靶向cd123的嵌合抗原受体及其用途
阅读说明：本技术 靶向cd123的嵌合抗原受体及其用途 (Chimeric antigen receptor targeting CD123 and uses thereof ) 是由 何霆 齐菲菲 鲁薪安 丁艳萍 梁梦梦 于 2020-12-16 设计创作，主要内容包括：本发明提供了特异性结合CD123的单链抗体scFv,其包含选自SEQ ID NO：56、57、58、59、60和61的氨基酸序列。还提供了靶向CD123的嵌合抗原受体,其包含选自SEQ ID NO：65、66、67、68、69和70的氨基酸序列。还提供了编码所述scFv或嵌合抗原受体的核酸分子,包含所述核酸分子的载体和细胞,以及所述抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途。(The present invention provides a single chain antibody scFv that specifically binds CD123 comprising a heavy chain variable region selected from the group consisting of SEQ ID NO: 56. 57, 58, 59, 60 and 61. Also provided is a CD123 targeting chimeric antigen receptor comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 65. 66, 67, 68, 69 and 70. Also provided are nucleic acid molecules encoding the scFv or chimeric antigen receptor, vectors and cells comprising the nucleic acid molecules, and use of the antibody, chimeric antigen receptor, nucleic acid molecule, vector or cell for the manufacture of a medicament for the treatment of a tumor expressing CD 123.)
技术领域
本发明涉及具有不同抗原识别域的靶向CD123的scFv序列，基于这些scFv序列设计的嵌合抗原受体(CAR)和CAR-T细胞，以及其用于治疗表达CD123的肿瘤、特别是急性髓系白血病的用途。
背景技术
急性髓系白血病(AML)是一种发病率不断上升的恶性肿瘤，主要影响中位年龄为66岁的老年人。美国国家癌症研究所(National Cancer Institute)估计，2018年的新病例将达到19520例，而其中可能会有10670人死于这种疾病[1]。尽管65-74岁的患者在过去连续三十年中，12个月生存率从20％提高到30％，但目前AML的治疗效果仍不令人满意，这些患者的5年生存率＜5％[2]。对于较年轻的AML患者，主要的治疗方法是强化化疗，对于复发风险较高的患者(治愈率35-40％)，采用异基因造血细胞移植(AHCT)，而老年患者的预后仍然很差[2]。复发的AML患者获得第二次缓解的几率小于50％，而在第二次抢救性化疗后，只有16％的患者能实现病情控制[3]。自20世纪70年代引入蒽环类和阿糖胞苷类联合化疗以来，它一直是主要的治疗方法[4，5]。因此，对于此疾病，需要新的治疗策略。
嵌合抗原受体T细胞(CAR-T细胞)的概念首先提出于20世纪80年代后期，其结合了抗体特异性和T细胞杀伤效应，形成过继性免疫效应[6，7]。嵌合抗原受体(CAR)分子结构主要包括抗体单链可变片段(scFv)、胞内信号域及连接scFv与胞内信号域的铰链区和跨膜区。近年来，利用抗CD19和抗CD20嵌合抗原受体T细胞(CAR-T细胞)治疗淋巴样恶性肿瘤(B细胞急性淋巴细胞白血病和非霍奇金淋巴瘤)取得了良好的效果[8]。目前，针对AML治疗的CAR-T靶点，有CD123、CD33、CLL1、FLT3等；其中，靶向CD123的CAR-T研究最多。
CD123是IL-3受体a链(IL-3Ra)。CD123与CD131一起组成高亲和力的IL-3受体，与IL-3结合后，能够促进细胞增殖分化，同时抑制造血细胞凋亡[9，10]。CD123的高表达与信号转导及转录激活因子5(STAT5)信号通路的活化相关。STAT5激活通路参与在多种恶性血液病中[11]。CD123主要在造血系统中表达，CD123在正常CD34+CD38-细胞中低表达或不表达[12]。此外，正常情况下在髓系祖细胞、B淋巴祖细胞中CD123有较高表达，而红系祖细胞和多向分化潜能祖细胞中低表达或不表达[13，14]。CD123在60％-90％以上的AML原始细胞中高表达[15-18]。Bras AE等人[19]检测了455例AML患者，其中约55％的患者AML细胞表达CD123，且阳性率达80％以上。
虽然，CAR-T细胞在肿瘤细胞治疗中已取得了较好效果，但是CAR-T的疗效和安全性依然存在较大问题，主要有脱靶效应、细胞因子风暴、神经毒性等[20]。CAR-T细胞的最佳疗效和安全性，取决于多方面因素，其中CAR-T细胞的有效活化具有重要作用。CAR-T细胞的活化程度受CAR分子scFv的亲和力、铰链区长度、共刺激信号域、靶细胞表面抗原密度等多种因素影响。CAR分子的scFv作为与靶细胞表面抗原直接结合的区域，其亲和力对整体CAR-T细胞的活化意义重大。CAR分子scFv亲和力较低时，CAR-T细胞不能得到足够的抗原识别，从而无法达到最佳激活状态；CAR分子scFv亲和力较高时，CAR-T细胞可能会在识别肿瘤细胞的同时也能够识别抗原密度相对较低的正常细胞，产生严重脱靶效应。
因此，为了进一步提高CAR-T细胞的安全性和有效性，保证CAR-T细胞的有效活化，避免脱靶效应，仍需要筛选针对靶点具有适中亲和力的scFv。
发明内容
第一方面，本发明提供了一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含SEQ ID NO：1(GYX1X2X3X4(D)YX5X6X7)的VH-CDR1氨基酸序列，SEQ ID NO：2
(X8X9X10(X11)X12X13X14X15X16X17X18NX19X20X21KX22X23X24)的VH-CDR2氨基酸序列和SEQID NO：3
(AX25X26X27X28X29X30X31(YD)X32X33X34X35X36X37X38X39X40)的VH-CDR3氨基酸序列；其中X1选自S、T；X2选自F、I；X3选自T、S、M；X4选自S、D、T、G；X5选自W、Y、V、A、N；X6选自M、V、I、W；X7选自N、H；X8选自Y、R；X9选自I、C；X10选自D、N、Y、S；X11选自P、C；X12选自Y、G；X13选自D、N、S；X14选自S、N、D、G；X15选自E、A、G、S；X16选自T、I、S；X17选自H、S、N；X18选自Y、S；X19选自Q、E、P；X20选自K、N、S；X21选自F、L；X22选自D、G、S；X23选自K、R；X24选自A、I；X25选自R、G；X26选自G、S、E；X27选自E、P、R；X28选自G、S；X29选自N、Y、L、W；X30选自W、Y、L；X31选自G、D；X32选自R、E；X33选自Y、S、T；X34选自D、Y、G；X35选自G、Y；X36选自Y、L；X37选自A、G、P；X38选自M、L；X39选自D、A；X40选自Y、C；所述轻链可变结构域包含SEQ ID NO：4(X41SX42X43X44X45X46X47X48X49(QK)X50X51X52X53W)的VL-CDR1氨基酸序列，SEQ ID NO：5(X54SX55X56X57X58X59)的VL-CDR2氨基酸序列和SEQ IDNO：6(X60X61X62X63X64(P)X65TF)的VL-CDR3氨基酸序列；其中X41选自A、S；X42选自Q、K、S；X43选自S、D；X44选自I、V、L；X45选自S、N、D、L；X46选自K、Y、S、D、N；X47选自D、S；X48选自G、S；X49选自D、N；X50选自S、N；X51选自D、Y；X52选自L、I、M；X53选自A、N、H；X54选自G、T、A；X55选自T、N、Q；X56选自L、S、R；X57选自Q、E、A、I、D；X58选自S、P；X59选自G、E；X60选自Q、N；X61选自H、Y、S、F、G；X62选自N、D、H、Y；X63选自K、L、E、R、S；X64选自Y、L、D、S、F、T；X65选自Y、R、W；优选地，所述抗体是scFv。
第二方面，本发明提供了一种靶向CD123的嵌合抗原受体，其包含：第一方面所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域。
第三方面，本发明提供了一种核酸分子，其包含编码第一方面的抗体或第二方面的嵌合抗原受体的核苷酸序列。
第四方面，本发明提供了一种载体，其包含第三方面的核酸分子。
第五方面，本发明提供了一种细胞，其经第四方面的载体转染，从而包含第三方面的核酸分子或第四方面的载体。
第六方面，本发明提供了一种药物组合物，其包含第一方面的抗体、第二方面的嵌合抗原受体、第三方面的核酸分子、第四方面的载体或第五方面的细胞，以及可药用载体。
第七方面，本发明提供了第一方面的抗体、第二方面的嵌合抗原受体、第三方面的核酸分子、第四方面的载体或第五方面的细胞用于制备治疗表达CD123的肿瘤的药物的用途。
本发明通过对靶点CD123的CAR分子的scFv进行亲和力筛选以及CAR-T细胞杀伤效应比较，以获得靶点CD123的最佳scFv序列，使得CAR-T细胞具有更优的安全性和有效性。与现有技术相比，本发明能有效提高靶向CD123的CAR-T细胞对肿瘤细胞系及原代肿瘤细胞的杀伤效率，提高细胞增殖能力。
附图说明
图1表示不同scFv的CD123 CAR分子在T细胞中的转导效率。
图2表示CD123 CAR-T在培养过程中的总细胞增殖情况。
图3表示靶细胞AML原代细胞和THP-1细胞分别刺激下，对CD123 CAR-T细胞分化的影响。
图4表示CD123 CAR-T对CD123阳性细胞系(THP-1)及CD123阴性细胞系(K562)的杀伤效率。
图5表示CD123 CAR-T对AML原代细胞的杀伤效率。
图6表示CD123 CAR-T在AML原代细胞刺激5小时后的细胞因子表达。
具体实施方式
本发明提供了一种特异性结合CD123的抗体，其包括重链可变结构域VH和轻链可变结构域VL，所述重链可变结构域包含VH-CDR1、VH-CDR2和VH-CDR3，所述轻链可变结构域包含VL-CDR1、VL-CDR2和VL-CDR3。
在一个具体实施方案中，所述VH-CDR1选自由SEQ ID NO：8、9、10、11、12组成的组中的氨基酸序列；所述VH-CDR2选自由SEQ ID NO：14、15、16、17、18组成的组中的氨基酸序列；所述VH-CDR3选自由SEQ ID NO：20、21、22、23、24组成的组中的氨基酸序列；所述VL-CDR1选自由SEQ ID NO：26、27、28、29、30组成的组中的氨基酸序列；所述VL-CDR2选自由SEQID NO：32、33、34、35、36组成的组中的氨基酸序列；所述VL-CDR3选自由SEQ ID NO：38、39、40、41、42组成的组中的氨基酸序列。
特别地，本发明的特异性结合CD123的抗体包括：
1)包含如SEQ ID NO：8所示的VH-CDR1、如SEQ ID NO：14所示的VH-CDR2和如SEQID NO：20所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：26所示的VL-CDR1、如SEQID NO：32所示的VL-CDR2和如SEQ ID NO：38所示的VL-CDR3的轻链可变结构域；和/或
2)包含如SEQ ID NO：9所示的VH-CDR1、如SEQ ID NO：15所示的VH-CDR2和如SEQID NO：21所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：27所示的VL-CDR1、如SEQID NO：33所示的VL-CDR2和如SEQ ID NO：39所示的VL-CDR3的轻链可变结构域；和/或
3)包含如SEQ ID NO：10所示的VH-CDR1、如SEQ ID NO：16所示的VH-CDR2和如SEQID NO：22所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：28所示的VL-CDR1、如SEQID NO：34所示的VL-CDR2和如SEQ ID NO：40所示的VL-CDR3的轻链可变结构域；和/或
4)包含如SEQ ID NO：11所示的VH-CDR1、如SEQ ID NO：17所示的VH-CDR2和如SEQID NO：23所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：29所示的VL-CDR1、如SEQID NO：35所示的VL-CDR2和如SEQ ID NO：41所示的VL-CDR3的轻链可变结构域；和/或
5)包含如SEQ ID NO：12所示的VH-CDR1、如SEQ ID NO：18所示的VH-CDR2和如SEQID NO：24所示的VH-CDR3的重链可变结构域；和包含如SEQ ID NO：30所示的VL-CDR1、如SEQID NO：36所示的VL-CDR2和如SEQ ID NO：42所示的VL-CDR3的轻链可变结构域。
在另一个具体实施方案中，本发明的抗体中的所述重链可变结构域包含选自由SEQ ID NO：44、45、46、47和48组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列；和/或所述轻链可变结构域包含选自由SEQID NO：50、51、52、53和54组成的组中的氨基酸序列，或包含与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
特别地，本发明提供了一种特异性结合CD123的抗体，其包含：
1)如SEQ ID NO：44所示的重链可变结构域和如SEQ ID NO：50所示的轻链可变结构域；和/或
2)如SEQ ID NO：45所示的重链可变结构域和如SEQ ID NO：51所示的轻链可变结构域；和/或
3)如SEQ ID NO：46所示的重链可变结构域和如SEQ ID NO：52所示的轻链可变结构域；和/或
4)如SEQ ID NO：47所示的重链可变结构域和如SEQ ID NO：53所示的轻链可变结构域；和/或
5)如SEQ ID NO：48所示的重链可变结构域和如SEQ ID NO：54所示的轻链可变结构域；
在另一个具体实施方案中，本发明提供了一种特异性结合CD123的抗体，其包含：选自由SEQ ID NO：56、57、58、59、60和61组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
优选地，本发明所述抗体为单链抗体scFv；更优选地，所述单链抗体scFv的重链可变结构域和轻链可变结构域由富含甘氨酸和丝氨酸的接头肽链相连，并且重链可变结构域和轻链可变结构域的顺序可互换。
本发明还提供了一种靶向CD123的嵌合抗原受体，其包含：本发明所述的抗体；铰链区；跨膜结构域；共刺激结构域；和CD3ζ胞内信号转导结构域。
在具体实施方案中，所述共刺激结构域选自CD27、CD28、4-1BB、OX-40、CD30、CD40、PD-1、ICOS、LFA-1、CD-2、CD7、LIGHT、NKG2C、B7-H3或其任意组合；优选地，所述共刺激结构域选自4-1BB或CD28，所述4-1BB包含如SEQ ID NO：62所述的氨基酸序列。
在一个具体实施方案中，所述CD3ζ胞内信号转导结构域包含如SEQ ID NO：63所述的氨基酸序列。
在一个具体实施方案中，所述铰链区和跨膜结构域选自IgG1、IgG4、CD8α、CD28、IL-2受体、IL-7受体、IL-11受体、PD-1或CD34的铰链区和跨膜结构域。
优选地，所述的嵌合抗原受体包含选自由SEQ ID NO：65、66、67、68、69和70组成的组中的氨基酸序列，或与其具有至少85％、至少90％、至少95％、至少99％同一性的氨基酸序列。
本发明还提供了一种核酸分子，其包含编码本发明的抗体或嵌合抗原受体的核苷酸序列。
本发明还提供了一种载体，其包含本发明的核酸分子；优选地，所述载体是病毒载体；更优选地，所述载体是慢病毒载体。
本发明还提供了一种细胞，其经本发明的载体转染，从而包含本发明的核酸分子或载体；优选地，所述细胞是T细胞。
本发明还提供了本发明的抗体、嵌合抗原受体、核酸分子、载体或细胞用于制备治疗表达CD123的肿瘤的药物的用途；优选地，所述表达CD123的肿瘤是急性髓系白血病(AML)。
除非特别指明，本文所述技术方案可以任意组合。
本发明进一步通过以下实施例来说明。
实施例1：CD123 CAR分子设计及CAR-T制备
本实施例以设计具有不同抗原识别域的CD123 CAR分子，及CAR-T细胞制备为例。
抗原识别域设计原则：CAR-T-1为对照组，其CD123的scFv序列来自宾夕法尼亚大学报道[21]，在本文中命名为scFv-01。CAR-T-2～7为改造组，其scFv来源于小鼠免疫CD123胞外区融合蛋白所得单克隆抗体序列，在本文中命名为scFv-02-07。
将不同抗原识别域的CD123 scFv的核苷酸序列通过基因合成的方法合成，然后通过同源重组方法与CD8α铰链区、CD8α跨膜区、4-1BB和CD3ζ胞内区序列连接形成完整的CAR分子，即CAR 1-7。将此CAR核酸片段通过限制性内切酶酶切和连接酶连接的方式，构建到慢病毒转移质粒pLenti6.3/V5(Thermo Fisher，Waltham，MA，USA)中。将慢病毒包装质粒pLP/VSVG，pLP1/MDK，pLP2/RSK(Thermo Fisher，Waltham，MA，USA)与上述获得的转移质粒，用Lipofectamine 3000(Thermo Fisher，Waltham，MA，USA)转染至HEK293T细胞，48小时后收集培养基，300g离心去除细胞碎片后，用超速离心机25000rpm离心3小时。将沉淀用1mL生理盐水溶解，即为所需的慢病毒载体。
从健康志愿者的外周血单个核细胞中，使用CD3/CD28 beads(Thermo Fisher，Cat#40203D)，分离出T细胞。将分离纯化后的T细胞以1.0×106个细胞/mL，接入含有500IU/mL IL-2(山东金泰生物工程有限公司，中国)的X-VIVO 15培养基(Lonza，Switzerland)中培养。在培养48小时后，用带有CD123 CAR分子的慢病毒载体以MOI＝0.5感染T细胞，以获得CAR-T-1-7。慢病毒感染24小时后，进行细胞换液，并接入新鲜培养体系X-VIVO 15加500IU/mL IL-2中继续培养。在慢病毒感染5天后，使用移液枪反复吹打培养体系中的细胞并收集至离心管，置于磁力架上去除CD3/CD28 beads。T细胞离心并计数，取部分细胞使用流式细胞仪(NovoCyte 2060R，ACEA Biosciences，San Diego，CA，USA)检测各组细胞的CAR分子转导效率。
图1所示为T细胞感染慢病毒5天、7天、9天、12天后，各组细胞的CAR分子转导效率。与对照组CAR-T-1相比，CAR-T-2、CAR-T-3、CAR-T-7组的CAR分子转导效率与其接近，CAR-T-4、CAR-T-5、CAR-T-6组明显高于对照组，说明各组CAR-T细胞的CAR蛋白表达情况均较好。
实施例2：改造后CD123 CAR-T的体外增殖能力
本实施例以检测具有不同抗原识别域的CD123 CAR-T的增殖能力为例。
在CD123 CAR-T细胞培养过程中，以慢病毒感染T细胞为培养第二天，之后每隔2至3天，收集CAR-T细胞并离心，加1-2毫升的培养体系重悬，取10微升的细胞稀释一定倍数后，用台盼蓝(Solarbio，Cat#C0040)进行染色，倒置显微镜(Ts2-FL，Nikon，日本)下分别计活细胞总数与死细胞总数，计算出各组细胞的增殖情况。
图2显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-2和CAR-T-7的总细胞增殖有明显提高。说明CAR-T-2和CAR-T-7的scFv设计能够改善CD123 CAR-T细胞增殖能力。
实施例3：改造后CD123 CAR-T在靶细胞刺激下的细胞分化情况
本实施例以检测具有不同抗原识别域的CD123 CAR-T，分别在两种靶细胞AML原代细胞或THP-1细胞(均表达CD123)的刺激下，其T细胞分化情况为例。
CAR-T细胞培养至第5天时，各组分别取两份5×105个细胞于12孔板中，以效靶比(E：T)＝5：1分别加入靶细胞AML原代细胞或THP-1细胞。在靶细胞刺激6天后，即CAR-T细胞培养至第11天时，各组分别取1×106个细胞并离心，100微升DPBS(HyClone，CAT#SH30028.02)重悬；然后每组CAR-T细胞分别加T细胞分化相关荧光抗体进行标记，使用全光谱流式细胞仪(N7-00008-0A，Cytek Biosciences，Inc.Fremont)进行检测。
图3的结果中，CD45RA+CD62L+细胞群表示初始T细胞群和干性记忆T细胞群的组合，CD45RA+CD62L-细胞群为效应T细胞群。具有不同抗原识别域的CAR-T-2和CAR-T-7的CD45RA+CD62L+细胞群占比高于对照组CAR-T-1，且CD45RA+CD62L-细胞群占比低于对照组CAR-T-1。说明改造后的CD123抗原识别域具有更好的CAR-T细胞持久性。
实施例4：改造后CD123 CAR-T在体外对细胞系的杀伤效率
本实施例以检测具有不同抗原识别域的CD123 CAR-T对表达CD123和不表达CD123的肿瘤细胞系的杀伤效率为例。
分别收集并离心表达CD123的细胞系THP-1，以及不表达CD123的细胞系K562，用1毫升生理盐水(河北天成药业有限公司，中国)重悬，加5微升Calcein-AM(浓度1μg/μL，Cat#C3100MP，ThermoFisher，USA)，轻轻混匀，置于37℃培养箱中孵育30分钟，以标记靶细胞。孵育结束后，使用生理盐水清洗2次，加X-VIVO 15重悬并计数。向48孔细胞培养板(CorningIncorporated，Corning，NY，USA)中，每孔加入1×105个上述标记的靶细胞，并以E：T＝10：1的比例加入各组CAR-T细胞，置于37℃、5％CO2培养箱中孵育5小时。孵育完成后，加2％浓度的Triton-X-100(Cat#T8787-100ML，Sigma，Germany)裂解阳性对照组，即1×105个上述标记的靶细胞；每孔分别取100微升杀伤体系上清于酶标板中，使用多功能酶标仪(VarioscanLux，ThermoFisher)检测荧光值(激发波长：495nm，发射波长：515nm)。
图4显示，对于CD123阳性细胞系THP-1，具有不同抗原识别域的CD123 CAR-T中CAR-T-3和CAR-T-5的杀伤效率，较对照组CAR-T-1显著升高。同时，各组改造后CD123 CAR-T和对照组CAR-T-1，对不表达CD123的K562细胞系的杀伤效率，与T细胞组一致。说明各组CD123 CAR-T细胞对靶细胞均有一定杀伤效应，并有较好的特异性，且CAR-T-3和CAR-T-5的scFv设计更有利于CAR-T杀伤肿瘤细胞。
实施例5：改造后CD123 CAR-T在体外对AML原代细胞的杀伤效率
本实施例以检测具有不同抗原识别域的CD123 CAR-T对AML原代细胞的杀伤效率为例。
取3个分别为高抗原密度(即AML 1#，CD123表达率为99.87％，MFI为13298)、中抗原密度(即AML 2#，CD123表达率为75.4％，MFI为7518)、低抗原密度(即AML 3#，CD123表达率为94.58％，MFI为5252)的CD123阳性AML患者骨髓血样(抗原密度水平定义如下，高抗原密度：中位荧光强度(Median Fluorescence Intensity，MFI)大于10000；中抗原密度：MFI大于6000小于10000；低抗原密度：MFI小于6000)，将AML患者的骨髓血样本分离单个核细胞，并使用CD34 MicroBeads(Cat#130-100-453，美天旎，德国)分选出CD34阳性AML原代细胞，用1毫升生理盐水重悬，加10微升Calcein-AM(浓度1μg/μL，ThermoFisher，USA)，轻轻混匀，置于37℃培养箱中孵育30分钟，以标记AML原代细胞。孵育结束后，使用生理盐水清洗2次，加X-VIVO 15重悬并计数。向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×105个上述标记的AML原代细胞，并以E∶T＝10∶1的比例，分别加入CD123 CAR-T细胞，置于37℃、5％CO2培养箱中孵育5小时。孵育完成后，加2％浓度的Triton-X-100裂解阳性对照组，即1×105个上述标记的AML原代细胞；每孔分别取100微升杀伤体系上清于酶标板中，使用多功能酶标仪(Varioscan Lux，ThermoFisher)检测荧光值(激发波长：495nm，发射波长：515nm)。
图5显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-3对3个高(即AML1#)、中(即AML 2#)、低(即AML 3#)抗原密度的CD123阳性AML患者的原代细胞均具有显著增强的杀伤效率。说明具有CAR-T-3scFv的CD123 CAR-T细胞，能够有效提高对原代细胞的杀伤效率。
实施例6：改造后CD123 CAR-T在靶细胞刺激下的细胞因子表达
本实施例以检测具有不同抗原识别域的CD123 CAR-T在靶细胞AML原代细胞刺激下的细胞因子表达水平为例。
向48孔细胞培养板(Corning Incorporated，Corning，NY，USA)中，每孔加入1×105个靶细胞AML原代细胞，此AML原代细胞的CD123表达率为99.87％、MFI为13298，并以E：T＝10：1的比例加入各组CAR-T细胞，置于37℃、5％CO2培养箱中孵育5小时。取细胞上清，使用CBA试剂盒(BD Biosciences，San Jose，CA，USA)和流式细胞仪(NovoCyte 2060R，ACEABiosciences，San Diego，CA，USA)检测各种细胞因子的表达水平。
图6显示，与对照组CAR-T-1相比，具有不同抗原识别域的CAR-T-6组，在靶细胞刺激下，IL-2、TNF、IFN-γ的表达水平明显提高；其余各组CAR-T细胞的细胞因子表达，与对照组CAR-T-1相近。说明CAR-T-6的scFv设计有利于CD123 CAR-T细胞因子的表达。
以下表1和表2中为本发明中涉及的具体氨基酸序列。
表1示例性抗体的CDR和scFv序列
表2示例性嵌合抗原受体序列
参考文献:
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Ala Arg Gly Met Asp Tyr
1 5
<210> 23
<211> 19
<212> PRT
<213> 合成序列
<400> 23
Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala
1 5 10 15
Met Asp Tyr
<210> 24
<211> 12
<212> PRT
<213> 合成序列
<400> 24
Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys
1 5 10
<210> 25
<211> 11
<212> PRT
<213> 合成序列
<400> 25
Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp
1 5 10
<210> 26
<211> 11
<212> PRT
<213> 合成序列
<400> 26
Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp
1 5 10
<210> 27
<211> 15
<212> PRT
<213> 合成序列
<400> 27
Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp
1 5 10 15
<210> 28
<211> 12
<212> PRT
<213> 合成序列
<400> 28
Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp
1 5 10
<210> 29
<211> 11
<212> PRT
<213> 合成序列
<400> 29
Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp
1 5 10
<210> 30
<211> 17
<212> PRT
<213> 合成序列
<400> 30
Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala
1 5 10 15
Trp
<210> 31
<211> 7
<212> PRT
<213> 合成序列
<400> 31
Gly Ser Thr Leu Gln Ser Gly
1 5
<210> 32
<211> 7
<212> PRT
<213> 合成序列
<400> 32
Thr Ser Thr Leu Gln Pro Gly
1 5
<210> 33
<211> 7
<212> PRT
<213> 合成序列
<400> 33
Ala Ser Asn Leu Glu Ser Gly
1 5
<210> 34
<211> 7
<212> PRT
<213> 合成序列
<400> 34
Thr Ser Asn Leu Ala Ser Gly
1 5
<210> 35
<211> 7
<212> PRT
<213> 合成序列
<400> 35
Ala Ser Gln Ser Ile Ser Glu
1 5
<210> 36
<211> 7
<212> PRT
<213> 合成序列
<400> 36
Ala Ser Thr Arg Asp Ser Gly
1 5
<210> 37
<211> 9
<212> PRT
<213> 合成序列
<400> 37
Gln His Asn Lys Tyr Pro Tyr Thr Phe
1 5
<210> 38
<211> 8
<212> PRT
<213> 合成序列
<400> 38
Gln Tyr Asp Asp Leu Tyr Thr Phe
1 5
<210> 39
<211> 9
<212> PRT
<213> 合成序列
<400> 39
Gln Ser Asn Glu Asp Pro Tyr Thr Phe
1 5
<210> 40
<211> 9
<212> PRT
<213> 合成序列
<400> 40
Gln Phe His Arg Ser Pro Arg Thr Phe
1 5
<210> 41
<211> 9
<212> PRT
<213> 合成序列
<400> 41
Asn Gly His Ser Phe Pro Tyr Thr Phe
1 5
<210> 42
<211> 9
<212> PRT
<213> 合成序列
<400> 42
Gln His Tyr Ser Thr Pro Trp Thr Phe
1 5
<210> 43
<211> 115
<212> PRT
<213> 合成序列
<400> 43
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser
115
<210> 44
<211> 123
<212> PRT
<213> 合成序列
<400> 44
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 45
<211> 124
<212> PRT
<213> 合成序列
<400> 45
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Met Thr Tyr
20 25 30
Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Phe
35 40 45
Gly Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 46
<211> 113
<212> PRT
<213> 合成序列
<400> 46
Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser
<210> 47
<211> 126
<212> PRT
<213> 合成序列
<400> 47
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala
100 105 110
Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser
115 120 125
<210> 48
<211> 119
<212> PRT
<213> 合成序列
<400> 48
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Ser Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Asn Ala Thr Ser Tyr Asn Gln Asn Phe
50 55 60
Lys Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 49
<211> 107
<212> PRT
<213> 合成序列
<400> 49
Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly
1 5 10 15
Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp
20 25 30
Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile
35 40 45
Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 50
<211> 106
<212> PRT
<213> 合成序列
<400> 50
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr
20 25 30
Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile
35 40 45
His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 51
<211> 111
<212> PRT
<213> 合成序列
<400> 51
Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn
85 90 95
Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys
100 105 110
<210> 52
<211> 108
<212> PRT
<213> 合成序列
<400> 52
Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Phe Leu Gly
1 5 10 15
Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser
20 25 30
Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp
35 40 45
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Phe His Arg Ser Pro
85 90 95
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 53
<211> 107
<212> PRT
<213> 合成序列
<400> 53
Asp Ile Gln Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly
1 5 10 15
Asp Arg Val Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr
20 25 30
Leu His Trp Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile
35 40 45
Lys Tyr Ala Ser Gln Ser Ile Ser Glu Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro
65 70 75 80
Glu Asp Val Gly Val Tyr Phe Cys Gln Asn Gly His Ser Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 54
<211> 113
<212> PRT
<213> 合成序列
<400> 54
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly
1 5 10 15
Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val
50 55 60
Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln
85 90 95
His Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
100 105 110
Lys
<210> 55
<211> 237
<212> PRT
<213> 合成序列
<400> 55
Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Trp Met Asn Trp Val Lys Gln Arg Pro Asp Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Tyr Asp Ser Glu Thr His Tyr Asn Gln Lys Phe
50 55 60
Lys Asp Lys Ala Ile Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn Trp Asp Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr
100 105 110
Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser
130 135 140
Pro Gly Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser
145 150 155 160
Lys Asp Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu
165 170 175
Leu Ile Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe
180 185 190
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
195 200 205
Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His Asn Lys Tyr
210 215 220
Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235
<210> 56
<211> 244
<212> PRT
<213> 合成序列
<400> 56
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr
20 25 30
Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro Lys Phe
50 55 60
Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln
130 135 140
Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr
145 150 155 160
Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His
165 170 175
Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu
180 185 190
Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gly Asp
195 200 205
Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr
210 215 220
Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr Phe Gly Gly Gly Thr Lys
225 230 235 240
Leu Glu Ile Lys
<210> 57
<211> 250
<212> PRT
<213> 合成序列
<400> 57
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Met Thr Tyr
20 25 30
Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Phe
35 40 45
Gly Tyr Cys Asn Pro Tyr Asn Asp Gly Ile Asn Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Pro Ser Tyr Tyr Gly Arg Ser Tyr Tyr Tyr Gly Met Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr
130 135 140
Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile
145 150 155 160
Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met
165 170 175
Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Val Leu Ile Tyr
180 185 190
Ala Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala Arg Phe Ser Gly Ser
195 200 205
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu Glu Glu
210 215 220
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn Glu Asp Pro Tyr Thr
225 230 235 240
Phe Gly Gly Gly Thr Lys Leu Asp Ile Lys
245 250
<210> 58
<211> 236
<212> PRT
<213> 合成序列
<400> 58
Asp Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp
20 25 30
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile Ser Tyr Ser Gly Ser Ser Asn Ser Asn Pro Ser Leu
50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Arg Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Val Phe Leu Gly
130 135 140
Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser Ser Ser
145 150 155 160
Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp
165 170 175
Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser
180 185 190
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
195 200 205
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Phe His Arg Ser Pro
210 215 220
Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
225 230 235
<210> 59
<211> 248
<212> PRT
<213> 合成序列
<400> 59
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Ser Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Asn Met His Trp Val Lys Gln Thr Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Tyr Pro Gly Asn Gly Gly Thr Asn Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Ile Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys
85 90 95
Ala Arg Glu Arg Gly Leu Tyr Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala
100 105 110
Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly
115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
130 135 140
Leu Thr Gln Ser Pro Ala Thr Leu Ser Val Thr Pro Gly Asp Arg Val
145 150 155 160
Ser Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Asp Tyr Leu His Trp
165 170 175
Tyr Gln Gln Lys Ser His Glu Ser Pro Arg Leu Leu Ile Lys Tyr Ala
180 185 190
Ser Gln Ser Ile Ser Glu Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser
195 200 205
Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Pro Glu Asp Val
210 215 220
Gly Val Tyr Phe Cys Gln Asn Gly His Ser Phe Pro Tyr Thr Phe Gly
225 230 235 240
Gly Gly Thr Lys Leu Glu Ile Lys
245
<210> 60
<211> 247
<212> PRT
<213> 合成序列
<400> 60
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Ser Gly Ala
1 5 10 15
Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr
20 25 30
Tyr Met His Trp Val Lys Gln Ser His Val Lys Ser Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asn Pro Tyr Asn Asn Ala Thr Ser Tyr Asn Gln Asn Phe
50 55 60
Lys Asp Lys Ala Ser Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Gly Glu Gly Trp Leu Leu Pro Leu Ala Cys Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Ser Ser
130 135 140
Leu Ala Met Ser Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Ser
145 150 155 160
Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr
165 170 175
Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser
180 185 190
Thr Arg Asp Ser Gly Val Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly
195 200 205
Thr Asp Phe Thr Leu Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala
210 215 220
Asp Tyr Phe Cys Gln Gln His Tyr Ser Thr Pro Trp Thr Phe Gly Gly
225 230 235 240
Gly Thr Lys Leu Glu Ile Lys
245
<210> 61
<211> 244
<212> PRT
<213> 合成序列
<400> 61
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr
20 25 30
Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile
35 40 45
His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp Leu Tyr Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser
115 120 125
Gly Pro Glu Leu Val Lys Thr Gly Ala Ser Val Lys Met Ser Cys Lys
130 135 140
Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His Trp Val Arg Gln
145 150 155 160
Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn
165 170 175
Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr
180 185 190
Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Phe Lys Ser Leu Thr
195 200 205
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr
210 215 220
Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
225 230 235 240
Thr Val Ser Ser
<210> 62
<211> 42
<212> PRT
<213> 合成序列
<400> 62
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
1 5 10 15
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
20 25 30
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
35 40
<210> 63
<211> 112
<212> PRT
<213> 合成序列
<400> 63
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
1 5 10 15
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
20 25 30
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
50 55 60
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
65 70 75 80
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
85 90 95
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 64
<211> 501
<212> PRT
<213> 合成序列
<400> 64
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Pro
35 40 45
Gly Ala Glu Leu Val Arg Pro Gly Ala Ser Val Lys Leu Ser Cys Lys
50 55 60
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Trp Met Asn Trp Val Lys Gln
65 70 75 80
Arg Pro Asp Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Tyr Asp
85 90 95
Ser Glu Thr His Tyr Asn Gln Lys Phe Lys Asp Lys Ala Ile Leu Thr
100 105 110
Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu Thr
115 120 125
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Asn Trp Asp Asp
130 135 140
Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Gln Ile Thr
165 170 175
Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly Glu Thr Ile Thr Ile
180 185 190
Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Asp Leu Ala Trp Tyr Gln
195 200 205
Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile Tyr Ser Gly Ser Thr
210 215 220
Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr
225 230 235 240
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Met
245 250 255
Tyr Tyr Cys Gln Gln His Asn Lys Tyr Pro Tyr Thr Phe Gly Gly Gly
260 265 270
Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
275 280 285
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
290 295 300
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
305 310 315 320
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
325 330 335
Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys
340 345 350
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
355 360 365
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
370 375 380
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
385 390 395 400
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
405 410 415
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
420 425 430
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
435 440 445
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
450 455 460
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
465 470 475 480
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
485 490 495
Ala Leu Pro Pro Arg
500
<210> 65
<211> 508
<212> PRT
<213> 合成序列
<400> 65
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser
35 40 45
Gly Pro Glu Leu Val Lys Thr Gly Ala Ser Val Lys Met Ser Cys Lys
50 55 60
Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Tyr Val His Trp Val Arg Gln
65 70 75 80
Ser His Gly Lys Ser Leu Glu Trp Ile Gly Tyr Ile Ser Cys Tyr Asn
85 90 95
Gly Ala Thr Asn Tyr Asn Pro Lys Phe Lys Gly Lys Ala Thr Phe Thr
100 105 110
Val Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Phe Lys Ser Leu Thr
115 120 125
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr
130 135 140
Gly Tyr Asp Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
145 150 155 160
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
165 170 175
Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala
180 185 190
Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile
195 200 205
Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg
210 215 220
Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg
225 230 235 240
Phe Ser Gly Ser Gly Ser Gly Gly Asp Tyr Ser Phe Ser Ile Ser Asn
245 250 255
Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asp
260 265 270
Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr
275 280 285
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
290 295 300
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
305 310 315 320
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
325 330 335
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
340 345 350
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
355 360 365
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
370 375 380
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
385 390 395 400
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
405 410 415
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
420 425 430
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
435 440 445
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
450 455 460
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
465 470 475 480
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
485 490 495
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505
<210> 66
<211> 514
<212> PRT
<213> 合成序列
<400> 66
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser
35 40 45
Gly Pro Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys
50 55 60
Ala Ser Gly Tyr Thr Phe Met Thr Tyr Val Ile His Trp Val Lys Gln
65 70 75 80
Lys Pro Gly Gln Gly Leu Glu Trp Phe Gly Tyr Cys Asn Pro Tyr Asn
85 90 95
Asp Gly Ile Asn Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr
100 105 110
Ser Asp Lys Ser Ser Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Thr
115 120 125
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Ser Pro Ser Tyr Tyr
130 135 140
Gly Arg Ser Tyr Tyr Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser
145 150 155 160
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
165 170 175
Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Ser Leu Ala
180 185 190
Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser
195 200 205
Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro
210 215 220
Gly Gln Pro Pro Lys Val Leu Ile Tyr Ala Ala Ser Asn Leu Glu Ser
225 230 235 240
Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
245 250 255
Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys
260 265 270
Gln Gln Ser Asn Glu Asp Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu
275 280 285
Asp Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
290 295 300
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
305 310 315 320
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
325 330 335
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
340 345 350
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
355 360 365
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
370 375 380
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
385 390 395 400
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys
405 410 415
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
420 425 430
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
435 440 445
Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
450 455 460
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
465 470 475 480
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
485 490 495
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
500 505 510
Pro Arg
<210> 67
<211> 500
<212> PRT
<213> 合成序列
<400> 67
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Lys Leu Gln Glu Ser
35 40 45
Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr
50 55 60
Val Thr Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn Trp Ile Arg
65 70 75 80
Gln Phe Pro Gly Asn Lys Leu Glu Trp Met Gly Tyr Ile Ser Tyr Ser
85 90 95
Gly Ser Ser Asn Ser Asn Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr
100 105 110
Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr
115 120 125
Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala Arg Gly Met Asp Tyr Trp
130 135 140
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
145 150 155 160
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser
165 170 175
Pro Ala Ile Met Ser Val Phe Leu Gly Glu Arg Val Thr Met Thr Cys
180 185 190
Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His Trp Tyr Gln Gln
195 200 205
Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu
210 215 220
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser
225 230 235 240
Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr
245 250 255
Tyr Cys His Gln Phe His Arg Ser Pro Arg Thr Phe Gly Gly Gly Thr
260 265 270
Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
275 280 285
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
290 295 300
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
305 310 315 320
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
325 330 335
Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys
340 345 350
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
355 360 365
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
370 375 380
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
385 390 395 400
Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
405 410 415
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
420 425 430
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
435 440 445
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
450 455 460
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
465 470 475 480
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
485 490 495
Leu Pro Pro Arg
500
<210> 68
<211> 512
<212> PRT
<213> 合成序列
<400> 68
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser
35 40 45
Gly Ala Glu Leu Val Arg Ser Gly Ala Ser Val Lys Met Ser Cys Lys
50 55 60
Ala Ser Gly Tyr Thr Phe Ser Ser Tyr Asn Met His Trp Val Lys Gln
65 70 75 80
Thr Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Tyr Pro Gly Asn
85 90 95
Gly Gly Thr Asn Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr
100 105 110
Ala Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln Ile Ser Ser Leu Thr
115 120 125
Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Glu Arg Gly Leu Tyr
130 135 140
Asp Tyr Asp Glu Thr Gly Tyr Tyr Ala Met Asp Tyr Trp Gly Gln Gly
145 150 155 160
Thr Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
165 170 175
Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ala Thr
180 185 190
Leu Ser Val Thr Pro Gly Asp Arg Val Ser Leu Ser Cys Arg Ala Ser
195 200 205
Gln Ser Ile Ser Asp Tyr Leu His Trp Tyr Gln Gln Lys Ser His Glu
210 215 220
Ser Pro Arg Leu Leu Ile Lys Tyr Ala Ser Gln Ser Ile Ser Glu Ile
225 230 235 240
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Ser Asp Phe Thr Leu Ser
245 250 255
Ile Asn Ser Val Glu Pro Glu Asp Val Gly Val Tyr Phe Cys Gln Asn
260 265 270
Gly His Ser Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
275 280 285
Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile
290 295 300
Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala
305 310 315 320
Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr
325 330 335
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
340 345 350
Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile
355 360 365
Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
370 375 380
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
385 390 395 400
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly
405 410 415
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
420 425 430
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
435 440 445
Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
450 455 460
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
465 470 475 480
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
485 490 495
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 510
<210> 69
<211> 511
<212> PRT
<213> 合成序列
<400> 69
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Lys Gln Ser
35 40 45
Gly Pro Glu Leu Val Lys Ser Gly Ala Ser Val Lys Ile Ser Cys Lys
50 55 60
Ala Ser Gly Tyr Ser Phe Thr Gly Tyr Tyr Met His Trp Val Lys Gln
65 70 75 80
Ser His Val Lys Ser Leu Glu Trp Ile Gly Arg Ile Asn Pro Tyr Asn
85 90 95
Asn Ala Thr Ser Tyr Asn Gln Asn Phe Lys Asp Lys Ala Ser Leu Thr
100 105 110
Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu His Ser Leu Thr
115 120 125
Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Glu Gly Trp Leu
130 135 140
Leu Pro Leu Ala Cys Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
145 150 155 160
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp
165 170 175
Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ala Met Ser Val Gly Gln
180 185 190
Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser
195 200 205
Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
210 215 220
Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Asp Ser Gly Val Pro
225 230 235 240
Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
245 250 255
Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln His
260 265 270
Tyr Ser Thr Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
275 280 285
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
290 295 300
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
305 310 315 320
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
325 330 335
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
340 345 350
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
355 360 365
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
370 375 380
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
385 390 395 400
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln
405 410 415
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
420 425 430
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
435 440 445
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
450 455 460
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
465 470 475 480
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
485 490 495
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 510
<210> 70
<211> 508
<212> PRT
<213> 合成序列
<400> 70
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 Asn Trp Ser His Pro Gln Phe Glu Lys Gly
20 25 30
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Leu Thr Gln Ser
35 40 45
Pro Ser Ser Leu Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys
50 55 60
Lys Ala Ser Gln Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys
65 70 75 80
Pro Gly Lys Gly Pro Arg Leu Leu Ile His Tyr Thr Ser Thr Leu Gln
85 90 95
Pro Gly Ile Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Gly Asp Tyr
100 105 110
Ser Phe Ser Ile Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr
115 120 125
Cys Leu Gln Tyr Asp Asp Leu Tyr Thr Phe Gly Gly Gly Thr Lys Leu
130 135 140
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Thr
165 170 175
Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr
180 185 190
Asp Tyr Tyr Val His Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu
195 200 205
Trp Ile Gly Tyr Ile Ser Cys Tyr Asn Gly Ala Thr Asn Tyr Asn Pro
210 215 220
Lys Phe Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr
225 230 235 240
Ala Tyr Met Gln Phe Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
245 250 255
Tyr Cys Ala Gly Gly Glu Gly Tyr Tyr Gly Tyr Asp Gly Tyr Ala Met
260 265 270
Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Thr Thr Thr
275 280 285
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
290 295 300
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
305 310 315 320
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
325 330 335
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
340 345 350
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
355 360 365
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
370 375 380
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
385 390 395 400
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu
405 410 415
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
420 425 430
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
435 440 445
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
450 455 460
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
465 470 475 480
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
485 490 495
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505
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