CN114752615A - 过表达atp5if1基因的靶向cd19的car-t细胞及其应用 - Google Patents
过表达atp5if1基因的靶向cd19的car-t细胞及其应用 Download PDFInfo
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Abstract
本发明属于细胞免疫治疗技术范畴,具体涉及一种过表达ATP5IF1基因的靶向CD19的CAR‑T细胞及其应用。具体的说,一方面,涉及一种融合基因,包括ATP5IF1基因和靶向CD19的嵌合抗原受体的编码基因,该融合基因的核苷酸序列如SEQ ID NO.1第2491位‑4338位所示。本发明还涉及一种慢病毒表达载体、一种重组慢病毒、一种过表达ATP5IF1基因的靶向CD19的CAR‑T细胞,经体内外实验表明,过表达ATP5IF1的靶向CD19的CAR‑T细胞(即CD19‑IF1‑CAR‑T细胞)能够特异性杀伤肿瘤细胞,增强抗B细胞恶性淋巴瘤疗效,明显延长荷瘤小鼠的生存期。
Description
技术领域
本发明属于细胞免疫治疗技术范畴,具体涉及过表达ATP5IF1基因的靶向CD19的CAR-T细胞及其应用。
背景技术
基于输注遗传重定向的自体T细胞的过继免疫疗法已被证明可用于治疗血液系统恶性肿瘤和实体瘤。因此,已经出现了多种基于T细胞受体(TCR)或嵌合抗原受体(CAR)赋予T细胞所需抗原受体的功能性获得策略。其中使用CAR已显示出有效的作用,可增强针对癌症的免疫反应。尽管CAR-T细胞疗法可对疾病清除产生明显影响,但仍面临毒副作用强、难以维持长期缓解等难题。
CD8+T细胞亚群在抵抗肿瘤的保护性免疫中起关键作用。在慢性感染或癌症的情况下,CD8+T细胞暴露于持久性抗原和/或炎症信号。这种过多的信号通常会导致CD8+T细胞功能逐渐衰减,这种状态即为T细胞耗竭。耗竭的T细胞表现出效应功能(细胞因子产生和杀伤功能)的逐渐丧失、多种抑制性受体(如PD-1、LAG3和TIM3)的表达增加等特征。为了提高过继性T细胞免疫治疗(包括CAR-T细胞治疗)的疗效,人们尝试了各种方法来调节T细胞分化和记忆形成。从终末耗尽的T细胞转化为预先耗尽的T细胞或更年轻的早期记忆T细胞的策略对于癌症免疫治疗非常重要,而通过调控T细胞的代谢重编程将有可能获得这种效果。研究发现,记忆性T细胞具有提高的氧化磷酸化水平,从而增强其在能量缺乏环境下的存活能力,特别是在肿瘤微环境中,由于葡萄糖和其他营养物质的缺乏,T细胞会遭受能量缺乏。与糖酵解代谢通路相比,氧化磷酸化循环能更高效的为T细胞提供能量(ATP),以支持细胞生存和功能。肿瘤的耗竭蛋白(PD-L1)会加剧能量短缺。代谢重编程是一种通过维持能量稳态来克服T细胞能量短缺的潜在策略。然而,该策略由于缺乏代谢重编程中的分子靶点而受到限制。
ATP5IF1作为线粒体中F1Fo-ATP合酶(ATP合酶,复合体V)的抑制蛋白,在能量底物富集的环境中,通过磷酸化ADP以牺牲线粒体膜电位来发挥催化产生ATP的作用。ATP合酶正常情况下产能,而在能量底物缺乏的条件下水解ATP,通过维持线粒体膜电位来保护细胞免于凋亡。ATP合酶的两种活性都受到ATP5IF1的调控,因此ATP5IF1似乎在维持线粒体的正常结构和功能中至关重要。近期研究表明,ATP5IF1在维持线粒体嵴的数量方面有着重要作用,与细胞的能量代谢调控密切相关。在T细胞的不同亚型中,ATP5IF1的表达水平存在差异,调控其表达量可影响T细胞的分化,可能有助于维持T细胞在某种分化阶段,从而更多的发挥其相应的生物学效用。
CAR-T细胞疗法目前在治疗血液系统恶性肿瘤中所面临的最大的问题之一就是CAR-T细胞在体内难以长期存活,导致其疗效难以长期维持,存在复发的风险。现阶段已显示出CAR-T细胞在治疗靶向CD19的恶性血液瘤中的良好疗效,并有4种针对CD19靶点的CAR-T药物获得FDA批准上市,用来治疗ALL白血病、DLBCL淋巴瘤和MCL套细胞淋巴瘤。但是CAR-T细胞输注时由于患者体内T细胞的大量扩增以及T细胞杀伤肿瘤过程中会分泌大量细胞因子,可能导致细胞因子释放综合症(Cytokine Release Syndrome,CRS)的出现,具体表现为发热、缺氧以及伴随着血清中某些细胞因子水平显着升高,如干扰素-γ(IFN-γ)等。此外,T细胞在抗肿瘤过程中会出现耗竭,难以维持长效。
发明内容
本发明的目的在于解决现有技术靶向CD19的CAR-T细胞药物现存在的问题,提供一种过表达ATP5IF1的靶向CD19的CAR-T细胞及其应用。
第一方面,本发明提供一种融合基因,包括ATP5IF1基因和靶向CD19的嵌合抗原受体的编码基因,该融合基因的核苷酸序列如SEQ ID NO.1第2491位-4338位所示。
第二方面,本发明提供一种慢病毒表达载体,该载体的核苷酸序列如SEQ ID NO.1所示。
第三方面,本发明提供一种重组慢病毒,通过以下制备方法得到:将所述慢病毒表达载体、Gag/Pol载体、Rev载体和VSV-G载体混合后转染至293T细胞中,转染后6h更换为DMEM完全培养基培养,收集转染24h和48h的细胞上清液,离心后取上清并用0.45μm滤头过滤,将滤液高速离心并弃去上清,保留沉淀,即得所述重组慢病毒。
第四方面,本发明提供一种过表达ATP5IF1基因的靶向CD19的CAR-T细胞,所述CAR-T细胞为转染所述重组慢病毒的T淋巴细胞。
第五方面,本发明提供所述的CAR-T细胞在制备治疗和/或辅助治疗恶性肿瘤的药物的应用。
进一步的,所述恶性肿瘤包括血液系统恶性肿瘤。
更进一步的,所述血液系统恶性肿瘤包括CD19阳性的B细胞淋巴瘤。
本发明具有如下有益效果:
1)本发明选用CD19为靶点,通过过表达ATP5IF1基因调控T细胞能量代谢,从而使得CAR-T细胞疗法更高效、更持久。通过在CAR-T结构中增加ATP5IF1片段,使得CAR-T细胞在表达CAR外,共表达影响线粒体能量代谢的ATP5IF1基因,过表达此基因可调节线粒体的代谢通路减弱T细胞耗竭,增加记忆型T细胞的比率,提高CAR-T细胞抗肿瘤疗法的持久性。令申请人惊奇的是,CD19-CAR-T细胞过表达ATP5IF1基因后,实验发现T细胞耗竭情况减缓,TCM和TSCM增加,荷瘤小鼠的生存期明显延长。
2)本发明的CD19-IF1-CAR-T细胞具有显著的肿瘤杀伤活性,能够用于制备治疗恶性淋巴的药物中,尤其能够以CD19为靶抗原,用于治疗CD19高表达的恶性淋巴瘤。
附图说明
图1为靶向CD19的CAR(B)和过表达ATP5IF1的靶向CD19的CAR(A)的结构示意图;
图2为CD19-CART-ATP5IF1-copGFP(A)和CD19-CART-copGFP(B)的质粒图谱;
图3为流式细胞术检测CAR-T细胞转染阳性率;
图4为CD19-IF1-CAR-T细胞、CD19-CAR-T细胞以及未转染的T细胞对于靶细胞NALM-6Luc+的杀伤效果;
图5为ELISA检测共培养条件下IFN-γ的表达水平;
图6为流式细胞术检测共培养条件下CAR-T细胞表型状态,其中,图A为CAR-T细胞中CD8和CD4阳性亚群的比例,图B为CD8阳性T细胞亚群中TSCM、TCM、TEM细胞的比例,图C为CD4阳性T细胞亚群中TSCM、TCM、TEM细胞的比例;
图7为流式细胞术检测共培养条件下抑制性受体PD-1、TIM-3、LAG-3以及GranzymeB表达水平;其中,图A为CD8阳性T细胞亚群PD-1、TIM-3、LAG-3表达量,图B为CD4阳性T细胞亚群PD-1、TIM-3、LAG-3表达量,图C为共培养环境的T细胞中Granzyme B的表达水平。
图8为CD19-IF1-CAR-T细胞、CD19-CAR-T细胞以及未感染的T细胞治疗22天(A)和28天(B)恶性淋巴瘤(NALM-6Luc+)模型小鼠体内的CD19+癌细胞的绝对数量;
图9为经治疗的模型小鼠的生存期示意图。
具体实施方式
下面结合附图和具体实施例对本发明进行详细说明,但不应理解为本发明的限制。如未特殊说明,下述实施例中所用的技术手段为本领域技术人员所熟知的常规手段,下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:CD19-IF1-CAR-T细胞的制备
1.慢病毒表达载体的构建
(1)表达针对CD19的嵌合抗原受体基因和ATP5IF1基因的慢病毒载体的构建
本实施例的融合基因包括ATP5IF1基因和靶向CD19的嵌合抗原受体的编码基因,该融合基因的核苷酸序列如SEQ ID NO.1第2491位-4338位所示,其中,SEQ ID NO.1第3949-4020位为P2A酶切位点,第4021位-4338位为ATP5IF1基因。
嵌合抗原受体(图1B)包括顺序连接的:
a.CD8前导肽,SEQ ID NO.1第2491位-2553位;
b.抗CD19 scFv,SEQ ID NO.1第2554位-3279位;
c.CD8铰链区(SEQ ID NO.1第3280位-3414位)、CD8跨膜区(SEQ ID NO.1第3415位-3477位)和CD8胞内区(SEQ ID NO.1第3478位-3489位);
d.4-1BB,SEQ ID NO.1第3490位-3612位;
e.胞内CD3ζ,SEQ ID NO.1第3613位-3948位;
所述融合基因由上海龙钱生物科技有限公司通过常规的基因工程手段人工合成,并克隆至质粒pLenti-EF1a-MCS-copGFP(上海毅乐生物科技有限公司,货号:LESTG00)的EcoRI和BamHI多克隆位点,质粒图谱如图2A所示,该慢病毒表达载体的核苷酸序列如SEQID NO.1所示,命名为CD19-CART-ATP5IF1-copGFP。
(2)表达针对CD19的嵌合抗原受体基因的慢病毒载体的构建
由上海龙钱生物科技有限公司通过常规的基因工程手段构建,命名为CD19-CART-copGFP,其嵌合抗原受体结构如图1A所示,质粒图谱如图2B所示,CD19-CART-copGFP核苷酸序列如SEQ ID NO.2所示。
(3)使用无内毒素质粒大提试剂盒(Endo-free Plasmid Maxi Kit,TIANGEN)进行目的质粒和慢病毒包装的辅助载体质粒(Gag/Pol、Rev和VSV-G)进行大量提取,用核酸分析仪测定浓度和纯度,然后置于-80℃冰箱中保存待用。
2.重组慢病毒的制备、包装与浓缩
(1)用含10%胎牛血清的DMEM高糖培养基,于37℃、5%二氧化碳培养箱中培养293T细胞,待细胞达对数生长期,用胰酶消化并收集1×106细胞接种于新的T175培养瓶中,混匀,改用RPMI-1640完全培养基(含10%FBS,简称为R10培养液)、37℃、5%CO2培养箱中培养过夜,使其生长到70%-80%融合度时,更换32mL新鲜的R10培养液,三小时后备用。
(2)将3个包装质粒Gag/Pol、Rev和VSV-G及慢病毒表达载体按表1比例于1mL无血清无双抗的RPMI-1640完全培养基中混合均匀,室温孵育5分钟。
表1慢病毒包装体系
Gag/Pol | 18μg |
Rev | 18μg |
VSV-G | 7μg |
目的质粒 | 15μg |
总计 | 58μg |
(3)将174μg转染试剂Express In(Express In和上述4种质粒质量比为3:1)逐滴加入至2mL无血清无双抗的RPMI-1640完全培养基中混合均匀,室温孵育30分钟。将最终的Express In及质粒混合液逐滴加到293T细胞的T175培养瓶中,混匀,37℃、5%CO2培养箱中培养。收集转染24小时和48小时的293T细胞上清液。
(4)将293T细胞上清液约35ml收集于50ml离心管中,2000转,10分钟离心后取上清,使用60ml注射器连接0.45μm针头滤器将所得上清液过滤于35ml超速离心管中。然后将离心管装入转头的金属离心管,置于冰上。抽真空,28,000转,2小时低温离心后去真空,轻轻拿出离心管置于冰上。于专用超净台中打开金属离心管,吸去部分离心后的病毒上清,用镊子取出超速离心管,吸去大部分上清,剩余约4ml。用5ml移液管反复吹打剩余的4ml病毒上清约10次。分装浓缩后的慢病毒上清每管l ml,转入-80℃冰箱中进行低温保存,其中包含CD19-CART-ATP5IF1-copGFP的重组慢病毒命名为CD19 scFV-IF1-GFP,包含CD19-CART-copGFP的重组慢病毒命名为CD19 scFV-GFP。
3.人外周血T细胞的激活及感染
(1)供者人脐带血,分别加入生理盐水进行1:1稀释,混匀;
(2)使用人淋巴细胞分离液(天津灏洋生物制品有限公司)分离单个核细胞,先向50mL离心管中加入15mL淋巴细胞分离液,然后将稀释的血液沿管壁缓慢加至分离液上层,室温,2000转离心20分钟;
(3)离心后轻轻吸取单个核细胞所在的中间白膜层,转移至新的离心管中,每管中加入15mL的无菌PBS,2000转,离心8分钟;
(4)弃上清,再重复以上操作2次;将沉淀用RPMI-1640完全培养基重悬并计数,所得细胞即为单个核细胞;
(5)将分离所得的单个核细胞用CD3 T cell提取试剂盒(Human CD3 PositiveSelection Kit,Invitrogen)磁铁吸附法分选T细胞,使用PBS缓冲液重悬T细胞,1300转,离心5分钟后取上清,然后加入抗CD3(1μg/mL)和抗CD28(5μg/mL)抗体(HuCD3/CD28/CD2 Tcell Act,Stemcell)作为刺激剂,并用含有hIL-2(200U/mL)和10%FBS(Gibco)的完全RPMI-1640培养液重悬并于37℃、5%CO2培养箱中进行激活培养。
(6)次日将包装好的慢病毒CD19 scFV-IF1-GFP和CD19 scFV-GFP转染T淋巴细胞,病毒感染细胞的比例MOI=5:1。对转染后的T淋巴细胞和未转染的T淋巴细胞(UT)进行扩增培养,培养温度为37℃,CO2浓度为5%,获得细胞终产品。过继转移后三天,使用流式细胞仪分选出GFP+的2种细胞,即CD19-CAR-T细胞(转染CD19 scFV-GFP)和CD19-IF1-CAR-T细胞(转染CD19 scFV-IF1-GFP),UT细胞(未转染病毒)作为对照,如图3所示,继续培养10天后,细胞成熟待用。
实施例2:CD19-IF1-CAR-T细胞体外抗肿瘤效果测定
1.CD19-IF1-CAR-T细胞对CD19+NALM-6luc+杀伤作用:
将实施例1制备好的CD19-CAR-T细胞、CD19-IF1-CAR-T细胞和UT细胞与1×104CD19+NALM-6Luc+细胞按照不同比例(E:T=5:1、3:1、1:1、1:2)于96孔板中共培养,24h后通过检测萤火虫荧光素酶报告基因(碧云天,One-LumiTM II萤火虫荧光素酶报告基因检测试剂盒)以肿瘤细胞溶解率代表相应CAR-T细胞的杀伤活性。
结果如图4所示,通过对于细胞培养上清液中Luci的测定发现,CD19-IF1-CAR-T细胞的肿瘤细胞溶解率更高(图4),即ATP5IF1过表达的CAR-T细胞的确增强了其杀伤肿瘤细胞的作用,且在靶向CD19时表现更佳。
2.共培养体系中测定IFN-γ的表达水平:
(1)约1×104CD19-CAR-T细胞、CD19-IF1-CAR-T细胞、UT细胞分别与1×104CD19+NALM-6Luc+在含150μl RPMI-1640完全培养基的96孔平板中共培养24小时;
(2)收集细胞上清,并通过ELISA检测试剂盒(Invitrogen)检测细胞因子如IFN-γ。
图5表明,ATP5IF1过表达的CAR-T细胞在与肿瘤细胞共培养过程中释放出了更多的IFN-γ,即提示ATP5IF1过表达的CAR-T细胞可能发挥更大的抗肿瘤作用。
3.共培养体系中T细胞记忆性状态表型的测定
(1)在检测T细胞表面标志物时,将细胞悬液收集并1000转离心后弃上清液;
(2)使用PBS缓冲液洗涤细胞1次后,流式抗体染色20分钟,
(3)每管用1mL PBS缓冲液清洗细胞1次,用300μl重悬染好的细胞后上机。
4.为了进一步评估细胞功能,进行多组效靶细胞的共培养实验:
(1)将约2×105CD19-CAR-T细胞、CD19-IF1-CAR-T细胞分别与2×105CD19+NALM-6Luc+细胞于含1.5mL RPMI-1640完全培养基的24孔板中共培养24h,收集细胞并用预冷的PBS清洗2遍;
(2)为研究过表达ATP5IF1的CAR-T细胞作用后的耗竭情况,流式细胞仪(BD)检测PD1/TIM3/LAG3在不同CAR-T细胞中的表达水平。将洗涤后的细胞收集至1.5mLEP管中,使用anti-human CD3-APC-CY7(Biolegend)、anti-human CD8-Pacific Blue(Biolegend)、anti-human PD1-APC(Biolegend)、anti-human TIM3-PerCP-Cy5.5(Biolegend)和anti-human LAG3-PE(BD)抗体染色20分钟;
(3)每管加入1mL PBS洗涤,1300转,离心5分钟,弃上清;重复洗涤2次;
(4)每管使用300μL PBS将细胞重悬后上机;
(5)为进一步分析T细胞活化的受抑制情况,将CD19-IF1-CAR-T细胞和CD19-CAR-T细胞与NALM-6Luc+细胞以1:1比例共培养24h,然后用流式细胞仪检测Granzyme B的表达水平。
(6)其中,检测前需要使用4%多聚甲醛先将细胞破膜固定,然后用PE-anti-Granzyme B抗体染色20分钟,PBS洗涤2次,重悬后上机检测。
结果如图7所示,在CD4+T亚群中,过表达ATP5IF1的CAR-T细胞组的T细胞耗竭相关的细胞因子PD-1、LAG-3和TIM-3的表达水平均降低,反之,在CD8+T细胞亚群中,三者的表达水平均增高。
实施例3:CD19-IF1-CAR-T细胞体内抗肿瘤效果进行评价
取18只6周龄NCG雌鼠,每只小鼠均通过鼠尾静脉注射1.2×106NALM-6Luc+细胞,然后将其随机分为3组:UT细胞组、CD19-CAR-T细胞组和CD19-IF1-CAR-T组。一周后尾静脉注射1.2×106UT细胞、1.2×106CD19-CAR-T细胞和1.2×106CD19-IF1-CAR-T细胞分别治疗三组小鼠。期间,在第22天(图8A)和28天(图8B)从小鼠鼠尾获得静脉血并用流式细胞仪分析肿瘤细胞在小鼠体内的CD19+NALM-6Luc+癌细胞的绝对数量。50天内观察小鼠状态并记录小鼠生存期,绘制生存曲线,图9为小鼠生存期示意图。
图8-9的结果显示,注射CD19-IF1-CAR-T细胞的荷瘤小鼠体内的CD19+肿瘤细胞清除效果最佳,且小鼠生存期明显延长,长达约50天。
尽管已描述了本发明的优选实施例,但本领域内的技术人员一旦得知了基本创造性概念,则可对这些实施例作出另外的变更和修改。所以,所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
序列表
<110> 新乡医学院
<120> 过表达ATP5IF1基因的靶向CD19的CAR-T细胞及其应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9078
<212> DNA
<213> 人工序列
<400> 1
gcgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60
acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120
cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180
attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240
tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300
agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360
ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420
cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480
tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540
gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600
aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660
aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720
agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780
atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840
gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900
taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960
acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020
cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080
ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140
tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200
gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260
aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320
gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380
aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560
ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680
tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740
tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800
aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860
aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920
atcgatacta gtggatctat gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc 1980
ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt 2040
ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 2100
ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 2160
ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc 2220
ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg 2280
cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt 2340
gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc 2400
tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc 2460
tgtgaccggc gcctacgctc tagagaattc atggccttac cagtgaccgc cttgctcctg 2520
ccgctggcct tgctgctcca cgccgccagg ccggacatcc agatgacaca gactacatcc 2580
tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 2640
agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 2700
catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 2760
gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 2820
cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 2880
ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 2940
ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 3000
tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 3060
ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 3120
accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 3180
gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 3240
gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccacgacgcc agcgccgcga 3300
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 3360
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 3420
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 3480
tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 3540
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 3600
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 3660
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 3720
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 3780
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 3840
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 3900
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgctt cgaaggaagc 3960
ggagctacta acttcagcct gctgaagcag gctggagacg tggaggagaa ccctggacct 4020
atggcagtga cggcgttggc ggcgcggacg tggcttggcg tgtggggcgt gaggaccatg 4080
caagcccgag gcttcggctc ggatcagtcc gagaatgtcg accggggcgc gggctccatc 4140
cgggaagccg gtggggcctt cggaaagaga gagcaggctg aagaggaacg atatttccga 4200
gcacagagta gagaacaact ggcagctttg aaaaaacacc atgaagaaga aatcgttcat 4260
cataagaagg agattgagcg tctgcagaaa gaaattgagc gccataagca gaagatcaaa 4320
atgctaaaac atgatgatgg atccggaagc ggagagggca gaggaagtct gctaacatgc 4380
ggtgacgtcg aggagaatcc tggacctatg gagagcgacg agagcggcct gcccgccatg 4440
gagatcgagt gccgcatcac cggcaccctg aacggcgtgg agttcgagct ggtgggcggc 4500
ggagagggca cccccaagca gggccgcatg accaacaaga tgaagagcac caaaggcgcc 4560
ctgaccttca gcccctacct gctgagccac gtgatgggct acggcttcta ccacttcggc 4620
acctacccca gcggctacga gaaccccttc ctgcacgcca tcaacaacgg cggctacacc 4680
aacacccgca tcgagaagta cgaggacggc ggcgtgctgc acgtgagctt cagctaccgc 4740
tacgaggccg gccgcgtgat cggcgacttc aaggtggtgg gcaccggctt ccccgaggac 4800
agcgtgatct tcaccgacaa gatcatccgc agcaacgcca ccgtggagca cctgcacccc 4860
atgggcgata acgtgctggt gggcagcttc gcccgcacct tcagcctgcg cgacggcggc 4920
tactacagct tcgtggtgga cagccacatg cacttcaaga gcgccatcca ccccagcatc 4980
ctgcagaacg ggggccccat gttcgccttc cgccgcgtgg aggagctgca cagcaacacc 5040
gagctgggca tcgtggagta ccagcacgcc ttcaagaccc ccattgcctt cgccagatcc 5100
cgcgctcagt cgtccaattc tgccgtggac ggcaccgccg gacccggctc caccggatct 5160
cgctaagtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 5220
aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 5280
attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 5340
tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 5400
gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgg gactttcgct 5460
ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 5520
ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatc atcgtccttt 5580
ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 5640
ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 5700
cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 5760
cctggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa 5820
agaaaagggg ggactggaag ggctaattca ctcccaacga aaataagatc tgctttttgc 5880
ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg 5940
gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg 6000
tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat 6060
ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg caaagaaatg 6120
aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca aataaagcaa 6180
tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc 6240
caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct aactccgccc 6300
agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag 6360
gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctagac 6420
ttttgcagag acggcccaaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 6480
tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 6540
gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 6600
ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 6660
cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 6720
cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 6780
aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 6840
gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 6900
tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 6960
agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 7020
ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 7080
taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 7140
gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 7200
gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 7260
ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg 7320
ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 7380
gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 7440
caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 7500
taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 7560
aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 7620
tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 7680
tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 7740
gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 7800
gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 7860
aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 7920
gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 7980
ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 8040
tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 8100
atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 8160
ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 8220
ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 8280
ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 8340
atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 8400
gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 8460
tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 8520
ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 8580
acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc 8640
tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa 8700
aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg 8760
agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac 8820
tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac 8880
agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt 8940
tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 9000
gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 9060
acggccagtg ccaagctg 9078
<210> 2
<211> 8694
<212> DNA
<213> 人工序列
<400> 2
gcgcgtgtag tcttatgcaa tactcttgta gtcttgcaac atggtaacga tgagttagca 60
acatgcctta caaggagaga aaaagcaccg tgcatgccga ttggtggaag taaggtggta 120
cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca tggattggac gaaccactga 180
attgccgcat tgcagagata ttgtatttaa gtgcctagct cgatacaata aacgggtctc 240
tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta 300
agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact 360
ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtggcg 420
cccgaacagg gacctgaaag cgaaagggaa accagagctc tctcgacgca ggactcggct 480
tgctgaagcg cgcacggcaa gaggcgaggg gcggcgactg gtgagtacgc caaaaatttt 540
gactagcgga ggctagaagg agagagatgg gtgcgagagc gtcagtatta agcgggggag 600
aattagatcg cgatgggaaa aaattcggtt aaggccaggg ggaaagaaaa aatataaatt 660
aaaacatata gtatgggcaa gcagggagct agaacgattc gcagttaatc ctggcctgtt 720
agaaacatca gaaggctgta gacaaatact gggacagcta caaccatccc ttcagacagg 780
atcagaagaa cttagatcat tatataatac agtagcaacc ctctattgtg tgcatcaaag 840
gatagagata aaagacacca aggaagcttt agacaagata gaggaagagc aaaacaaaag 900
taagaccacc gcacagcaag cggccactga tcttcagacc tggaggagga gatatgaggg 960
acaattggag aagtgaatta tataaatata aagtagtaaa aattgaacca ttaggagtag 1020
cacccaccaa ggcaaagaga agagtggtgc agagagaaaa aagagcagtg ggaataggag 1080
ctttgttcct tgggttcttg ggagcagcag gaagcactat gggcgcagcc tcaatgacgc 1140
tgacggtaca ggccagacaa ttattgtctg gtatagtgca gcagcagaac aatttgctga 1200
gggctattga ggcgcaacag catctgttgc aactcacagt ctggggcatc aagcagctcc 1260
aggcaagaat cctggctgtg gaaagatacc taaaggatca acagctcctg gggatttggg 1320
gttgctctgg aaaactcatt tgcaccactg ctgtgccttg gaatgctagt tggagtaata 1380
aatctctgga acagattgga atcacacgac ctggatggag tgggacagag aaattaacaa 1440
ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag aaaagaatga 1500
acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta acataacaaa 1560
ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag gtttaagaat 1620
agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac cattatcgtt 1680
tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag aagaagaagg 1740
tggagagaga gacagagaca gatccattcg attagtgaac ggatctcgac ggtatcggtt 1800
aacttttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 1860
aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaat tcaaaatttt 1920
atcgatacta gtggatctat gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc 1980
ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt 2040
ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 2100
ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 2160
ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc 2220
ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg 2280
cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt 2340
gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc 2400
tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc 2460
tgtgaccggc gcctacgctc tagagaattc atggccttac cagtgaccgc cttgctcctg 2520
ccgctggcct tgctgctcca cgccgccagg ccggacatcc agatgacaca gactacatcc 2580
tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 2640
agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 2700
catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 2760
gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 2820
cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 2880
ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 2940
ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 3000
tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 3060
ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 3120
accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 3180
gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 3240
gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccacgacgcc agcgccgcga 3300
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 3360
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 3420
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 3480
tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 3540
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 3600
ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 3660
cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 3720
aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 3780
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 3840
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 3900
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgctt cgaaggatcc 3960
ggaagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga 4020
cctatggaga gcgacgagag cggcctgccc gccatggaga tcgagtgccg catcaccggc 4080
accctgaacg gcgtggagtt cgagctggtg ggcggcggag agggcacccc caagcagggc 4140
cgcatgacca acaagatgaa gagcaccaaa ggcgccctga ccttcagccc ctacctgctg 4200
agccacgtga tgggctacgg cttctaccac ttcggcacct accccagcgg ctacgagaac 4260
cccttcctgc acgccatcaa caacggcggc tacaccaaca cccgcatcga gaagtacgag 4320
gacggcggcg tgctgcacgt gagcttcagc taccgctacg aggccggccg cgtgatcggc 4380
gacttcaagg tggtgggcac cggcttcccc gaggacagcg tgatcttcac cgacaagatc 4440
atccgcagca acgccaccgt ggagcacctg caccccatgg gcgataacgt gctggtgggc 4500
agcttcgccc gcaccttcag cctgcgcgac ggcggctact acagcttcgt ggtggacagc 4560
cacatgcact tcaagagcgc catccacccc agcatcctgc agaacggggg ccccatgttc 4620
gccttccgcc gcgtggagga gctgcacagc aacaccgagc tgggcatcgt ggagtaccag 4680
cacgccttca agacccccat tgccttcgcc agatcccgcg ctcagtcgtc caattctgcc 4740
gtggacggca ccgccggacc cggctccacc ggatctcgct aagtcgacaa tcaacctctg 4800
gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta 4860
tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt 4920
ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc 4980
aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt 5040
gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg 5100
gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac 5160
aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc 5220
acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac 5280
cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct 5340
cagacgagtc ggatctccct ttgggccgcc tccccgcctg gtacctttaa gaccaatgac 5400
ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac tggaagggct 5460
aattcactcc caacgaaaat aagatctgct ttttgcttgt actgggtctc tctggttaga 5520
ccagatctga gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata 5580
aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta 5640
gagatccctc agaccctttt agtcagtgtg gaaaatctct agcagtagta gttcatgtca 5700
tcttattatt cagtatttat aacttgcaaa gaaatgaata tcagagagtg agaggaactt 5760
gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa 5820
agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca 5880
tgtctggctc tagctatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat 5940
ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc tgagctattc 6000
cagaagtagt gaggaggctt ttttggaggc ctagactttt gcagagacgg cccaaattcg 6060
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 6120
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 6180
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 6240
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 6300
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 6360
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 6420
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 6480
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 6540
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 6600
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 6660
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 6720
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 6780
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 6840
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 6900
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 6960
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 7020
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 7080
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 7140
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 7200
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 7260
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 7320
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 7380
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 7440
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 7500
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 7560
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 7620
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 7680
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 7740
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 7800
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 7860
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 7920
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 7980
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 8040
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 8100
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 8160
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 8220
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 8280
ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg 8340
gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg 8400
tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta 8460
ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc 8520
atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc 8580
tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta 8640
acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gctg 8694
Claims (7)
1.一种融合基因,其特征在于,包括ATP5IF1基因和靶向CD19的嵌合抗原受体的编码基因,该融合基因的核苷酸序列如SEQ ID NO.1第2491位-4338位所示。
2.一种慢病毒表达载体,其特征在于,该载体的核苷酸序列如SEQ ID NO.1所示。
3.一种重组慢病毒,其特征在于,通过以下步骤制备得到:
将权利要求2所述慢病毒表达载体、Gag/Pol载体、Rev载体和VSV-G载体混合后转染至293T细胞中,转染后6h更换为DMEM完全培养基培养,收集转染24h和48h的细胞上清液,离心后取上清并用0.45μm滤头过滤,将滤液超高速离心3小时后,弃去上清,保留底部沉淀,即得所述重组慢病毒。
4.一种过表达ATP5IF1基因的靶向CD19的CAR-T细胞,其特征在于,所述CAR-T细胞为转染权利要求3所述重组慢病毒的T淋巴细胞。
5.权利要求4所述的CAR-T细胞在制备治疗和/或辅助治疗恶性肿瘤的药物的应用。
6.根据权利要求5所述的应用,其特征在于,所述恶性肿瘤包括血液系统恶性肿瘤。
7.根据权利要求6所述的应用,其特征在于,所述血液系统恶性肿瘤包括CD19阳性的B细胞淋巴瘤。
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