野生型HIV-1感染的原代CD4+T细胞方面的有效性,利用野生型HIV-1NL4-3-EGFP和NDK-EGEP两个毒株分别感染健康人血液样本中分离的CD4+T淋巴细胞,感染后3小时换液。感染后的第8天,该细胞与3BNC-CAR改造的同源CD8+T淋巴细胞以1:4的比例混合,在24孔板中进行细胞杀伤实验。靶细胞数量为106/孔,RMPI1640完全培养基体积为500μl/孔。48小时后,通过流式细胞术检测EGFP+CD4+T淋巴细胞的比例,验证3BNC-CAR-T细胞的杀伤作用,实验结果如图5所示。
结果表明,以未经CAR分子改造的CD8+T细胞组为参照,3BNC-CAR-T细胞组能清除其中99%的HIV-1感染的细胞,表现出显著的杀伤效果,充分展示了3BNC-CAR-T细胞的特异性和高效性。
实施例6:CAR-T淋巴细胞在体内杀灭感染HIV病毒细胞的活性检测
为了进一步证明Anti-gp120CAR-T淋巴细胞能否在体内清除HIV感染的细胞,我们将带有荧光基因的NL4-3-EGFP病毒(1x106pg p24/mouse)静脉注射到人源化小鼠BLT体内,感染小鼠同时,从健康志愿者分离PBMC然后分离CD8+T细胞,转导3BNC-CAR慢病毒,体外扩增10天后,计数后用500μl PBS重悬,按1x107CD8+T/kg的剂量静脉回输。两周后,从小鼠体内收集脾脏,放入包埋剂中制备冷冻切片。制备大于20张的10μm厚度的冷冻切片,在荧光共聚焦显微镜下照相,结果如图6A所示,将相片利用Velocity 5.0软件进行量化分析,分析结果如图6B所示。同时,以收集到的部分脾脏细胞制备单细胞悬液,取5x106细胞于DNAzol来提取基因组DNA,利用Nested-QPCR定量前病毒的拷贝数来评估体内所有感染HIV的细胞数目(如图6C)。
结果表明,与未接收CAR-T细胞治疗的对照组相比,病毒蛋白表达水平和病毒基因组水平显著性分别减少99.3%和99.1%,证实了CAR-T细胞能在体内进行有效地裂解和清除HIV感染的细胞,为CAR-T细胞进行人体临床测试奠定理论基础。
序列表
<213>人工序列(Artificial Sequence)
<400>3
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cccactgctg 60
atcccccagg tccaattgtt acagtctggg gcagcggtga cgaagcccgg ggcctcagtg 120
agagtctcct gcgaggcttc tggatacaac attcgtgact actttattca ttggtggcga 180
caggccccag gacagggcct tcagtgggtg ggatggatca atcctaagac aggtcagcca 240
aacaatcctc gtcaatttca gggtagagtc agtctgactc gacacgcgtc gtgggacttt 300
gacacatttt ccttttacat ggacctgaag gcactaagat cggacgacac ggccgtttat 360
ttctgtgcgc gacagcgcag cgactattgg gatttcgacg tctggggcag tggaacccag 420
gtcactgtct cgtcagcgtc gaccaagggc ccaggcggag ggggttcagg tggaggaggc 480
tctggcggtg gcggaagcga catccagatg acccagtctc catcctccct gtctgcctct 540
gtgggagata ccgtcactat cacttgccag gcaaacggct acttaaattg gtatcaacag 600
aggcgaggga aagccccaaa actcctgatc tacgatgggt ccaaattgga aagaggggtc 660
ccatcaaggt tcagtggaag aagatggggg caagaatata atctgaccat caacaatctg 720
cagcccgaag acattgcaac atatttttgt caagtgtatg agtttgtcgt ccctgggacc 780
agactggatt tgaaacgtac ggtggctgca ccaaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgattt ttgggtgctg 960
gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 1020
ttctgggtga ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 1080
cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 1140
gcctatcgct ccaaacgggg cagaaagaaa ctcctgtatatattcaaaca accatttatg 1200
agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1260
序列表
<110> 武汉云谷生物医药科技有限公司
<120> 一种3BNC-CAR分子的构建及其在杀灭HIV-1感染细胞中的应用
<130> 2017
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 813
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cccactgctg 60
atcccccagg tccaattgtt acagtctggg gcagcggtga cgaagcccgg ggcctcagtg 120
agagtctcct gcgaggcttc tggatacaac attcgtgact actttattca ttggtggcga 180
caggccccag gacagggcct tcagtgggtg ggatggatca atcctaagac aggtcagcca 240
aacaatcctc gtcaatttca gggtagagtc agtctgactc gacacgcgtc gtgggacttt 300
gacacatttt ccttttacat ggacctgaag gcactaagat cggacgacac ggccgtttat 360
ttctgtgcgc gacagcgcag cgactattgg gatttcgacg tctggggcag tggaacccag 420
gtcactgtct cgtcagcgtc gaccaagggc ccaggcggag ggggttcagg tggaggaggc 480
tctggcggtg gcggaagcga catccagatg acccagtctc catcctccct gtctgcctct 540
gtgggagata ccgtcactat cacttgccag gcaaacggct acttaaattg gtatcaacag 600
aggcgaggga aagccccaaa actcctgatc tacgatgggt ccaaattgga aagaggggtc 660
ccatcaaggt tcagtggaag aagatggggg caagaatata atctgaccat caacaatctg 720
cagcccgaag acattgcaac atatttttgt caagtgtatg agtttgtcgt ccctgggacc 780
agactggatt tgaaacgtac ggtggctgca cca 813
<210> 2
<211> 271
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Leu Gln Ser Gly Ala Ala
20 25 30
Val Thr Lys Pro Gly Ala Ser Val Arg Val Ser Cys Glu Ala Ser Gly
35 40 45
Tyr Asn Ile Arg Asp Tyr Phe Ile His Trp Trp Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Gln Trp Val Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro
65 70 75 80
Asn Asn Pro Arg Gln Phe Gln Gly Arg Val Ser Leu Thr Arg His Ala
85 90 95
Ser Trp Asp Phe Asp Thr Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu
100 105 110
Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp
115 120 125
Tyr Trp Asp Phe Asp Val Trp Gly Ser Gly Thr Gln Val Thr Val Ser
130 135 140
Ser Ala Ser Thr Lys Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
165 170 175
Leu Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn
180 185 190
Gly Tyr Leu Asn Trp Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu
195 200 205
Leu Ile Tyr Asp Gly Ser Lys Leu Glu Arg Gly Val Pro Ser Arg Phe
210 215 220
Ser Gly Arg Arg Trp Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu
225 230 235 240
Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val
245 250 255
Val Pro Gly Thr Arg Leu Asp Leu Lys Arg Thr Val Ala Ala Pro
260 265 270
<210> 3
<211> 1617
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgctgctgc tggtgaccag cctgctgctg tgcgagctgc cccaccccgc cccactgctg 60
atcccccagg tccaattgtt acagtctggg gcagcggtga cgaagcccgg ggcctcagtg 120
agagtctcct gcgaggcttc tggatacaac attcgtgact actttattca ttggtggcga 180
caggccccag gacagggcct tcagtgggtg ggatggatca atcctaagac aggtcagcca 240
aacaatcctc gtcaatttca gggtagagtc agtctgactc gacacgcgtc gtgggacttt 300
gacacatttt ccttttacat ggacctgaag gcactaagat cggacgacac ggccgtttat 360
ttctgtgcgc gacagcgcag cgactattgg gatttcgacg tctggggcag tggaacccag 420
gtcactgtct cgtcagcgtc gaccaagggc ccaggcggag ggggttcagg tggaggaggc 480
tctggcggtg gcggaagcga catccagatg acccagtctc catcctccct gtctgcctct 540
gtgggagata ccgtcactat cacttgccag gcaaacggct acttaaattg gtatcaacag 600
aggcgaggga aagccccaaa actcctgatc tacgatgggt ccaaattgga aagaggggtc 660
ccatcaaggt tcagtggaag aagatggggg caagaatata atctgaccat caacaatctg 720
cagcccgaag acattgcaac atatttttgt caagtgtatg agtttgtcgt ccctgggacc 780
agactggatt tgaaacgtac ggtggctgca ccaaccacga cgccagcgcc gcgaccacca 840
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 900
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgattt ttgggtgctg 960
gtggtggttg gtggagtcct ggcttgctat agcttgctag taacagtggc ctttattatt 1020
ttctgggtga ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 1080
cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 1140
gcctatcgct ccaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1200
agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1260
gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtaccag 1320
cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1380
ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1440
caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1500
gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1560
acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 1617
<210> 4
<211> 538
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gln Val Gln Leu Leu Gln Ser Gly Ala Ala
20 25 30
Val Thr Lys Pro Gly Ala Ser Val Arg Val Ser Cys Glu Ala Ser Gly
35 40 45
Tyr Asn Ile Arg Asp Tyr Phe Ile His Trp Trp Arg Gln Ala Pro Gly
50 55 60
Gln Gly Leu Gln Trp Val Gly Trp Ile Asn Pro Lys Thr Gly Gln Pro
65 70 75 80
Asn Asn Pro Arg Gln Phe Gln Gly Arg Val Ser Leu Thr Arg His Ala
85 90 95
Ser Trp Asp Phe Asp Thr Phe Ser Phe Tyr Met Asp Leu Lys Ala Leu
100 105 110
Arg Ser Asp Asp Thr Ala Val Tyr Phe Cys Ala Arg Gln Arg Ser Asp
115 120 125
Tyr Trp Asp Phe Asp Val Trp Gly Ser Gly Thr Gln Val Thr Val Ser
130 135 140
Ser Ala Ser Thr Lys Gly Pro Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
165 170 175
Leu Ser Ala Ser Val Gly Asp Thr Val Thr Ile Thr Cys Gln Ala Asn
180 185 190
Gly Tyr Leu Asn Trp Tyr Gln Gln Arg Arg Gly Lys Ala Pro Lys Leu
195 200 205
Leu Ile Tyr Asp Gly Ser Lys Leu Glu Arg Gly Val Pro Ser Arg Phe
210 215 220
Ser Gly Arg Arg Trp Gly Gln Glu Tyr Asn Leu Thr Ile Asn Asn Leu
225 230 235 240
Gln Pro Glu Asp Ile Ala Thr Tyr Phe Cys Gln Val Tyr Glu Phe Val
245 250 255
Val Pro Gly Thr Arg Leu Asp Leu Lys Arg Thr Val Ala Ala Pro Thr
260 265 270
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
275 280 285
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
290 295 300
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Phe Trp Val Leu
305 310 315 320
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
325 330 335
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
340 345 350
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
355 360 365
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
370 375 380
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
385 390 395 400
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
405 410 415
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
420 425 430
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
435 440 445
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
450 455 460
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
465 470 475 480
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
485 490 495
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
500 505 510
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
515 520 525
Ala Leu His Met Gln Ala Leu Pro Pro Arg
530 535
<210> 5
<211> 9251
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagcgcgtt ttgcctgtac tgggtctctc 840
tggttagacc agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag 900
cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct 960
ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc 1020
cgaacaggga cctgaaagcg aaagggaaac cagagctctc tcgacgcagg actcggcttg 1080
ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga 1140
ctagcggagg ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa 1200
ttagatcgcg atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa 1260
aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag 1320
aaacatcaga aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat 1380
cagaagaact tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga 1440
tagagataaa agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta 1500
agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac 1560
aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca 1620
cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct 1680
ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcctc aatgacgctg 1740
acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg 1800
gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag 1860
gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt 1920
tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa 1980
tctctggaac agatctggaa tcacacgacc tggatggagt gggacagaga aattaacaat 2040
tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa 2100
caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat 2160
tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata 2220
gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt 2280
cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt 2340
ggagagagag acagagacag atccattcga ttagtgaacg gatcttccat cgaattcctg 2400
cagcccgggg gatctaaaag aaaagggggg attggggggt acagtgcagg ggaaagaata 2460
gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat tacaaaaatt 2520
caaaattttc gggtttatta cagggacagc agagatccag tttatcgatg agtaattcat 2580
acaaaaggac tcgcccctgc cttggggaat cccagggacc gtcgttaaac tcccactaac 2640
gtagaaccca gagatcgctg cgttcccgcc ccctcacccg cccgctctcg tcatcactga 2700
ggtggagaag agcatgcgtg aggctccggt gcccgtcagt gggcagagcg cacatcgccc 2760
acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg 2820
cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg 2880
ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc 2940
gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat 3000
ggcccttgcg tgccttgaat tacttccacg cccctggctg cagtacgtga ttcttgatcc 3060
cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag gagccccttc 3120
gcctcgtgct tgagttgagg cctggcttgg gcgctggggc cgccgcgtgc gaatctggtg 3180
gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa atttttgatg 3240
acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc aagatctgca 3300
cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt cccagcgcac 3360
atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg ggtagtctca 3420
agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc cgccctgggc 3480
ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc ttcccggccc 3540
tgctgcaggg agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg gtgagtcacc 3600
cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact ccacggagta 3660
ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt cgtctttagg 3720
ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg agactgaagt 3780
taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg agtttggatc 3840
ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat ttcaggtgtc 3900
gtgaggatct atttccggtg aattcgccac cacgcgtctg gaacaatcaa cctctggatt 3960
acaaaatttg tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg 4020
gatacgctgc tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct 4080
cctccttgta taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc 4140
aacgtggcgt ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca 4200
ccacctgtca gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac 4260
tcatcgccgc ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt 4320
ccgtggtgtt gtcggggaag ctgacgtcct ttccatggct gctcgcctgt gttgccacct 4380
ggattctgcg cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc 4440
cttcccgcgg cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga 4500
cgagtcggat ctccctttgg gccgcctccc cgcatcgata ccgtcgagac ctggaaaaac 4560
atggagcaat cacaagtagc aacacagcag ctaccaatgc tgcttgtgcc tggctagaag 4620
cacaagagga ggaggaggtg ggttttccag tcacacctca ggtaccttta agaccaatga 4680
cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 4740
taattcactc ccaacgaaga caagatatcc ttgatctgtg gatctaccac acacaaggct 4800
acttccctga ttggcagaac tacacaccag ggccagggat cagatatcca ctgacctttg 4860
gatggtgcta caagctagta ccagttgagc aagagaaggt agaagaagcc aatgaaggag 4920
agaacacccg cttgttacac cctgtgagcc tgcatgggat ggatgacccg gagagagaag 4980
tattagagtg gaggtttgac agccgcctag catttcatca catggcccga gagctgcatc 5040
cggactgtac tgggtctctc tggttagacc agatctgagc ctgggagctc tctggctaac 5100
tagggaaccc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg 5160
cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga 5220
aaatctctag cagggcccgt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc 5280
cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc 5340
actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct 5400
attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg 5460
catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct 5520
agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg 5580
cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct 5640
tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg catcccttta 5700
gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt 5760
tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg 5820
ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat 5880
tcttttgatt tataagggat tttggggatt tcggcctatt ggttaaaaaa tgagctgatt 5940
taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt 6000
ccccaggctc cccaggcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc 6060
aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat 6120
tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt 6180
tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc 6240
gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt 6300
tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca cgtgttgaca 6360
attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg aactaaacca 6420
tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg 6480
agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg 6540
tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca 6600
acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg 6660
tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc 6720
cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg 6780
aggagcagga ctgacacgtg ctacgagatt tcgattccac cgccgccttc tatgaaaggt 6840
tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc ggggatctca 6900
tgctggagtt cttcgcccac cccaacttgt ttattgcagc ttataatggt tacaaataaa 6960
gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt 7020
tgtccaaact catcaatgta tcttatcatg tctgtatacc gtcgacctct agctagagct 7080
tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac 7140
acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac 7200
tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc 7260
tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg 7320
cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc 7380
actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt 7440
gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc 7500
ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa 7560
acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc 7620
ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg 7680
cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc 7740
tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc 7800
gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca 7860
ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact 7920
acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg 7980
gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt 8040
ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct 8100
tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga 8160
gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa 8220
tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac 8280
ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga 8340
taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc 8400
cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca 8460
gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta 8520
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg 8580
tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc 8640
gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg 8700
ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt 8760
ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt 8820
cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata 8880
ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc 8940
gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac 9000
ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa 9060
ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct 9120
tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat 9180
ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc 9240
cacctgacgt c 9251
Claims (7)
1.一种单链抗体scFv,其特征在于:该单链抗体序列作为整个CAR分子的胞外结合结构域能够识别HIV病毒感染细胞表面的gp120,其氨基酸序列如SEQ ID NO.2所示。
2.编码权利要求1所述的单链抗体ScFv的基因,其特征在于:其核苷酸序列如SEQ IDNO.1所示。
3.一种杀灭HIV-1感染细胞的嵌合型抗原受体3BNC-CAR,其特征在于:该嵌合型抗原受体是通过N端到C端顺次拼接信号肽、权利要求1所述的单链抗体ScFv、CD8hinge、白细胞抗原分化群分子跨膜区CD28-TM及其细胞内结构域(ICD)、4-1BB和白细胞抗原分化群3的ζ链CD3得到的,所得到的嵌合型抗原受体的氨基酸序列如SEQ ID NO.4所示。
4.编码权利要求3所述的嵌合型抗原受体3BNC-CAR的基因,其特征在于:其核苷酸序列如SEQ ID NO.3所示。
5.一种基因修饰的CD8+T淋巴细胞,其特征在于:所述基因修饰的CD8+T淋巴细胞是使用权利要求3所述的嵌合型抗原受体3BNC-CAR分子转导至CD8+T淋巴细胞中制备得到的。
6.根据权利要求5所述的基因修饰的CD8+T细胞,其特征在于:由以下方法制备得到:
(1)从外周血分离PBMC后再用磁珠阳选获得CD8+T细胞,经anti-CD3/28磁珠(细胞与磁珠比例为1:3)激活CD8+T细胞;
(2)细胞激活12小时后,加入重组有3BNC-CAR分子的慢病毒感染;
(3)从病毒感染后第三天开始,细胞计数并根据细胞状态和增殖情况补加培养基,细胞浓度调整至0.6x106/ml,并补充IL-2100U/Ml,进一步扩增细胞直到满足回输的细胞数。
7.权利要求5所述的基因修饰的CD8+T淋巴细胞的用途,其特征在于:所述基因修饰的CD8+T淋巴细胞应用于制备清除HIV-1感染细胞的制剂。
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