CN108148862B - 一种封闭pdl1的用于抑制免疫逃脱的car-t转基因载体及其构建方法和应用 - Google Patents
一种封闭pdl1的用于抑制免疫逃脱的car-t转基因载体及其构建方法和应用 Download PDFInfo
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Abstract
本发明公开了一种封闭PDL1的用于抑制免疫逃脱的CAR‑T转基因载体,包括:含氨苄青霉素抗性基因AmpR序列(SEQ ID NO.1);原核复制子pUC Ori序列(SEQ ID NO.2);病毒复制子SV40Ori序列(SEQ ID NO.3);eWPRE增强型土拨鼠乙肝病毒转录后调控元件(SEQ ID NO.11);人EF1α启动子(SEQ ID NO.12);用于慢病毒包装的慢病毒包装顺式元件;人PDL1的人源化单链抗体PDL1scFv1(SEQ ID NO.21)、PDL1scFv2(SEQ ID NO.22)、或PDL1scFv3;(SEQ ID NO.23);IRES核糖体结合序列(SEQ ID NO.25);IL6信号肽(SEQ ID NO.26);人源抗体Fc段(SEQ ID NO.27);以及用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体。此外,本发明还公开了该载体的构建方法及其在制备抑制免疫逃脱的药物中的应用。
Description
技术领域
本发明属于医学生物领域,具体涉及一种载体,尤其涉及一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体。此外,本发明还涉及该载体的构建方法和应用。
背景技术
肿瘤免疫治疗的理论基础是免疫系统具有识别肿瘤相关抗原、调控机体攻击肿瘤细胞(高度特异性的细胞溶解)的能力。1950年代,Burnet和Thomas提出了“免疫监视”理论,认为机体中经常会出现的突变的肿瘤细胞可被免疫系统所识别而清除,为肿瘤免疫治疗奠定了理论基础[Burnet FM.Immunological aspects of malignant disease.Lancet,1967;1:1171-4]。随后,各种肿瘤免疫疗法包括细胞因子疗法、单克隆抗体疗法、过继免疫疗法、疫苗疗法等相继应用于临床。
2013年一种更先进的肿瘤免疫疗法---CAR-T疗法成功用于临床,并表现了前所未有的临床疗效。CAR-T,全称是Chimeric Antigen Receptor T-Cell Immunotherapy,嵌合抗原受体T细胞免疫疗法。CAR-T疗法在临床上最领先的有诺华的CLT019,采用CLT019治疗复发难治急性淋巴细胞白血病患者,六个月的肿瘤无进展生存率达到67%,其中最长的应答时间达到两年多。总部位于中国上海的上海优卡迪生物医药科技有限公司与医院合作,共治疗复发难治急性淋巴细胞白血病患者36例,其中完全24例,缓解比例达到66.6%。这是抗癌研究的颠覆性突破。CAR-T细胞疗法可能是最有可能治愈癌症的手段之一,并被《Science》杂志评为2013年度十大科技突破之首。
CAR-T疗法虽然效果显著,但是在治疗实体肿瘤的过程中遇到很多困难,其中一个很重要的原因就是PD1/PDL1抑制性免疫检查点(如图1A所示),它们结合传递抑制信号,抑制了T细胞的免疫活性,在免疫耐受中发挥了重要作用,同时也是促使肿瘤细胞逃逸的重要原因。
PD-1(又称CD279)是一种免疫抑制性受体,属于CD28家族成员的Ⅰ型跨膜蛋白,程序性细胞死亡分子-1受体1992年由Ishida等[Ishida Y,Agata Y,Shibahara K,etal.Induced expression of PD-1,a novel member of the immunoglobulin genesuperfamily,upon programmed cell death〔J〕.EMBO J,1992,11(11):3887-3895.]采用消减杂交方法于凋亡的T细胞杂交瘤中得到并命名。人PD-1基因位于2q37.35染色体上,编码一个约55kD的跨膜糖蛋白。PD-1在激活的T细胞、B细胞、单核细胞和树突状细胞表面广泛表达,PD-1结构上与CTLA-4有30%的同源性,胞内区存在两个酪氨酸残基,分别参与构成了N端的一个免疫受体酪氨酸抑制基序(immunoreceptor tyrosine-based inhibitorymotif,ITIM)和C端的一个免疫受体酪氨酸依赖的转换基序(immunoreceptor tyrosin-based switch motif,ITSM);胞外区则是由一个IgV样结构域组成,含有多个糖基化位点并被重度糖基化,该结构域可以与配体结合,从而发挥抑制T细胞活化的功能[李瑛,焦顺昌等.PD-1/PD-L1信号通路在肿瘤免疫逃逸中的作用及临床意义[J].Acad J Chin PLA MedSch,Jul 2015,36(7).]。
PD-L1在多数癌症组织中过量表达,包括NSCLC、黑色素瘤、乳腺癌、胶质瘤、淋巴瘤、白血病及各种泌尿系肿瘤、消化道肿瘤、生殖系肿瘤等[Intlekofer AM,ThompsonCB.At the bench:preclinical rationale for CTLA-4and PD-1blockade as cancerimmunotherapy[J].J Leukoc Biol,2013,94(1):25-39.].Parsa在鼠和人的肿瘤细胞中,发现T细胞异常分泌的IFN-γ,IFN-γ可以诱导肿瘤细胞上的PD-L1高表达[Ding H,Wu X,Wu J,et al.Delivering PD-1inhibitory signal concomitant with blocking ICOSco-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice[J].ClinImmunol,2006,118(2/3):258-267.]。PD-L1高表达,可以通过抑制RAS及PI3K/AKT信号通路,进而调控细胞周期检查点蛋白和细胞增殖相关蛋白表达,最终导致T细胞增殖的抑制[11]。Dong等体外实验和小鼠模型还发现,PD-1/PD-L1信号通路的激活可以诱导特异性CTL调亡,使CTL的细胞毒杀伤效应敏感性下降,促使肿瘤细胞发生免疫逃逸[Dong H,StromeSE,Salomao DR,et al.Tumor-associated B7-H1promotes T-cell apoptosis:apotential mechanism of immune evasion[J].Nat Med,2002,8(8):793-800.]。
目前临床上主要是是使用商品化的PD1单抗,作为免疫检查点抑制剂,来抑制肿瘤细胞的免疫逃脱。2014年9月3日,百时美施贵宝公司抗PD-1药物Opdivo(Nivolumab)在日本正式上市,在日本国内此药物目前仍然只限于黑色素瘤患者。药物被设定为每100mg,729849日元(约合人民币43000元)的价格。体重每1kg就需要2mg的使用量,因此体重50kg的人则需要100mg的使用量。并且,体重每增加10kg用药剂量需要增加20mg(150200日元,约合人民币8778元),每3周为一个疗程。价格相当昂贵,普通家庭难以承受。
因此,如何采用低成本的方法来抑制肿瘤细胞免疫逃脱的发生,不影响CAR-T治疗的疗效,成为CAR-T治疗的一个技术难题。
发明内容
本发明要解决的技术问题之一是提供一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体。首先,节约了成本,省去了购买抗体药物的昂贵费用。其次,避免了scFv基因的在体递送效率低的问题。第三,通过慢病毒转导的PDL1scFv基因,能够有效利用细胞内的蛋白质翻译体系,大量表达出相应的PDL1scFv,经过体液循环,达到良好的PDL1封闭效果,不影响CAR-T治疗的疗效。
本发明要解决的技术问题之二是提供该载体的构建方法。
本发明要解决的技术问题之三是提供该载体的应用。
为解决上述技术问题,本发明采用如下技术方案:
在本发明的一方面,提供一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体,包括:
用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列,如SEQ ID NO.1所示;
用于质粒复制的原核复制子pUC Ori序列,如SEQ ID NO.2所示;
用于增强真核细胞内的复制的病毒复制子SV40Ori序列,如SEQ ID NO.3所示;
用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件,如SEQ ID NO.11所示;
用于嵌合抗原受体基因的真核转录的人EF1α启动子,如SEQ ID NO.12所示;
用于慢病毒包装的慢病毒包装顺式元件;
人PDL1的人源化单链抗体,所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1、或者如SEQ ID NO.22所示的PDL1scFv2、或者如SEQ ID NO.23所示的PDL1scFv3;
用于共同转录表达蛋白质的IRES核糖体结合序列,如SEQ ID NO.25所示;
IL6信号肽,如SEQ ID NO.26所示;
人源抗体Fc段,如SEQ ID NO.27所示;
以及用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体。
作为本发明优选的技术方案,所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1。
所述慢病毒包装顺式元件可以采用第二代慢病毒载体或者第三代慢病毒载体,优选第三代慢病毒载体。所述第二代慢病毒载体包括:如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件。所述第三代慢病毒载体包括:如SEQID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件,以及如SEQID NO.4所示的RSV启动子。
作为本发明优选的技术方案,所述用于组成集识别、传递、启动于一体的二代CAR的嵌合抗原受体包括:如SEQ ID NO.13所示的CD8leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8Hinge嵌合受体铰链、如SEQ IDNO.18所示的CD8Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域;所述用于组成集识别、传递、启动于一体的三代CAR的嵌合抗原受体包括:如SEQ ID NO.13所示的CD8leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的OptimalLinker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8Transmembrane嵌合受体跨膜区、如SEQ IDNO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域、以及如SEQ ID NO.28所示的CD28嵌合受体共刺激因子。
作为本发明优选的技术方案,所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变,具体为:g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
在本发明的第二方面,提供一种上述封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体的构建方法,包括以下步骤:
(1)将如SEQ ID NO.1所示的含氨苄青霉素抗性基因AmpR序列、如SEQ ID NO.2所示的原核复制子pUC Ori序列、如SEQ ID NO.3所示的病毒复制子SV40Ori序列、用于慢病毒包装的慢病毒包装顺式元件、如SEQ ID NO.11所示的eWPRE增强型土拨鼠乙肝病毒转录后调控元件存储于慢病毒骨架质粒上;
(2)将如SEQ ID NO.12所示的人EF1α启动子、用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体组合成二代CAR或三代CAR设计方案,经过酶切、连接、重组反应克隆至慢病毒骨架质粒中,得到二代CAR或三代CAR设计的重组慢病毒质粒;
(3)将人PDL1的人源化单链抗体PDL1scFv1、PDL1scFv2、或PDL1scFv3,IRES核糖体结合序列、IL6信号肽以及人源抗体Fc段分别克隆至重组慢病毒质粒中,得到PDL1阻断重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3;
(4)将得到的重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞,在HEK293T/17细胞中进行基因转录表达后,包装成功重组慢病毒载体会释放到细胞培养上清中,收集包含的重组慢病毒载体的上清液;
(5)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的柱纯化方式进行纯化,分别得到重组慢病毒载体。
作为本发明优选的技术方案,步骤(3)中,由人EF1α启动子启动整个CAR基因表达;CAR蛋白定位于细胞膜表面,识别BCMA抗原,刺激T细胞增殖和细胞因子分泌,激活下游信号通路的表达;当scfv区域与BCMA抗原结合时,信号通过嵌合受体传递至细胞内,从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞一系列生物学效应;由IRES核糖体结合序列共表达PDL1scFv与Fc的融合蛋白,并在IL6信号肽的引导下分泌到细胞外,通过与PDL1的结合,阻断PD1与PDL1的结合,从而阻断PD1/PDL1的信号通路,达到抑制免疫逃脱的效果。
作为本发明优选的技术方案,步骤(5)中,所述抽滤步骤要控制上清体积在200ml~2000ml,控制真空度在-0.5MPA~-0.9MPA,防止由于堵孔带来的载体损失;所述吸附步骤要控制溶液的PH值在6~8,防止PH的变化导致载体失活;所述洗脱步骤要控制洗脱液的离子强度在0.5M~1.0M,防止离子强度的变化导致洗脱不完全或者载体失活。
在本发明的第三方面,提供上述载体在制备抑制免疫逃脱的药物中的应用。
与现有技术相比,本发明具有如下有益效果:
本发明所采用的载体骨架为第三代慢病毒载体(如图2A所示)(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开),3’SIN LTR去除了U3区域,消除了慢病毒载体自我复制的可能性,大大提高了安全性;增加了cPPT和WPRE元件,提高了转导效率和转基因的表达效率;采用RSV启动子保证了慢病毒载体包装时核心RNA的持续高效转录;采用人自身的EF1α启动子,使CAR基因能够在人体内长时间持续表达。
本发明所采用的第三代慢病毒骨架质粒(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开)采用eWPRE元件,与常规WPRE相比,能够增强初级转录产物的多聚腺苷化,增加细胞内mRNA的含量,增强转基因的表达效率。
本发明所采用的慢病毒载体柱纯化方式(如图7所示)(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开),不同于通常采用的超速离心或者高速离心的方式,半自动化操作,避免人工操作的繁琐和失误,所回收的慢病毒载体在内毒素、支原体、宿主DNA残留等指标上完全达到临床标准。
本发明所述的重组慢病毒载体系统(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开),为第三代慢病毒载体,3’SIN LTR去除了U3区域,消除了慢病毒载体自我复制的可能性,大大提高了安全性;增加了cPPT和eWPRE元件,提高了转导效率和转基因的表达效率;采用RSV启动子保证了慢病毒载体包装时核心RNA的持续高效转录;采用人自身的EF1α启动子,使CAR基因能够在人体内长时间持续表达。
本发明采用的scfv段的Linker设计(已在2016年3月17日申请的“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”发明专利中公开),能够显著提高细胞因子的分泌、CAR-T细胞的体外杀伤作用以及临床治疗效果。
本发明所采用的是针对PDL1的单链抗体封闭技术,单链抗体(single chainantibody fragment,scFv),是由抗体重链可变区和轻链可变区通过15~20个氨基酸的短肽(linker)连接而成。scFv能较好地保留其对抗原的亲和活性,并具有分子量小、穿透力强和抗原性弱等特点。
本发明所采用的人PDL1阻断单链抗体设计,能够有效在T细胞中过表达并分泌至胞外,能够有效阻断PD1与PDL1结合,阻断PD1/PDL1信号通路的传递抑制信号,在T细胞杀伤实验中,经过QPCR检测,能有效提高T细胞中IL2、TNFα、IFNγ的mRNA的转录水平,解除对T细胞活化相关基因的抑制作用,将来可以在体内阻断PD1/PDL1的信号通路,达到抑制免疫逃脱的效果,提高CAR-T细胞治疗针对实体肿瘤的疗效。
本发明中采用的scFv片段和抗体Fc段,均经过人源化改造,能有有效减少体内人抗鼠抗体(Human anti-mouse antibodies,HAMA)的产生,提高scFv的半衰期和作用效果。
本发明采用PDL1scFv的作用方式(如图1B所示)。首先,节约了成本,省去了购买抗体药物的昂贵费用。其次,避免了scFv基因的在体递送效率低的问题。第三,通过慢病毒转导的PDL1scFv基因,能够有效利用细胞内的蛋白质翻译体系,大量表达出相应的PDL1scFv,经过体液循环,达到良好的PDL1封闭效果。本发明筛选了一系列PDL1抗体的基因序列、氨基酸序列等生物信息学方面的信息,预测了PDL1scFv的重链及轻链的可变区,分析了PDL1scFv组合的二级结构及其物理化学性质,通过可溶性表达和间接ELISA法测定了PDL1scFv的亲和力常数,从中优选出3条scFv进行细胞功能水平的检测,最终确定了PDL1scFv1作为最佳选择,未来可进入临床研究阶段。本发明的重组慢病毒载体骨架可以搭载不同的治疗性基因并广泛的用于过继性细胞治疗领域,搭载PDL1scFv基因用于封闭PDL1,抑制免疫负调节信号通路,从而抑制肿瘤的免疫逃脱。本发明的慢病毒载体可以实现在人T淋巴细胞上表达BCMA嵌合抗原受体,引导并激活T淋巴细胞对BCMA阳性细胞的杀伤作用,在临床上用于治疗多发性骨髓瘤(MM)。在人T淋巴细胞内表达细胞程式死亡配体1(Programmed cell death 1ligand 1,PDL1)的scFv,有效封闭PDL1,阻断免疫负调节信号通路,临床上可用于抑制肿瘤的免疫逃脱,提高CAR-T细胞免疫治疗的疗效。
可见,本发明所述的重组慢病毒载体在给多发性骨髓瘤(MM)的CAR-T治疗提供可靠转基因保障的同时,可以阻断肿瘤的免疫逃脱机制,提高CAR-T细胞治疗的疗效,大大降低患者承担的医疗成本,解决了本领域的技术难题,且达到了预料不到的技术效果。
本发明所述的PDL1单链抗体表达框及其基因表达产物,不仅可以应用于CAR-T治疗多发性骨髓瘤(MM)中用于消除或减轻肿瘤的免疫逃脱机制,还可应用于抑制CAR-T治疗诸如胰腺癌、脑胶质瘤、骨髓瘤等等类型肿瘤时的免疫逃脱机制。
附图说明
图1A是本发明的PD1/PDL1信号通路示意图。
图1B是本发明的PDL1scFv的作用方式示意图;
图2是本发明的慢病毒载体结构示意图;其中图2A是本发明采用的第三代慢病毒载体结构示意图,图2B是第二代和第三代慢病毒载体结构比较示意图;
图3为本发明实施例1中构建本发明所述的重组慢病毒载体的构建流程图。其中,(A)图是慢病毒骨架质粒pLenti-3G Basic2的结构示意图;(B)图是pCARmm-Basic2质粒的结构示意图;(C)图是pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、pCARmm-PDL1scFv3、pCARmm-scFv0质粒的示意图;(D)图是慢病毒包装质粒pPac-GP的结构示意图;(E)图是慢病毒包装质粒pPac-R的结构示意图;(F)图是膜蛋白pEnv-G的结构示意图;
图4是本发明实施例1中慢病毒骨架质粒pLenti-3G Basic2的酶切预测及酶切琼脂糖凝胶电泳图;图4A是慢病毒骨架质粒pLenti-3G Basic2的酶切预测示意图,其中,lane1是pLenti-3G Basic2的Cla I+BamH I酶切预测,条带从上到下依次为:5854bp;lane 2是1kb DNA ladder Marker预测,条带从上到下依次为:10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;图4B是慢病毒骨架质粒pLenti-3G Basic2的酶切琼脂糖凝胶电泳图,其中,lane 1是pLenti-3G Basic2的Cla I+BamH I酶切电泳结果;lane 2是1kb DNA ladder Marker的电泳结果;
图5是本发明实施例1中重组慢病毒质粒pCARmm-Basic2的酶切预测及酶切琼脂糖凝胶电泳图;图5A是重组慢病毒质粒pCARmm-Basic2的酶切预测图,其中,lane 1是1kb DNAladder Marker,条带从上到下依次为:10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;lane 2是pCARmm-Basic2的Xba I+Xho I酶切预测,条带从上到下依次为:6839bp、1692bp;图5B是重组慢病毒质粒pCARmm-Basic2的酶切琼脂糖凝胶电泳图,其中,lane1是1kb DNA ladder Marker的电泳结果;lane2是pCARmm-Basic2的XbaI+Xho I酶切电泳结果;
图6是本发明实施例1中重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2和pCARmm-PDL1scFv3、pCARmm-scFv0的酶切预测及酶切琼脂糖凝胶电泳图;图6A是pCARmm-PDL1scFv1的酶切预测图,其中,lane1是1kb DNA ladder Marker,条带从上到下依次为:10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;lane2是pCARmm-PDL1scFv1的BsrG I酶切预测,条带从上到下依次为:9592bp、1298bp;图6B是pCARmm-PDL1scFv1的酶切琼脂糖凝胶电泳图,其中,lane1是1kb DNA ladder Marker的电泳结果,lane2是pCARmm-PDL1scFv1的BsrG I酶切电泳结果;图6C是pCARmm-PDL1scFv2的酶切预测图,其中,lane1是1kb DNA ladder Marker,条带从上到下依次为:10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;lane2是pCARmm-PDL1scFv2的Sal I酶切预测,条带从上到下依次为:8484bp、1588bp、818bp;图6D是pCARmm-PDL1scFv2的酶切琼脂糖凝胶电泳图,其中lane1是1kb DNA ladder Marker的电泳结果;lane2是pCARmm-PDL1scFv2的Sal I酶切电泳结果;图6E是pCARmm-PDL1scFv3的酶切预测图,其中,lane1是1kb DNA ladder Marker,条带从上到下依次为:10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;lane2是pCARmm-PDL1scFv3的PvuI I酶切预测:条带从上到下依次为:3354bp、2364bp、1920bp、1460bp、823bp、733bp;图6F是pCARmm-PDL1scFv3的酶切琼脂糖凝胶电泳图,其中lane1是1kb DNA ladder Marker的电泳结果,lane2是pCARmm-PDL1scFv3的Pvu I I酶切电泳结果;图6G是pCARmm-scFv0的酶切预测图,其中,lane1是1kb DNA ladder Marker,条带从上到下依次为:10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp;lane2是pCARmm-scFv0的KpnI酶切预测,条带从上到下依次为:7028bp、3626bp、895bp、347bp;图6H是pCARmm-scFv0的酶切琼脂糖凝胶电泳图,其中,lane1是1kb DNA ladder Marker的电泳结果,lane2是pCARmm-scFv0的Kpn I酶切电泳结果。
图7是本发明实施例2中离子交换色谱法纯化重组慢病毒载体的流程图;
图8是本发明实施例2中重组慢病毒载体的滴度检测结果示意图;
图9是本发明实施例2中重组慢病毒载体的不同纯化方式的支原体检测结果示意图,其中,lane1为DL2000marker,从上到下条带条带从上到下依次为:2kb、1kb、750bp、500bp、250bp、100bp;lane2为阳性对照;lane3为阴性对照;lane4为PBS;lane5为水;lane6为lvCARmm-PDL1scFv1;lane7为lvCARmm-PDL1scFv2;lane8为lvCARmm-PDL1scFv3;lane9为lvCARmm-scFv0;
图10是本发明实施例3中mRNA相对表达量的柱状图;其中,图10A是RT-QPCR结果示意图,表明CAR基因在PBMC细胞内高效转录;图10B是RT-QPCR结果示意图,表明scFv基因在PBMC细胞内高效转录;
图11是本发明实施例3中CAR蛋白表达量的WB检测图,结果表明CAR蛋白在PBMC细胞内高效表达,图11A中,M为蛋白Marker,lane1为PBMC空细胞,lane2为对照病毒MOCK,lane3为lvCARmm-PDL1scFv1,lane4为lvCARmm-PDL1scFv2,lane5为lvCARmm-PDL1scFv3,lane6为lvCARmm-scFv0;图11B是beta-actin内参条带;
图12是本发明实施例3中scFv蛋白表达量的ELISA检测结果图,结果表明scFv蛋白在PBMC细胞中高效表达;
图13是本发明实施例3中重组慢病毒载体转导的PBMC,在不同效靶比条件下共培养,24h后检测对靶细胞的杀伤情况示意图;
图14是本发明实施例3中不同效应细胞分别与靶细胞共培养条件下,24h PD1mRNA转录水平的变化情况示意图;
图15是本发明实施例3中不同效应细胞分别与靶细胞共培养条件下,24h细胞因子转录水平的变化情况示意图;其中,图15A代表RT-QPCR结果,IL2基因在各实验组PBMC细胞内mRNA转录水平;图15B代表RT-QPCR结果,TNFα基因在各实验组PBMC细胞内mRNA转录水平;图15C代表RT-QPCR结果,IFNγ基因在各实验组PBMC细胞内mRNA转录水平。
具体实施方式
下面结合具体实施例进一步阐述此发明。应理解的是,在此描述的特定实施方式通过举例的方式来表示,并不作为对本发明的限制。在不偏离本发明范围的情况下,本发明的主要特征可以用于各种实施方式。实施例1构建重组慢病毒载体
一、材料
1、慢病毒骨架质粒pLenti-3G Basic2,慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G,HEK293T/17细胞、同源重组酶由世翱(上海)生物医药科技有限公司提供;
2、引物:根据引物设计原则设计扩增DNA片段和靶位点所需的引物,该引物由上海生物公司合成,具体为:
EF1α-F:5’-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3’(SEQ ID NO.29)
EF1α-R:5’-TCACGACACCTGAAATGGAAGA-3’(SEQ ID NO.30)
CD8 leader-F:5’-GGTGTCGTGAGGATCCGCCACCATGGCCTTACCAGTGACCGC-3’
(SEQ ID NO.31)
CD8 leader-R:5’-GGTCATCTGGATGTCCGGCCTGGCGGCGTG-3’(SEQ ID NO.32)
VL-F:5’-CACGCCGCCAGGCCGGACATCCAGATGACCCAGAGCC-3’(SEQ ID NO.33)
VL-R:5’-ACGCTTGATCTCCAGTTTGGT-3’(SEQ ID NO.34)
OLC-VH-F:5’-ACTGGAGATCAAGCGTGGTGGCGGTGGCTCGGGCGGTGGTGGGTCGG
GTGGCGGCGGATCTCAGGTGCAGCTGGTCCAGAG-3’(SEQ ID NO.35)
VH-R:5’-GCTGGACACGGTCACTAGTGTG-3’(SEQ ID NO.36)
CD8 Hinge-F:5’-AGTGACCGTGTCCAGCACCACGACGCCAGCGCC-3’(SEQ ID NO.37)
CD8 Hinge-R:5’-GTAGATATCACAGGCGAAGTCCA-3’(SEQ ID NO.38)
CD8 Transmembrane-F:5’-CGCCTGTGATATCTACATCTGGGCGCCCTTGGC-3’(SEQ IDNO.39)
CD8 Transmembrane-R:5’-TCTTTCTGCCCCGTTTGCAGTAAAGGGTGATAACCAGTG-3’(SEQID NO.40)
CD137-F:5’-AAACGGGGCAGAAAGAAACTC-3’(SEQ ID NO.41)
CD137-R:5’-TGCTGAACTTCACTCTCAGTTCACATCCTCCTTCTTCTTC-3’(SEQ ID NO.42)
TCR-F:5’-AGAGTGAAGTTCAGCAGGAGCG-3’(SEQ ID NO.43)
TCR-R:5’-GGAGAGGGGCGTCGACTTAGCGAGGGGGCAGGGC-3’(SEQ ID NO.44)
IRES-F:5’-GCCCTGCCCCCTCGCTAAGCCCCTCTCCCTCCCC-3’(SEQ ID NO.45)
IRES-R:5’-CCAGGGAGAAGGCAACTGGACCGAAGGCGCTTGTGGAGAAGGAGTTC
ATGGTGGCATTATCATCGTGTTTTTCAAAGGA-3’(SEQ ID NO.46)
PDL1s1-F:5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCAGATATTGTGCTGACCCAGAG-3’(SEQ ID NO.47)
PDL1s1-R:5’-GCAGCTTTTCGGTTCGCTGCTCACGGTCACCAGGGT-3’(SEQ ID NO.48)
PDL1s2-F:5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCAGATATTCAGATGACCCAGAGC-3’(SEQ ID NO.49)
PDL1s2-R:5’-GCAGCTTTTCGGTTCGCTGCTCACGGTCACCAGGGT-3’(SEQ ID NO.50)
PDL1s3-F:5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCAGATATTGTGCTGACCCAGAGC-3’(SEQ ID NO.51)
PDL1s3-R:5’-GCAGCTTTTCGGTTCCGCGCTCGCGGTCACCAGGGT-3’(SEQ ID NO.52)
s0-F:5’-GTTGCCTTCTCCCTGGGGCTGCTCCTGGTGTTGCCTGCTGCCTTCCCTG
CCCCATTGTTCTGGATTCCTGCTTCCA-3’(SEQ ID NO.53)
s0-R:5’-GCAGCTTTTCGGTTCTGCAGAGACAGAGACCAGAGT-3’(SEQ ID NO.54)
Fc-F:5’-GAACCGAAAAGCTGCGATAAAAC-3’(SEQ ID NO.55)
Fc-R:5’-CTAGCAATCTAGAGGTTATTTGCCCGGGCTCAGGCTCA-3’(SEQ ID NO.56)
WPRE-QPCR-F:5’-CCTTTCCGGGACTTTCGCTTT-3’(SEQ ID NO.57)
WPRE-QPCR-R:5’-GCAGAATCCAGGTGGCAACA-3’(SEQ ID NO.58)
Actin-QPCR-F:5’-CATGTACGTTGCTATCCAGGC-3’(SEQ ID NO.59)
Actin-QPCR-R:5’-CTCCTTAATGTCACGCACGAT-3’(SEQ ID NO.60)
CAR-QPCR-F:5’-GACTTGTGGGGTCCTTCTCCT-3’(SEQ ID NO.61)
CAR-QPCR-R:5’-GCAGCTACAGCCATCTTCCTC-3’(SEQ ID NO.62)
PD1-QPCR-F:5’-TGCAGCTTCTCCAACACAT-3’(SEQ ID NO.63)
PD1-QPCR-R:5’-CTTGTCCGTCTGGTTGCT-3’(SEQ ID NO.64)
IL2-QPCR-F:5’-CACCAGGATGCTCACATTTAAGT-3’(SEQ ID NO.65)
IL2-QPCR-R:5’-GTCCCTGGGTCTTAAGTGAAAGT-3’(SEQ ID NO.66)
Fc-QPCR-F:5’-GACATTGGAAATGTGAACATGT-3’(SEQ ID NO.67)
Fc-QPCR-R:5’-CACAGCTGGGGTTTGGTGA-3’(SEQ ID NO.68)
TNFα-QPCR-F:5’-TCTCTAATCAGCCCTCTG-3’(SEQ ID NO.69)
TNFα-QPCR-R:5’-GGGTTTGCTACAACATGG-3’(SEQ ID NO.70)
IFNγ-QPCR-F:5’-GACTAATTATTCGGTAACTGA-3’(SEQ ID NO.71)
IFNγ-QPCR-R:5’-GATGCTCTTCGACCTCGAAACA-3’(SEQ ID NO.72)
3、SEQ ID NO.15~SEQ ID NO.72所示的DNA序列由上海捷瑞生物工程有限公司合成,并以寡核苷酸干粉或者质粒形式保存;
4、工具酶Xba I、Xho I、Pvu II、Sal I、BsrG I、BamH I、Kpn I、Cla I、T4DNA连接酶均购自NEB公司;
5、高保真酶PrimeSTAR、RN购自Takara公司;
6、0.22μm-0.8μm PES滤器购自millipore公司;
7、质粒抽提试剂盒、琼脂糖凝胶回收试剂盒均购自MN公司;
8、感受态细胞TOP10购自tiangen公司;
9、NaCl、KCl、Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2、NaOH、PEG6000均购自上海生工;
10、Opti-MEM、FBS、DMEM、1640、Pen-Srep、Hepes、购自invitrogen公司;
11、Biotinylated protein L、proteinG-HRP购自GeneScript公司;
12、辣根过氧化物酶标记的二抗、DAB工作液均购自北京中杉金桥;
13、ECL+plusTM Western blotting system购自Amersham公司;
14、DNeasy试剂盒购自上海捷瑞公司;
15、淋巴细胞分离液购自深圳达科为公司;
16、phycoerythrin(PE)-conjugated streptavidin购自BD Bioscience公司;
17、SA-HRP、TMB底物溶液、ELISA反应终止液购自上海翊圣公司;
18、支原体检测试剂盒、内毒素检测试剂盒、BCMA-K562细胞、BCMA-PDL1-K562细胞购自世翱(上海)公司;
19、LDH检测试剂盒购自promega公司。
二、重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的构建方法。
参见图3,本发明所述重组慢病毒载体的构建方法如下:
1、将人EF1α启动子、CD8leader嵌合受体信号肽、BCMA单链抗体轻链VL、OptimalLinker C、BCMA单链抗体重链VH、CD8Hinge嵌合受体铰链、CD8Transmembrane嵌合受体跨膜区、CD137嵌合受体共刺激因子、TCR嵌合受体T细胞激活域片段克隆至慢病毒骨架质粒pLenti-3G Basic2,得到重组慢病毒质粒pCARmm-Basic2,再将scFv片段与Fc片段分别连接到pCARmm-Basic2中,得到IL-6R阻断重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、pCARmm-PDL1scFv3以及对照pCARmm-scFv0。
(1)将慢病毒骨架质粒pLenti-3G Basic2使用Cla I和BamH I限制性内切酶进行双酶切,产物经过1.5%的琼脂糖凝胶电泳,确认5854bp的片段V1(如图4所示),并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
1、溶胶 | 按200μl NTI/100mg gel比例加入溶胶液,50℃水浴放置5-10分钟。 |
2、结合DNA | 11000g离心30秒,弃去滤液。 |
3、洗膜 | 加入700μl NT3,11000g离心30秒,弃去滤液。 |
4、洗膜 | 重复第三步一次 |
5、晾干 | 11000g离心1分钟,换新的收集管,室温放置1分钟。 |
6、洗脱DNA | 加入15-30μl NE,室温放置1分钟,11000g离心1分钟,收集滤液。 |
表1琼脂糖凝胶回收步骤
(2)用引物EF1α-F和EF1α-R以合成的SEQ ID NO.12为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认1208bp的片段a,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
试剂 | 体积(μl) |
H<sub>2</sub>O | 32.5 |
5×Buffer(with Mg2+) | 10 |
dNTP(各2.5mM) | 4 |
Primer1(+)(10μM) | 1 |
Primer2(-)(10μM) | 1 |
Template | 1 |
PrimeSTAR | 0.5 |
表2 50μl PCR反应体系
(3)用引物CD8leader-F和CD8leader-R以合成的SEQ ID NO.13为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认101bp的片段b,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(4)用引物VL-F和VL-R以合成的SEQ ID NO.14为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认336bp的片段c,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(5)用引物OLC-VH-F和VH-R以合成的SEQ ID NO.16为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认421bp的片段d,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(6)用引物CD8Hinge-F和CD8Hinge-R以合成的SEQ ID NO.17为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认147bp的片段e,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(7)用引物CD8Transmembrane-F和CD8Transmembrane-R以合成的SEQ ID NO.18为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认100bp的片段f,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(8)用引物CD137-F和CD137-R以合成的SEQ ID NO.19为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认142bp的片段g,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(9)用引物TCR-F和TCR-R以合成的SEQ ID NO.20为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃30sec)*35cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认355bp的片段h,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(10)将DNA片段b、c、d各1μl作为模板,使用表3中的体系,除引物外加入Eppendorf管内,PCR循环条件为:98℃3min,(98℃10sec,60℃10sec,72℃30sec)*6cycle,加入引物CD8leader-F/VH-R,(98℃10sec,60℃10sec,72℃40sec)*24cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认814bp的片段i,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
试剂 | 体积(μl) |
H<sub>2</sub>O | 33.5-1*模板数 |
5×Buffer(with Mg2+) | 10 |
dNTP(各2.5mM) | 4 |
Primer1(+)(10μM) | 1 |
Primer2(-)(10μM) | 1 |
Template | 1*模板数 |
PrimeSTAR | 0.5 |
表3 50μl重叠PCR反应体系
(11)将DNA片段e、f、g、h各1μl作为模板,使用表3中的体系,除引物外加入Eppendorf管内,PCR循环条件为:98℃3min,(98℃10sec,60℃10sec,72℃30sec)*6cycle,加入引物CD8Hinge-F/TCR-R,(98℃10sec,60℃10sec,72℃30sec)*24cycle,72℃5min。产物经过1.5%的琼脂糖凝胶电泳,确认704bp的片段j,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(12)将DNA片段V1、a、i、j以5μl总体积且摩尔比1:1:1:1的比例加入Eppendorf管内,加入同源重组酶反应液15μl,混匀后在42℃孵育30分钟,转移至冰上放置2-3分钟,将反应液加入50μl TOP10中,轻轻旋转以混匀内容物,在冰中放置30分钟,将管放到预加温到42℃的恒温水浴锅中热激90秒,快速将管转移到冰浴中,使细胞冷却2-3分钟,每管加900μlLB培养液,然后将管转移到37℃摇床上,温育1小时使细菌复苏,取100μl的转化菌液涂布于Amp LB琼脂平板上,倒置平皿,于恒温培养箱中37℃培养,16小时。
挑取克隆进行菌落PCR鉴定,鉴定正确的克隆即为重组慢病毒质粒pCARmm-Basic2,对正确的克隆进行酶切鉴定(见图5);
(13)将重组慢病毒质粒pCARmm-Basic2使用Sal I和Nhe I限制性内切酶进行双酶切,产物经过1.5%的琼脂糖凝胶电泳,确认8491bp的片段V2,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(14)用引物IRES-F和IRES-R以合成的SEQ ID NO.25为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认605bp的片段k,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(15)用引物PDL1s1-F和PDL1s1-R以合成的SEQ ID NO.21为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认754bp的片段l,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(16)用引物PDL1s2-F和PDL1s2-R以合成的SEQ ID NO.22为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认777bp的片段m,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(17)用引物PDL1s3-F和PDL1s3-R以合成的SEQ ID NO.23为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认774bp的片段n,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(18)用引物s0-F和s0-R以合成的SEQ ID NO.24为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认729bp的片段o,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(19)用引物Fc-F和Fc-R以合成的SEQ ID NO.27为模板,使用表2中的体系,PCR循环条件为:98℃3min,(98℃10sec,55℃15sec,72℃2min)*35cycle,72℃10min。产物经过1.5%的琼脂糖凝胶电泳,确认726bp的片段p,并割胶回收置于Eppendorf管内,用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1),并测定产物的纯度和浓度;
(20)将DNA片段(V2、k、l、p)、(V2、k、m、p)、(V2、k、n、p)、(V2、k、o、p)分别以5μl总体积且摩尔比1:1:1:1的比例加入Eppendorf管内,加入同源重组酶反应液15μl,混匀后在42℃孵育30分钟,转移至冰上放置2-3分钟,将反应液加入50μl TOP10中,轻轻旋转以混匀内容物,在冰中放置30分钟,将管放到预加温到42℃的恒温水浴锅中热激90秒,快速将管转移到冰浴中,使细胞冷却2-3分钟,每管加900μl LB培养液,然后将管转移到37℃摇床上,温育1小时使细菌复苏,取100μl的转化菌液涂布于Amp LB琼脂平板上,倒置平皿,于恒温培养箱中37℃培养,16小时。挑取克隆进行菌落PCR鉴定,鉴定正确的克隆即为重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、pCARmm-PDL1scFv3以及对照pCARmm-scFv0,对正确的克隆进行酶切鉴定(见图6);
2、重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的包装。
(1)完全培养基:取出预热好的新鲜培养基,加入10%FBS+5ml Pen-Srep,上下颠倒混匀即可;
(2)1XPBS溶液:称量NaCl 8g,KCl 0.2,Na2HPO4.12H2O 3.58g,KH2PO4 0.24g置于1000ml烧杯中,加入900ml Milli-Q grade超纯水溶解,溶解完成后,使用1000ml量筒定容至1000ml,121℃高温湿热灭菌20min;
(3)0.25%Trypsin溶液:称量Trypsin 2.5g,EDTA 0.19729g置于1000ml烧杯中,加入900ml 1XPBS溶解,溶解完成后,使用1000ml量筒定容至1000ml,0.22μM过滤除菌,长期使用可保存至-20℃冰箱;
(4)0.5M CaCl2溶液:称量36.75g CaCl2用400ml Milli-Q grade超纯水溶解;用Milli-Q grade超纯水将总体积定容至500ml,混匀;0.22μm过滤除菌,分装保存到50ml离心管中,每管45ml左右,4℃保存。
(5)2XHBS溶液:称量4.09g NaCl,0.269g Na2HPO4,5.96g Hepes,用400ml Milli-Q grade超纯水溶解;校准pH仪后,用2M NaOH溶液将HBS溶液的pH调到7.05。调整每瓶HBS的PH消耗2M NaOH为3ml左右;
(6)从液氮罐中取出冻存的HEK293T/17细胞,迅速转移到37℃水浴中,1~2min后转移到超净台中,无菌操作将冻存管中的液体全部转移至10cm2培养皿中,补足含10%FBS的DMEM至8mL/10cm2dish,24h后显微镜观察细胞,细胞汇合的程度大于80%进行传代;
(7)选择细胞状态良好、无污染的HEK293T/17细胞,每2-6个培养皿为一组,将细胞胰酶消化后,用电动移液器吸取4-12ml完全培养基,向每个消化后的培养皿中加2ml,避免培养皿变干;使用1ml移液器将所有细胞吹打成单细胞悬液,转移到培养基瓶中;
(8)将上述2-6个培养皿中的剩余细胞转移到培养基瓶中,并用培养基再冲洗一便培养皿;
(9)盖紧培养基瓶盖,上下颠倒10次左右充分混匀细胞悬液,将细胞传到8-24个10cm2培养皿中,每皿的细胞密度应当约4×106个/10ml完全培养基左右。如果细胞密度和预期的相差较大,则需要对细胞进行计数,然后按照4×106个/皿的量接种;
(10)每6个培养皿整理为一摞,注意保持上下皿之间的配合。将培养皿左右,前后晃动数次,使细胞充分铺开,然后放入5%CO2培养箱。剩余细胞做同样处理;
(11)检查所传代细胞,细胞汇合度应当为70-80%,轮廓饱满,贴壁良好,在细胞培养皿中均匀分布;
(12)为细胞换液,将培养基替换为新鲜完全培养基,每皿9ml,并将培养箱的CO2浓度设定值提高到8%;
(13)按照N+0.5配DNA/CaCl2溶液。每皿HEK293T/17细胞转染质粒量按照下列比例使用:重组慢病毒质粒(20μg),pPac-GP(15μg),pPac-R(10μg),pEnv-G(7.5μg)。取一个新的5ml离心管,加入0.5M CaCl2:0.25ml,重组慢病毒质粒20μg:pPac-GP 15μg:pPac-R 10μg:pEnv-G 7.5μg,补充超纯水至0.5ml盖上盖子,充分混匀;
(14)另取一支5ml离心管,加入0.5ml DNA/CaCl2溶液。打开涡旋振荡器,一只手拿住5ml离心管的上端,使管底接触振荡头,使液体在管壁上散开流动,另一只手拿一把1mL移液枪,吸取0.5mL 2×HBS溶液,缓慢滴加进入离心管,控制流速,以半分钟滴完为宜。2×HBS加入后,继续振荡5秒钟,停止振荡,可直接加入需要转染的细胞中;
(15)取一皿细胞,将离心管中的1mL钙转液滴加进去,尽可能使钙转试剂分布到整个培养皿中;
(16)钙转液加入后,在皿盖上做好标记,将培养皿放还到另一个5%CO2培养箱中。确保培养皿水平放置,每摞培养皿不要超过6个。在5%CO2培养箱中放置(6–8h);
(17)将第一个培养箱的CO2浓度设定值调回到5%;
(18)24小时后,检查细胞状态。细胞汇合度应当为80–85%左右,状态良好。将培养基吸走,更换10ml新鲜的DMEM完全培养基;
(19)48小时后,观察转染效率。绝大多数细胞仍然是贴壁的。可以看到超过95%细胞都会带有绿色荧光。将同一个病毒包装上清液收集到一起,并向培养皿中继续添加10mL新鲜培养基;
(20)72小时后,再次将同一个病毒上清液收集到一起,两次收集的病毒可以放在一起,丢弃培养皿;此时收集的上清里包含了重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0。
实施例2重组慢病毒载体的浓缩及检测
一、离子交换色谱法纯化重组慢病毒载体(如图7所示);
(1)将收集的上清液使用Thermo真空泵,经0.22μm-0.8μm的PES滤器抽滤,除去杂质;
(2)按1:1~1:10的比例往上清中加入1.5M NaCl 250mM Tris-HCl(pH 6-8);
(3)将2个离子交换柱串联放置,用4ml 1M NaOH、4ml 1M NaCl、5ml 0.15M NaCl25mM Tris-HCl(pH6-8)溶液依次过柱;
(4)将步骤2中获得的溶液通过蠕动泵以1-10ml/min的速度给离子交换柱上样;
(5)全部上清液过柱后,使用10ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液清洗一遍;
(6)根据上样量使用1-5ml 1.5M NaCl 25mM Tris-HCl(pH 6-8)进行洗脱,收集洗脱液;
(7)将洗脱液分成25到50μl一管,冻存到-80℃冰箱,进行长期保存;
二、滴度测定;
(1)取24孔板接种293T细胞。每孔细胞为5×104个,所加培养基体积为500ul,不同种类的细胞生长速度有所差异,进行病毒感染时的细胞融合率为40%-60%;
(2)准备3个无菌EP管,在每个管中加入90ul的新鲜完全培养基(高糖DMEM+10%FBS)接种细胞24小时后,取两个孔的细胞用血球计数板计数,确定感染时细胞的实际数目,记为N;
(3)取待测定的病毒原液10ul加入到第一个管中,轻轻混匀后,取10ul加入到第二个管中,然后依次操作直到最后一管;在每管中加入410ul完全培养基(高糖DMEM+10%FBS),终体积为500ul;
(4)感染开始后20小时,除去培养上清,更换为500μl完全培养基(高糖DMEM+10%FBS),5%CO2继续培养48小时;
(5)72小时后,观察荧光表达情况,正常情况下,荧光细胞数随稀释倍数增加而相应减少,并拍照;
(6)用0.2ml 0.25%胰酶-EDTA溶液消化细胞,在37℃放置1分钟。用培养基吹洗整个细胞面,离心收集细胞。按照DNeasy试剂盒的说明抽提基因组DNA。每个样品管中加入200μl洗脱液洗下DNA并定量;
(7)准备目的DNA检测qPCRmix总管Ⅰ(QPCR引物序列为SEQ ID NO.57---SEQ IDNO.58):
n=number of reactions.例如:总反应数为40,将1ml 2×TaqMan UniversalPCR Master Mix,4μl forward primer,4μl reverse primer,4μl probe和788μl H2O混和。震荡后放在冰上;
(8)准备内参DNA检测qPCRmix管Ⅱ(QPCR引物序列为SEQ ID NO.59---SEQ IDNO.60):
2×TaqMan Master Mix 25μl×n
10×RNaseP primer/probe mix 2.5μl×n
H2O 17.5μl×n
n=number of reactions.例如:总反应数为40,将1ml 2×TaqMan UniversalPCR Master Mix,100μl 10×RNaseP primer/probe mix和700μl H2O混和。震荡后放在冰上;
(9)在预冷的96孔PCR板上完成PCR体系建立。从总管Ⅰ中各取45μl加入到A-D各行的孔中,从总管Ⅱ中各取45μl加入到E-G各行的孔中。
(10)分别取5μl质粒标准品和待测样品基因组DNA加入到A-D行中,每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(11)分别取5μl基因组标准品和待测样品基因组DNA加入到E-G行中,每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(12)所使用定量PCR仪为ABI PRISM 7500定量系统。循环条件设定为:50℃2分钟,95℃10分钟,然后是95℃15秒,60℃1分钟的40个循环。
数据分析:测得的DNA样品中整合的慢病毒载体拷贝数用基因组数加以标定,得到每基因组整合的病毒拷贝数。
滴度(integration units per ml,IU ml-1)的计算公式如下:
IU ml-1=(C×N×D×1000)/V
其中:C=平均每基因组整合的病毒拷贝数
N=感染时细胞的数目(约为1×105)
D=病毒载体的稀释倍数
V=加入的稀释病毒的体积数
(13)重组慢病毒载体1vCARmm-PDL1scFv1、1vCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、1vCARmm-scFv0的滴度结果(如图8所示);
三、内毒素测定;
(1)、内毒素工作标准品为15EU/支;
(2)、鲎试剂灵敏度λ=0.25EU/ml,0.5ml/管
(3)、内毒素标准品稀释:取内毒素标准品一支,分别用BET水按比例稀释成4λ和2λ的溶解,封口膜封口,震荡溶解15min;稀释时每稀释一步均应在漩涡混合器上混匀30s;
(4)、加样:取鲎试剂若干支,每支加入BET水0.5ml溶解,分装至若干支无内毒素试管中,每管0.1ml。其中2支为阴性对照管,加入BET水0.1ml;
2支为阳性对照管,加入2λ浓度的内毒素工作标准品溶液0.1ml;
2支为样品阳性对照管,加入0.1ml含2λ内毒素标准品的样品溶液(稀释20倍的待测样品1ml+4λ的内毒素标准品溶液1ml=2ml含2λ内毒素标准品的稀释40倍样品)。
样品管中加入0.1ml样品,稀释比例见表5,37±1℃水浴(或培养箱)保温60±1min;
表5内毒素稀释比例及对应内毒素含量
(5)、重组慢病毒载体1vCARmm-PDL1scFv1、1vCARmm-PDL1scFv2、1vCARmm-PDL1scFv3、lvCARmm-scFv0的内毒素检测结果(如表6所示),内毒素含量在0~2.5EU/ml之间,符合要求;
稀释倍数 | 原液 | 5 | 10 | 20 | 40 | 80 | 160 |
对应EU/ml | 0.25 | 1.25 | 2.5 | 5 | 10 | 20 | 40 |
lvCARmm-PDLlscFvl | (+) | (-) | (-) | (-) | (-) | (-) | (-) |
lvCARmm-PDLlscFv2 | (+) | (-) | (-) | (-) | (-) | (-) | (-) |
lvCARmm-PDLlscFv3 | (+) | (-) | (-) | (-) | (-) | (-) | (-) |
lvCARmm-scFv0 | (+) | (-) | (-) | (-) | (-) | (-) | (-) |
表6重组慢病毒载体的内毒素检测结果
四、支原体测定及比较;
(1)在实验前三日,细胞样品用无抗生素培养基进行培养;
(2)收集1ml细胞悬浮液(细胞数大于1*105),置于1.5ml离心管中;
(3)13000×g离心1min,收集沉淀,弃去培养基;
(4)加入500ul PBS用枪头吹吸或涡旋振荡,重悬沉淀。13000×g离心5min;
(5)步骤4重复一次;
(6)加入50μl Cell Lysis Buffer,用枪头吹吸,充分混匀后,在55℃水浴中孵育20min;
(7)将样品置于95℃中加热5min;
(8)13000×g离心5min后,取5μl上清作为模板,25μlPCR反应体系为:ddH20 6.5μl、Myco Mix 1μl、2x Taq Plus Mix Master(Dye Plus)12.5μl、模板5μl;PCR循环条件为:95℃30sec,(95℃30sec,56℃30sec,72℃30sec)*30cycle,72℃5min。
(9)支原体检测结果显示(如图9所示),重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0均不含支原体。
实施例3重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的功能检测。
一、CAR基因的细胞水平表达检测:
(1)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染PBMC细胞后,收集细胞采用RT-PCR进行CAR基因和scFv基因mRNA转录水平的检测,验证CAR基因和scFv基因的表达,如果CAR基因和scFv基因mRNA转录水平增高,则说明CAR基因和scFv基因的转录水平表达成功;
(2)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染PBMC细胞后,收集细胞采用western blot进行CAR蛋白表达水平的检测,验证CAR基因的表达,如果CAR蛋白表达水平增高,则说明CAR基因的翻译水平表达成功;
(3)重组慢病毒载体lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染PBMC细胞后,收集细胞培养上清采用ELISA进行scFv蛋白表达水平的检测,验证scFv基因的表达,如果scFv蛋白表达水平增高,则说明scFv基因的翻译水平表达成功;
(4)分别将MOI=15的lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0和对照病毒Mock感染细胞,48h后提取6孔板中细胞的总RNA和总蛋白分别进行荧光定量PCR实验和免疫印迹实验。具体步骤:包被6孔板的四个孔,每个孔加入相应的PBS和RN,4℃过夜。12小时后按MOI=15包被病毒,37℃培养箱放置5h;取出的6孔板,弃掉病毒上清,用PBS洗两遍,按1*106/孔,包被PBMC(用淋巴细胞分离液从人血中分离),加入500ul培养基(含10%血清、20U/ml IL-2、Polybrene 8ug/ml)。静置20min,1000g20℃离心30min,37℃培养48h。
(5)Trizol法提取6孔板中PBMC细胞的总RNA,逆转录扩增cDNA,用CAR基因QPCR引物(序列为SEQ ID NO.61---SEQ ID NO.62)和scFv基因QPCR引物(序列为SEQ ID NO.67---SEQ ID NO.68)进行荧光定量PCR实验,反应体系见表7,以内参Actin为对照组,验证其mRNA的转录情况。
试剂 | 体积(μl) |
SYBR premix ex taq: | 10μl |
ROX Reverse Dye(50x) | 0.4μl |
上游引物(2.5μM): | 0.5μl |
下游引物(2.5μM): | 0.5μl |
cDNA | 1.0μl |
ddH<sub>2</sub>O | 7.6μl |
表7 20μl qPCR反应体系
(6)蛋白免疫印迹(Western Blot)通过聚丙烯酰胺凝胶电泳将从PBMC中提取的总蛋白质按相对分子质量分离。采用湿转(4℃,400mA,120min),将蛋白转移到PVDF膜上。用封闭液(含5%脱脂牛奶的TBST溶液)室温封闭PVDF膜1h,封闭液1:1000稀释Biotinylatedprotein L,然后与封闭好的PVDF膜室温孵育4℃过夜。TBST洗膜3次,每次10min。封闭液1:500稀释相应的SA-HRP,室温下孵育PVDF膜2h,TBST洗膜3次,每次10min。采用Amersham公司ECL+plusTM Western blotting system试剂盒进行显色。X光显影获得显示条带的胶片。
(7)酶联免疫吸附测定(Enzyme Linked ImmunoSorbent Assay,ELISA)通过将1:2、1:5、1:10稀释的细胞培养上清包被至96孔板中,同时设置阴性对照、阳性对照和空白孔,4℃过夜。次日洗涤3次,于反应孔中,加入新鲜1:10000稀释的proteinG-HRP 0.1ml,37℃孵育30-60分钟,洗涤,最后一遍用纯水洗涤。于各反应孔中加入的TMB底物溶液0.1ml,37℃孵育10~30分钟。于各反应孔中加入ELISA反应终止液0.05ml。在酶标仪上,于405nm处,测各孔OD值。
(8)RT-QPCR检测显示,重组慢病毒载体感染PBMC后的CAR基因和scFv基因的转录水平比空细胞有明显升高(如图10所示),说明CAR基因和scFv基因的转录水平表达成功。
(9)蛋白免疫印迹(Western Blot)的结果表明,重组慢病毒载体感染PBMC后CAR蛋白的表达水平比对照病毒MOCK和空细胞有明显升高(如图11所示),说明CAR基因的翻译水平表达成功。
(10)酶联免疫吸附测定(ELISA)的结果表明,重组慢病毒载体感染PBMC后scFv蛋白的表达水平比对照病毒MOCK和空细胞有明显升高(如图12所示),说明scFv基因的翻译水平表达成功。
二、细胞杀伤实验效果评估。
(1)分别培养BCMA-K562细胞和PBMC细胞;
(2)实验开始前4天,分别用MOI=15的lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的病毒感染PBMC细胞,培养72-96h后可安排开始实验;
(3)收集靶细胞(BCMA-K562)4x105cells和效应细胞(lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0分别转导的PBMC细胞)2.8x106cells,800g,6min离心,弃上清;
(4)用1ml 1xPBS溶液分别重悬靶细胞和效应细胞,800g,6min离心,弃上清;
(5)重复步骤3一次;
(6)用700ul培养基(1640培养基+10%FBS)重悬效应细胞,用2ml培养基(1640培养基+10%FBS)重悬靶细胞;
(7)设置效靶比为1:1、5:1、10:1的实验孔,并设置对照组,每组3个复孔;
(8)250xg,5min平板离心;
(9)37℃5%CO2培养箱中培养24小时;
(10)250xg,5min平板离心;
(11)取每个孔的50ul上清到新96孔板中,并且每孔加入50ul底物溶液(避光操作);
(12)避光孵育25分钟;
(13)每孔加入50ul终止液;
(14)酶标仪检测490nm吸光度;
(15)将3个复孔取平均值;将所有实验孔、靶细胞孔和效应细胞孔的吸光值减去培养基背景吸光值的均值;将靶细胞最大值的吸光值减去体积校正对照吸光值的均值。
(16)将步骤15中获得的经过校正的值带入下面公式,计算每个效靶比所产生的细胞毒性百分比。结果如图13所示,lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0重组慢病毒载体转导的PBMC细胞在几个效靶比条件下杀伤效率明显高于PBMC空细胞和对照病毒,说明scFv基因的表达对CAR基因的功能影响较小。
杀伤效率=(实验孔-效应细胞孔-靶细胞孔)/(靶细胞最大孔-靶细胞孔)X100%
三、PDL1阻断效果评估(PD1、IL2、TNFα、IFNγmRNA转录水平)。
(1)分别培养BCMA-PDL1-K562细胞和PBMC细胞;
(2)实验开始前4天,分别用MOI=15的lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0的病毒感染PBMC细胞,培养72-96h后可安排开始实验;
(3)收集靶细胞(BCMA-PDL1-K562)4x105cells和效应细胞(lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0分别转导的PBMC细胞)2.8x106cells,800g,6min离心,弃上清;
(4)用1ml 1xPBS溶液分别重悬靶细胞和效应细胞,800g,6min离心,弃上清;
(5)重复步骤4一次;
(6)用700ul培养基(1640培养基+10%FBS)重悬效应细胞,用2ml培养基(1640培养基+10%FBS)重悬靶细胞;
(7)设置效靶比为10:1的实验孔,并设置对照组;
(8)250xg,5min平板离心;
(9)37℃5%CO2培养箱中共培养24小时,1000xg,2min平板离心,收集细胞抽总mRNA,反转cDNA,检测PD1、IL2、TNFα、IFNγmRNA转录水平;
(10)用PD1基因QPCR引物(序列为SEQ ID NO.63---SEQ ID NO.64),IL2基因QPCR引物(序列为SEQ ID NO.65---SEQ ID NO.66),TNFα基因QPCR引物(序列为SEQ IDNO.69---SEQ ID NO.70),IFNγ基因QPCR引物(序列为SEQ ID NO.71---SEQ ID NO.72),进行荧光定量PCR实验,反应体系见表6,以内参Actin为对照组,验证其mRNA的转录情况。
(11)RT-QPCR检测结果显示,lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0转导的PBMC并与靶细胞孵育后,PD1基因的mRNA水平与Mock组和空细胞组相比明显升高,lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3、lvCARmm-scFv0四组之间的PD1基因的mRNA水平则没有太大差别(如图14所示),说明T细胞被激活后,PD1的表达水平也同步上升。而其后通过检测IL2、TNFα、IFNγmRNA转录水平发现,lvCARmm-PDL1scFv1、lvCARmm-PDL1scFv2、lvCARmm-PDL1scFv3三组与对照病毒lvCARmm-scFv0组相比IL2、TNFα、IFNγmRNA转录水平明显升高,其中lvCARmm-PDL1scFv1组的IL2、TNFα、IFNγ基因的转录mRNA水平升高最为明显(如图15所示),IL2、TNFα、IFNγ基因的表达水平越高说明T细胞越处于活化状态,说明lvCARmm-PDL1scFv1组能有效阻断PD1/PDL1信号通路,解除对T细胞活化相关基因的抑制作用,将来可以在体内阻断PD1/PDL1的信号通路,达到抑制免疫逃脱的效果,提高CAR-T细胞治疗针对实体肿瘤的疗效。
序列表
<110>上海优卡迪生物医药科技有限公司
<120>一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体及其构建方法和应用
<130> HJ16-12296
<160> 72
<170> PatentIn version 3.5
<210> 1
<211> 861
<212> DNA
<213>人工序列
<400> 1
atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60
gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120
cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180
gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240
cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300
gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360
tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420
ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480
gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540
cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct 600
tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc 660
tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct 720
cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac 780
acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc 840
tcactgatta agcattggta a 861
<210> 2
<211> 674
<212> DNA
<213>人工序列
<400> 2
cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 60
ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 120
actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 180
gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 240
ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 300
gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 360
acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 420
tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 480
gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 540
cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 600
cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 660
ccttttgctc acat 674
<210> 3
<211> 147
<212> DNA
<213>人工序列
<400> 3
atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60
tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120
ggcttttttg gaggcctaga cttttgc 147
<210> 4
<211> 228
<212> DNA
<213>人工序列
<400> 4
gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 60
cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 120
tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 180
gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacg 228
<210> 5
<211> 180
<212> DNA
<213>人工序列
<400> 5
ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 60
tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 120
gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 180
<210> 6
<211> 234
<212> DNA
<213>人工序列
<400> 6
tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac 60
acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca 120
gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc 180
cagtacaagc aaaaagcaga tcttattttc gttgggagtg aattagccct tcca 234
<210> 7
<211> 353
<212> DNA
<213>人工序列
<400> 7
atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 60
ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 120
agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 180
tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 240
atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 300
ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caa 353
<210> 8
<211> 233
<212> DNA
<213>人工序列
<400> 8
aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcctcaat 60
gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120
gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180
gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcc 233
<210> 9
<211> 489
<212> DNA
<213>人工序列
<400> 9
tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 60
gttggagtaa taaatctctg gaacagattg gaatcacacg acctggatgg agtgggacag 120
agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 180
agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 240
taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 300
aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc agggatattc 360
accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 420
agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg 480
acggttaac 489
<210> 10
<211> 119
<212> DNA
<213>人工序列
<400> 10
ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60
agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttta 119
<210> 11
<211> 592
<212> DNA
<213>人工序列
<400> 11
aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60
ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120
atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180
tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240
ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300
attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360
ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420
gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480
aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540
cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592
<210> 12
<211> 1178
<212> DNA
<213>人工序列
<400> 12
gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60
gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120
atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180
tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240
tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300
cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360
agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420
ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480
tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540
aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600
cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660
cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720
gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780
ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840
cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900
cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960
agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020
agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080
tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140
tggttcaaag tttttttctt ccatttcagg tgtcgtga 1178
<210> 13
<211> 63
<212> DNA
<213>人工序列
<400> 13
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 14
<211> 324
<212> DNA
<213>人工序列
<400> 14
gacatccaga tgacccagag ccctagctca ctgagcgcca gcgtgggcga cagggtgacc 60
attacctgct ccgccagcca ggacatcagc aactacctga actggtacca gcagaagccc 120
ggcaaggccc ccaagctgct gatctactac acctccaacc tgcactccgg cgtgcccagc 180
aggttcagcg gaagcggcag cggcaccgat ttcaccctga ccatctccag cctgcagccc 240
gaggacttcg ccacctacta ctgccagcag tacaggaagc tcccctggac tttcggccag 300
ggcaccaaac tggagatcaa gcgt 324
<210> 15
<211> 45
<212> DNA
<213>人工序列
<400> 15
ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45
<210> 16
<211> 363
<212> DNA
<213>人工序列
<400> 16
caggtgcagc tggtccagag cggcgccgaa gtgaagaagc ccggcagctc cgtgaaagtg 60
agctgcaagg ccagcggcgg caccttcagc aactactgga tgcactgggt gaggcaggcc 120
cccggacagg gcctggagtg gatgggcgcc acctacaggg gccacagcga cacctactac 180
aaccagaagt tcaagggccg ggtgaccatc accgccgaca agagcaccag caccgcctac 240
atggaactga gcagcctcag gagcgaggac accgctgtgt attactgcgc caggggcgcc 300
atctacgacg gctacgacgt gctggacaac tggggccagg gcacactagt gaccgtgtcc 360
agc 363
<210> 17
<211> 141
<212> DNA
<213>人工序列
<400> 17
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgatatcta c 141
<210> 18
<211> 66
<212> DNA
<213>人工序列
<400> 18
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60
tactgc 66
<210> 19
<211> 126
<212> DNA
<213>人工序列
<400> 19
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120
gaactg 126
<210> 20
<211> 336
<212> DNA
<213>人工序列
<400> 20
agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180
gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240
cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300
tacgacgccc ttcacatgca ggccctgccc cctcgc 336
<210> 21
<211> 726
<212> DNA
<213>人工序列
<400> 21
gatattgtgc tgacccagag cccggcgagc ctggcggtga gcccgggcca gcgcgcgacc 60
attacctgcc gcgcgagcca gagcgtgagc accagcagca gcagctttat gcattggtat 120
cagcagaaac cgggccagcc gccgaaactg ctgattaaat atgcgagcaa cctggaaagc 180
ggcgtgccgg cgcgctttag cggcagcggc agcggcaccg attttaccct gaccattaac 240
ccggtggaag cgaacgatac cgcgaactat tattgccagc atagctggga aattccgtat 300
acctttggcc agggcaccaa actggaaatt aaaggtggcg gtggctcggg cggtggtggg 360
tcgggtggcg gcggatctga agtgcagctg gtggaaagcg gcggcggcct ggtgaaaccg 420
ggcggcagcc tgcgcctgag ctgcgcggcg agcggcttta tttttcgcag ctatggcatg 480
agctgggtgc gccaggcgcc gggcaaaggc ctggaatggg tggcgagcat tagcagcggc 540
ggcagcacct attatccgga tagcgtgaaa ggccgcttta ccattagccg cgataacgcg 600
aaaaacagcc tgtatctgca gatgaacagc ctgcgcgcgg aagataccgc ggtgtatgat 660
tgcgcgcgcg gctatgatag cggctttgcg tattggggcc agggcaccct ggtgaccgtg 720
agcagc 726
<210> 22
<211> 717
<212> DNA
<213>人工序列
<400> 22
gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60
attagctgcc gcgcgagcca gagcgtgagc accagcagct atagctatgt gcattggtat 120
cagcaggcgc cgaaactgct gatttattat gcggcgaacc gctataccgg cgtgccggat 180
cgctttagcg cgcgctttag cggcagcggc agcggcaccg attttaccct gaacattcat 240
ccggtggaag aagaatattt ttgccagcag gattatacca gcccgtatac ctttggccag 300
ggcaccaaac tggaaattaa aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc 360
ggatctcaga ttaccctgaa agaaagcggc ccgaccctgg tgaaaccgac ccagaccctg 420
accctgacct gcgcggcgag cggctttagc tttagcagct atggcatgag ctgggtgcgc 480
cagaccccgg aagcgctgga atggctgggc gtgatttggc gcggcgtgac caccgattat 540
aacgcggcgt ttaaaggccg ctttaccatt agccgcgata acgcgcgcaa cattctgtat 600
ctgcagatga gcagcctgaa catggatccg gtggataccg cgacctatta ttgcgcgcgc 660
ctgggctttt atgcgatgga ttattggggc cagggcaccc tggtgaccgt gagcagc 717
<210> 23
<211> 723
<212> DNA
<213>人工序列
<400> 23
gatattgtgc tgacccagag cccggcgagc ctggcggtga gcctgggcca gcgcgcgacc 60
attacctgca aagcgagcca gagcgtgagc aacgatgtgg cgtggtatca gcagaaaccg 120
ggcaaaaaac cgggccagcc gccgaaactg ctgattaaat atgcgagcaa cctggaaagc 180
ggcgtgccgg gcagcggcta tggcaccgat tttaccttta ccattagcag cctgcagccg 240
gaagatattg cgaccgatac cgcgacctat tattgccagc atagctggga aattccgtat 300
acctttggcg gcggcaccaa actggaaatt aaaggtggcg gtggctcggg cggtggtggg 360
tcgggtggcg gcggatctga agaaaaactg gtggaaagcg gcggcggcct ggtgaaaccg 420
ggcggcagcc tgaaactgag ctgcaccgtg agcggcttta gcctgagcac ctatggcgtg 480
cattggattc gccagccgcc gggcaaaaaa cgcctggaat gggtggcgag cattagcagc 540
ggcggcagca tttattatcc ggatagcgtg atgagccgcc tgaccattac caaagataac 600
agcaaaaacc aggtggtgct gaccatgaac cgcagcgaag ataccgcgat gtattattgc 660
gcgcgcggct atgatgcggg ctttgcgttt tggggccagg gcaccctggt gaccgcgagc 720
gcg 723
<210> 24
<211> 699
<212> DNA
<213>人工序列
<400> 24
ttgttctgga ttcctgcttc catcagtgat gttgtgatga cccaaactgt cagtcttgga 60
gatcaagctt ccatctcttg cagatctagt cagaaccttg tacacaacaa tggaaacacc 120
tatttatatt ggttcctgca gaagtcaggc cagtctccaa agctcctgat ttatagggct 180
tccatccgat tttctggggt cccagacagg ttcagtggca gtggatcaga gacagatttc 240
acactcaaga tcagcagagt ggaggcttat ttctgctttc aaggtacaca tgttccgtgg 300
acgttcggtg gaggcaccaa gctggaaatc aaaggtggcg gtggctcggg cggtggtggg 360
tcgggtggcg gcggatctga ggtgctgctg caacagtctg gacctgagct ggtgaagata 420
ccctgcaagg cttctggata cacattcact gactacaaca tggactggat gaagcagagc 480
catggaaaga gccttgagtg gattggagat attaatccta agagtggtaa ttccatctac 540
aaccagaagt tcaagggcaa ggccacactg actgtagaca agtcctccag cacagcctac 600
atggagctcc gcagcctgac atctgaggac actgcagtct atgactggtc tgcctggttt 660
gctttctggg gccaagggac tctggtctct gtctctgca 699
<210> 25
<211> 575
<212> DNA
<213>人工序列
<400> 25
gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60
gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120
ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180
gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240
aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300
tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360
acgttgtgag ttggatagtt gtggaaagag tcaaatggct cacctcaagc gtattcaaca 420
aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480
gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataat 575
<210> 26
<211> 87
<212> DNA
<213>人工序列
<400> 26
atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg gctgctcctg 60
gtgttgcctg ctgccttccc tgcccca 87
<210> 27
<211> 696
<212> DNA
<213>人工序列
<400> 27
gaaccgaaaa gctgcgataa aacccatacc tgcccgccgt gcccggcgcc ggaactgctg 60
ggcggcccga gcgtgtttct gtttccgccg aaaccgaaag ataccctgat gattagccgc 120
accccggaag tgacctgcgt ggtggtggat gtgagccatg aagatccgga agtgaaattt 180
aactggtatg tggatggcgt ggaagtgcat aacgcgaaaa ccaaaccgcg cgaagaacag 240
tataacagca cctatcgcgt ggtgagcgtg ctgaccgtgc tgcatcagga ttggctgaac 300
ggcaaagaat ataaatgcaa agtgagcaac aaagcgctgc cggcgccgat tgaaaaaacc 360
attagcaaag cgaaaggcca gccgcgcgaa ccgcaggtgt ataccctgcc gccgagccgc 420
gaagaaatga ccaaaaacca ggtgagcctg acctgcctgg tgaaaggctt ttatccgagc 480
gatattgcgg tggaatggga aagcaacggc cagccggaaa acaactataa aaccaccccg 540
ccggtgctgg atagcgatgg cagctttttt ctgtatagca aactgaccgt ggataaaagc 600
cgctggcagc agggcaacgt gtttagctgc agcgtgatgc atgaagcgct gcataaccat 660
tatacccaga aaagcctgag cctgagcccg ggcaaa 696
<210> 28
<211> 123
<212> DNA
<213>人工序列
<400> 28
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 29
<211> 36
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 29
attcaaaatt ttatcgatgc tccggtgccc gtcagt 36
<210> 30
<211> 22
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 30
tcacgacacc tgaaatggaa ga 22
<210> 31
<211> 42
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 31
ggtgtcgtga ggatccgcca ccatggcctt accagtgacc gc 42
<210> 32
<211> 30
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 32
ggtcatctgg atgtccggcc tggcggcgtg 30
<210> 33
<211> 37
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 33
cacgccgcca ggccggacat ccagatgacc cagagcc 37
<210> 34
<211> 21
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 34
acgcttgatc tccagtttgg t 21
<210> 35
<211> 81
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 35
actggagatc aagcgtggtg gcggtggctc gggcggtggt gggtcgggtg gcggcggatc 60
tcaggtgcag ctggtccaga g 81
<210> 36
<211> 22
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 36
gctggacacg gtcactagtg tg 22
<210> 37
<211> 33
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 37
agtgaccgtg tccagcacca cgacgccagc gcc 33
<210> 38
<211> 23
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 38
gtagatatca caggcgaagt cca 23
<210> 39
<211> 33
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 39
cgcctgtgat atctacatct gggcgccctt ggc 33
<210> 40
<211> 39
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 40
tctttctgcc ccgtttgcag taaagggtga taaccagtg 39
<210> 41
<211> 21
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 41
aaacggggca gaaagaaact c 21
<210> 42
<211> 40
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 42
tgctgaactt cactctcagt tcacatcctc cttcttcttc 40
<210> 43
<211> 22
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 43
agagtgaagt tcagcaggag cg 22
<210> 44
<211> 34
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 44
ggagaggggc gtcgacttag cgagggggca gggc 34
<210> 45
<211> 34
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 45
gccctgcccc ctcgctaagc ccctctccct cccc 34
<210> 46
<211> 79
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 46
ccagggagaa ggcaactgga ccgaaggcgc ttgtggagaa ggagttcatg gtggcattat 60
catcgtgttt ttcaaagga 79
<210> 47
<211> 74
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 47
gttgccttct ccctggggct gctcctggtg ttgcctgctg ccttccctgc cccagatatt 60
gtgctgaccc agag 74
<210> 48
<211> 36
<212> DNA
<213>人工序列
<220>
<221> misc_feature
<223>引物
<400> 48
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Claims (9)
1.一种封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体,其特征在于,包括:
用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列,如SEQ ID NO.1所示;
用于质粒复制的原核复制子pUC Ori序列,如SEQ ID NO.2所示;
用于增强真核细胞内的复制的病毒复制子SV40 Ori序列,如SEQ ID NO.3所示;
用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件,如SEQID NO.11所示;
用于嵌合抗原受体基因的真核转录的人EF1α启动子,如SEQ ID NO.12所示;
用于慢病毒包装的慢病毒包装顺式元件;
人PDL1的人源化单链抗体,所述人PDL1的人源化单链抗体为如SEQ ID NO.21所示的PDL1scFv1、或者如SEQ ID NO.22所示的PDL1scFv2、或者如SEQ ID NO.23所示的PDL1scFv3;
用于共同转录表达蛋白质的IRES核糖体结合序列,如SEQ ID NO.25所示;
IL6信号肽,如SEQ ID NO.26所示;
人源抗体Fc段,如SEQ ID NO.27所示;
以及用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体;
所述用于组成集识别、传递、启动于一体的二代CAR的嵌合抗原受体包括:如SEQ IDNO.13所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域;
所述用于组成集识别、传递、启动于一体的三代CAR的嵌合抗原受体包括:如SEQ IDNO.13所示的CD8 leader嵌合受体信号肽、如SEQ ID NO.14所示的BCMA单链抗体轻链VL、如SEQ ID NO.15所示的Optimal Linker C、如SEQ ID NO.16所示的BCMA单链抗体重链VH、如SEQ ID NO.17所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.18所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.19所示的CD137嵌合受体共刺激因子、如SEQ ID NO.20所示的TCR嵌合受体T细胞激活域、以及如SEQ ID NO.28所示的CD28嵌合受体共刺激因子。
2.如权利要求1所述的载体,其特征在于,所述人PDL1的人源化单链抗体为如SEQ IDNO.21所示的PDL1scFv1。
3.如权利要求1所述的载体,其特征在于,所述慢病毒包装顺式元件采用第二代慢病毒载体包括:如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件。
4.如权利要求1所述的载体,其特征在于,所述慢病毒包装顺式元件采用第三代慢病毒载体包括:如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件,以及如SEQ ID NO.4所示的RSV启动子。
5.如权利要求1-4任一项所述的载体,其特征在于,所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变,具体为:g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
6.一种如权利要求1、3-5任一项所述的封闭PDL1的用于抑制免疫逃脱的CAR-T转基因载体的构建方法,其特征在于,包括以下步骤:
(1)将如SEQ ID NO.1所示的含氨苄青霉素抗性基因AmpR序列、如SEQ ID NO.2所示的原核复制子pUC Ori序列、如SEQ ID NO.3所示的病毒复制子SV40 Ori序列、用于慢病毒包装的慢病毒包装顺式元件、如SEQ ID NO.11所示的eWPRE增强型土拨鼠乙肝病毒转录后调控元件存储于慢病毒骨架质粒上;
(2)将如SEQ ID NO.12所示的人EF1α启动子、用于组成集识别、传递、启动于一体的二代CAR或三代CAR的嵌合抗原受体组合成二代CAR或三代CAR设计方案,经过酶切、连接、重组反应克隆至慢病毒骨架质粒中,得到二代CAR或三代CAR设计的重组慢病毒质粒;
(3)将人PDL1的人源化单链抗体PDL1scFv1、PDL1scFv2、或PDL1scFv3,与IRES核糖体结合序列、IL6信号肽以及人源抗体Fc段克隆至重组慢病毒质粒中,得到PDL1阻断重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3;
(4)将得到的重组慢病毒质粒pCARmm-PDL1scFv1、pCARmm-PDL1scFv2、或pCARmm-PDL1scFv3分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞,在HEK293T/17细胞中进行基因转录表达后,包装成功重组慢病毒载体会释放到细胞培养上清中,收集包含的重组慢病毒载体的上清液;
(5)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的柱纯化方式进行纯化,分别得到重组慢病毒载体。
7.如权利要求6所述的方法,其特征在于,步骤(3)中,由人EF1α启动子启动整个CAR基因表达;CAR蛋白定位于细胞膜表面,识别BCMA抗原,刺激T细胞增殖和细胞因子分泌,激活下游信号通路的表达;scfv区域与BCMA抗原结合,信号通过嵌合受体传递至细胞内,从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞一系列生物学效应;由IRES核糖体结合序列共表达PDL1scFv与Fc的融合蛋白,并在IL6信号肽的引导下分泌到细胞外,通过与PDL1的结合,阻断PD1与PDL1的结合,从而阻断PD1/PDL1的信号通路。
8.如权利要求6所述的方法,其特征在于,步骤(5)中,所述抽滤步骤要控制上清体积在200ml~2000ml,控制真空度在-0.5MPA~-0.9MPA,防止由于堵孔带来的载体损失;所述吸附步骤要控制溶液的pH值在6~8,防止pH的变化导致载体失活;所述洗脱步骤要控制洗脱液的离子强度在0.5M~1.0M,防止离子强度的变化导致洗脱不完全或者载体失活。
9.如权利要求1-5任一项所述的载体在制备抑制免疫逃脱的药物中的应用。
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EP17879118.2A EP3550024A4 (en) | 2016-12-05 | 2017-11-13 | TRANSGENIC PD-L1 KNOCKOUT CAR T VECTOR FOR THE SUPPRESSION OF IMMUNEVASION, MANUFACTURING METHOD FOR IT AND APPLICATION THEREOF |
US16/464,682 US10736920B2 (en) | 2016-12-05 | 2017-11-13 | PDL1 block CAR-T transgenic vector for suppressing immune escape, preparation method thereof, and application of the same |
PCT/CN2017/110656 WO2018103503A1 (zh) | 2016-12-05 | 2017-11-13 | 一种封闭pdl1的用于抑制免疫逃脱的car-t转基因载体及其构建方法和应用 |
KR1020197017093A KR102157197B1 (ko) | 2016-12-05 | 2017-11-13 | 일종의 pdl1 차단을 통한 면역회피 억제에 사용되는 car-t 형질전환 벡터 및 그의 구축방법과 용도 |
JP2019530074A JP6736109B2 (ja) | 2016-12-05 | 2017-11-13 | Pdl1がブロッキングされた免疫逃避を抑制するためのcar−t遺伝子組換えベクターおよびその構築方法と使用 |
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