CN113388022A - 一种输卵管癌靶标抗原、输卵管癌靶标抗原刺激培养的ctl细胞及其应用 - Google Patents
一种输卵管癌靶标抗原、输卵管癌靶标抗原刺激培养的ctl细胞及其应用 Download PDFInfo
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Abstract
本发明提供了一种输卵管癌靶标抗原、输卵管癌靶标抗原刺激培养的CTL细胞及其应用，属于肿瘤治疗技术领域。本发明中所述输卵管癌靶标抗原的氨基酸序列如SEQ ID No.23所示；本发明所述的输卵管癌靶标抗原是基于患者肿瘤组织的全外显子检测结果，通过预测分析、筛选获得的有效的肿瘤抗原，利用所述输卵管癌靶标抗原培养获得的CTL细胞，对肿瘤具有特异性的杀伤作用；本发明提供的输卵管癌靶标抗原可以应用于输卵管癌的治疗。
Description
本申请是申请日为2020年03月18日、申请号为202010190785.9、发明名称为《一种输卵管癌靶标抗原组合、输卵管癌靶标抗原组合刺激培养的CTL细胞及其应用》的分案申请。
技术领域
本发明属于肿瘤治疗技术领域，尤其涉及一种输卵管癌靶标抗原组合、输卵管癌靶标抗原组合刺激培养的CTL细胞及其应用。
背景技术
原发性输卵管癌是一种少见的女性生殖道恶性肿瘤。其发病率占妇科恶性肿瘤的0.5％。以40～65岁居多，多发生于绝经后妇女。目前对于输卵管癌的治疗原则是以手术为主，辅助放疗、化疗的综合治疗。
随着精准治疗的推广，常规的化疗、放疗和靶向已无法满足肿瘤的个性化和多变性，因此，寻找个性化的肿瘤新抗原，培养识别个性化肿瘤新抗原的CTL，将是彻底清除肿瘤的有效方法。
发明内容
有鉴于此，本发明的目的在于提供一种输卵管癌靶标抗原组合、输卵管癌靶标抗原组合刺激培养的CTL细胞及其应用；本发明提供的输卵管癌靶标抗原组合是基于患者肿瘤组织的全外显子检测结果，通过预测分析、筛选获得的有效的肿瘤抗原，利用所述输卵管癌靶标抗原组合培养获得的CTL细胞，对肿瘤具有特异性的杀伤作用；本发明提供的输卵管癌靶标抗原特异性针对同一种HLA类型的输卵管癌，实现个性化治疗。
本发明提供了一种输卵管癌靶标抗原组合，包括氨基酸序列如SEQ ID No.7、SEQID No.8、SEQ ID No.9、SEQ ID No.16、SEQ ID No.23、SEQ ID No.31和SEQ ID No.45所示的抗原中的一种或几种。
本发明提供了所述的输卵管癌靶标抗原组合在制备治疗输卵管癌的药物中的应用。
本发明提供了所述的输卵管癌靶标抗原组合在制备治疗输卵管癌的CTL细胞中的应用。
本发明提供了一种CTL细胞，所述CTL细胞为所述的输卵管癌靶标抗原组合刺激培养的CTL细胞。
本发明提供了所述的CTL细胞的制备方法，包括以下步骤：
1)将CTL细胞浓度调整至2～3×105cells/mL；
2)将调整细胞浓度后的CTL细胞、IL-2和所述的输卵管癌靶标抗原组合混合获得培养体系；
3)将所述培养体系进行培养获得输卵管癌靶标抗原组合刺激培养的CTL细胞。
优选的，IL-2在培养体系中的终浓度为150～250U/mL。
优选的，所述的输卵管癌靶标抗原组合中每一种输卵管癌靶标抗原在培养体系中的终浓度为40～60ng/mL。
优选的，步骤3)中所述培养的温度为36～38℃，所述培养的时间为22～26h。
优选的，步骤1)中所述的CTL细胞是通过PBMC细胞与APC细胞共培养获得。
本发明提供了所述的CTL细胞在制备治疗输卵管癌药物中的应用。
本发明的有益效果：本发明提供的输卵管癌靶标抗原组合是基于患者肿瘤组织的全外显子测序结果，通过预测分析、筛选获得的有效的肿瘤抗原，利用所述输卵管癌靶标抗原组合刺激培养获得的CTL细胞，对肿瘤具有特异性的杀伤作用；对所述CTL细胞培养后，流式分析细胞表型，结果显示NKT(CD56+CD3+)比例为29.31％，CD8+T细胞的比例为50.93％。
附图说明
图1为输卵管癌靶标抗原1～26号筛选结果；
图2为输卵管癌靶标抗原27～50号筛选结果
图3为输卵管癌靶标抗原组合9号和16号刺激培养的CTL对负载抗原多肽的靶细胞和只负载HLA的靶细胞的杀伤效率对比；
图4为输卵管癌靶标抗原组合31号和45号刺激培养的CTL对负载抗原多肽的靶细胞和只负载HLA的靶细胞的杀伤效率对比；
图5为输卵管癌靶标抗原组合7号、8号和23号刺激培养的CTL对负载抗原多肽的靶细胞和只负载HLA的靶细胞的杀伤效率对比；
图6为输卵管癌靶标抗原组合刺激培养的CTL流式分析细胞表型结果。
具体实施方式
本发明提供了一种输卵管癌靶标抗原组合，包括氨基酸序列如SEQ ID No.7、SEQID No.8、SEQ ID No.9、SEQ ID No.16、SEQ ID No.23、SEQ ID No.31和SEQ ID No.45所示的抗原中的一种或几种；具体如下所示：
| 多肽编号 | HLAAllele | MTEpitopeSeq | |
| 7 | NDUFS1 | HLA-B*15:01 | RLSVAGNCRMY(SEQ ID No.7) |
| 8 | C17orf97 | HLA-B*15:01 | RYQCLALKGF(SEQ ID No.8) |
| 9 | RAP2C | HLA-A*26:01 | YHKEIEVD(SEQ ID No.9) |
| 16 | PARP10 | HLA-A*26:01 | TVYGTGVYF(SEQ ID No.16) |
| 23 | C17orf97 | HLA-B*15:01 | QCLALKGF(SEQ ID No.23) |
| 31 | GPNMB | HLA-A*02:01 | VDVEEMCLLTV(SEQ ID No.31) |
| 45 | TRGC2 | HLA-A*02:01 | LLGRTAFC(SEQ ID No.45) |
在本发明中，所述输卵管癌靶标抗原组合优选的通过对患者肿瘤组织进行全外显子测序，通过分析测序结果、筛选获得的有效的肿瘤抗原。本发明对所述外显子测序的方法没有特殊限定，采用本领域常规的外显子测序方法即可。本发明在获得所述外显子测序的结果后，优选的采用肿瘤新抗原分析技术对外显子测序结果进行分析，在本发明中，所述分析优选的采用ImmunoMining肿瘤个体化新抗原分析预测软件进行，所述软件著作权的登记号:2019SR0130720。
本发明提供了所述的输卵管癌靶标抗原组合在制备治疗输卵管癌的药物中的应用。在本发明中，所述输卵管癌靶标抗原组合通过刺激培养CTL细胞实现对输卵管癌的治疗。
本发明还提供了所述的输卵管癌靶标抗原组合在制备治疗输卵管癌的CTL细胞中的应用。在本发明中，利用所述输卵管癌靶标抗原组合刺激培养CTL细胞，使获得的CTL细胞对于负载所述输卵管癌靶标抗原组合的细胞具有特异性的杀伤作用。
本发明提供了一种CTL细胞，所述CTL细胞为所述的输卵管癌靶标抗原组合刺激培养的CTL细胞。
本发明还提供了所述的CTL细胞的制备方法，包括以下步骤：1)将CTL细胞浓度调整至2～3×105cells/mL；2)将调整细胞浓度后的CTL细胞、IL-2和所述的输卵管癌靶标抗原组合混合获得培养体系；3)将所述培养体系进行培养获得输卵管癌靶标抗原组合刺激培养的CTL细胞。
在本发明中，所述CTL细胞优选的是通过PBMC细胞与APC细胞共培养获得。在本发明中，所述APC细胞的培养步骤优选的如下：将PBMC细胞复苏培养后，以输卵管癌靶标抗原溶液重悬培养获得。在本发明中，所述复苏培养的培养基优选为1640+10％(v/v)FBS；所述复苏培养的时间优选为22～26h，更优选为24h；所述复苏培养的温度优选为37℃，所述复苏培养的环境二氧化碳浓度优选为5％；所述复苏培养前PBMC细胞的浓度优选为1×106cells/mL；所述复苏培养前PBMC细胞的浓度优选的通过以复苏培养基稀释调整。本发明在所述复苏培养后，优选的进行离心收集细胞，所述离心的转速优选为1400～1600rpm，更优选为1500rpm，所述离心的时间优选为4～6min，更优选为5min。
在本发明中，所述输卵管癌靶标抗原溶液优选的包括以下组分：1640+10％FBS+150～250U/mL IL-2+1500～1700U/mL GM-CSF+40～60ng/mL输卵管癌靶标抗原(每一种输卵管癌靶标抗原的浓度)，更优选为1640+10％FBS+200U/mL IL-2+1600U/mL GM-CSF+50ng/mL输卵管癌靶标抗原。在本发明中，所述IL-2为细胞生长因子，能使T细胞长期存活，刺激T细胞进人细胞分裂周期；所述GM-CSF在体外可刺激中性粒细胞和巨噬细胞的集落形成；本发明对所述IL-2和GM-CSF的来源没有特殊限定，采用本领域常规的IL-2和GM-CSF即可。
在本发明中，以所述输卵管癌靶标抗原溶液重悬上述离心收集的细胞进行重悬培养。在本发明中，重悬后的细胞密度优选为1×106cells/mL，所述重悬培养的时间优选为22～26h，更优选为24h；所述重悬培养的温度优选为37℃，所述复苏培养的环境二氧化碳浓度优选为5％。本发明在所述重悬培养后获得APC细胞。
在本发明中，将上述培养获得的APC细胞和PBMC细胞共培养获得CTL细胞。在本发明中，所述APC细胞和PBMC细胞的数量比例优选为1:(90～110)，更优选为1:100。在本发明中，所述共培养的时间优选为20～22d，更优选为21d。在本发明中，当共培养48h后，每天向所述共培养体系中添加IL-2，使所述IL-2的终浓度为400～600U/mL，更优选为500U/mL。
本发明在获得所述CTL细胞后，将所述CTL细胞浓度调整至2～3×105cells/mL，更优选为2.5×105cells/mL。
在本发明中，将调整细胞浓度后的CTL细胞、IL-2和所述的输卵管癌靶标抗原组合混合获得培养体系。在本发明中，IL-2在培养体系中的终浓度优选为150～250U/mL，更优选为200U/mL；所述的输卵管癌靶标抗原组合中每一种输卵管癌靶标抗原在培养体系中的终浓度优选为40～60ng/mL，更优选为50ng/mL。
在本发明中，将所述培养体系进行培养获得输卵管癌靶标抗原组合刺激培养的CTL细胞。在本发明中，所述培养的温度优选为36～38℃，更优选为37℃，所述培养的时间优选为22～26h，更优选为24h。本发明在所述培养后，优选的收集所述输卵管癌靶标抗原组合刺激培养的CTL细胞；所述收集优选的采用离心的方法，所述离心的转速优选为1000～1500rpm，所述离心的时间优选为5～10min。
本发明提供了所述的CTL细胞在制备治疗输卵管癌药物中的应用。在本发明中，所述CTL细胞能够特异性杀伤肿瘤细胞，对负载所述输卵管癌靶标抗原组合的肿瘤细胞的杀伤力远远大于未负载所述输卵管癌靶标抗原的肿瘤细胞。本发明对所述药物的剂型没有特殊限定，采用本领域常规的细胞药物剂型即可。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
肿瘤组织的全外显子测序结果，采用ImmunoMining肿瘤个体化新抗原分析预测软件进行肿瘤新抗原分析，所述软件著作权的登记号:2019SR0130720。鼎成肽源生物信息技术有限公司，获得50条预测抗原；对预测的50条抗原进行合成(由南京金斯瑞多肽合成部合成)。
表1多肽序列及对应编号
1.共培养制备APC：
1)复苏冻存PBMC(2.5×107cells)，以1640+10％FBS稀释冻存液，1500rpm离心5min，以10mL 1640+10％FBS重悬，1×106cells/mL，37℃、5％CO2复苏培养24h；
2)分别配制抗原多肽溶液，1640+10％FBS+200U/mL IL-2+1600U/mL GM-CSF，每一种抗原多肽的终浓度为50ng/mL，混匀后备用；
3)1500rpm离心5min，离心收集细胞，以多肽溶液重悬细胞，密度调整至1×106cells/mL；
4)37℃、5％CO2培养24h后，细胞为APC，轻轻吹打细胞混匀备用。
2.共培养
1)离心收集PBMC，1640+10％FBS重悬，计数调整至1×106cells/mL；
2)按照PBMC：APC数量比为100:1的比例加入APC，混匀，记为Day 0；
3)37℃、5％CO2培养48h后，每天按照总体积补加IL-2，确保IL-2的终浓度为200U/mL；
4)培养至Day 21时，收集细胞即为CTL；
3.肿瘤新抗原的筛选：
1)收集上述培养获得的CTL细胞，以1640+10％FBS重悬，计数调整至2×106cells/mL；
2)10mL CTL细胞+10mL 1640+10％FBS将细胞调整至1×106cells/mL，再加入8μL500U/μL IL-2，IL-2终浓度为200U/mL，再分至96孔板中(平底)，200μL/孔；
3)每孔加入不同的抗原，即上述1～50号抗原，体积为5μL，浓度为2μg/mL多肽；设置阳性对照OKT3167 ng/mL、medium对照和CTL；
4)37℃5％CO2培养24h后，1500rpm离心10min，转移170μL上清至新的96孔板中；
5)为避免吸到细胞，再将步骤6)中新的96孔板进行1500rpm离心10min，转移110μL上清至新的96孔板；
6)以R&D ELISA试剂盒检测细胞上清中IFN-γ的释放量，以此来进行抗原的筛选。
筛选结果如图1和图2所示，IFN-γ的释放水平超过对照的，认为是有效的新抗原，其中7号、8号、9号、16号、23号、31号和45号多肽为筛选获得的有效的新的输卵管癌特异性抗原。
4.靶细胞的构建
1)通过人工合成的方法，进行HLA-A2601基因、HLA-A0201基因和HLA-B1501基因的合成(委托金维智生物科技有限公司)，分别将合成的HLA-A2601基因、HLA-A0201基因和HLA-B1501基因构建到pCDH表达载体上NheⅠ和Bam HⅠ酶切位点之间。
HLA-A2601(SEQ ID No.51)：
atggccgtcatggcgccccgaaccctcgtcctgctactctcgggggccctggccctgacccagacctgggcgggctcccactccatgaggtatttctacacctccgtgtcccggcccggccgcggggagccccgcttcatcgccgtgggctacgtggacgacacgcagttcgtgcggttcgacagcgacgccgcgagccagaggatggagccgcgggcgccgtggatagagcaggaggggccggagtattgggaccggaacacacggaatgtgaaggcccactcacagactgaccgagcgaacctggggaccctgcgcggctactacaaccagagcgaggacggttctcacaccatccagaggatgtatggctgcgacgtggggccggacgggcgcttcctccgcgggtaccagcaggacgcttacgacggcaaggattacatcgccctgaacgaggacctgcgctcttggaccgcggcggacatggcggctcagatcacccagcgcaagtgggagacggcccatgaggcggagcagtggagagcctacctggagggccggtgcgtggagtggctccgcagatacctggagaacgggaaggagacgctgcagcgcacggacgcccccaagacgcatatgactcaccacgctgtctctgaccatgaggccaccctgaggtgctgggccctgagcttctaccctgcggagatcacactgacctggcagcgggatggggaggaccagacccaggacacggagctcgtggagaccaggcctgcaggggatgggaccttccagaagtgggcgtctgtggtggtgccttctggacaggagcagagatacacctgccatgtgcagcatgagggtctgcccaagcccctcaccctgagatgggagccgtcttcccagcccaccatccccatcgtgggcatcattgctggcctggttctctttggagctgtgatcgctggagctgtggtcgctgctgtgatgtggaggaggaagagctcagatagaaaaggagggagctactctcaggctgcaagcagtgacagtgcccagggctctgatatgtctctcacagcttgtaaagtgtga
HLA-A0201(SEQ ID No.52)：
atggccgtcatggcgccccgaaccctcgtcctgctactctcgggggctctggccctgacccagacctgggcgggctctcactccatgaggtatttcttcacatccgtgtcccggcccggccgcggggagccccgcttcatcgcagtgggctacgtggacgacacgcagttcgtgcggttcgacagcgacgccgcgagccagaggatggagccgcgggcgccgtggatagagcaggagggtccggagtattgggacggggagacacggaaagtgaaggcccactcacagactcaccgagtggacctggggaccctgcgcggctactacaaccagagcgaggccggttctcacaccgtccagaggatgtatggctgcgacgtggggtcggactggcgcttcctccgcgggtaccaccagtacgcctacgacggcaaggattacatcgccctgaaagaggacctgcgctcttggaccgcggcggacatggcagctcagaccaccaagcacaagtgggaggcggcccatgtggcggagcagttgagagcctacctggagggcacgtgcgtggagtggctccgcagatacctggagaacgggaaggagacgctgcagcgcacggacgcccccaaaacgcatatgactcaccacgctgtctctgaccatgaagccaccctgaggtgctgggccctgagcttctaccctgcggagatcacactgacctggcagcgggatggggaggaccagacccaggacacggagctcgtggagaccaggcctgcaggggatggaaccttccagaagtgggcggctgtggtggtgccttctggacaggagcagagatacacctgccatgtgcagcatgagggtttgcccaagcccctcaccctgagatgggagccgtcttcccagcccaccatccccatcgtgggcatcattgctggcctggttctctttggagctgtgatcactggagctgtggtcgctgctgtgatgtggaggaggaagagctcagatagaaaaggagggagctactctcaggctgcaagcagtgacagtgcccagggctctgatgtgtctctcacagcttgtaaagtgtga
HLA-B1501(SEQ ID No.53)：
atgcgggtcacggcgccccgaaccgtcctcctgctgctctcgggagccctggccctgaccgagacctgggccggctcccactccatgaggtatttctacaccgccatgtcccggcccggccgcggggagccccgcttcatcgcagtgggctacgtggacgacacccagttcgtgaggttcgacagcgacgccgcgagtccgaggatggcgccccgggcgccatggatagagcaggaggggccggagtattgggaccgggagacacagatctccaagaccaacacacagacttaccgagagagcctgcggaacctgcgcggctactacaaccagagcgaggccgggtctcacaccctccagaggatgtacggctgcgacgtggggccggacgggcgcctcctccgcgggcatgaccagtccgcctacgacggcaaggattacatcgccctgaacgaggacctgagctcctggaccgcggcggacacggcggctcagatcacccagcgcaagtgggaggcggcccgtgaggcggagcagtggagagcctacctggagggcctgtgcgtggagtggctccgcagatacctggagaacgggaaggagacgctgcagcgcgcggaccccccaaagacacatgtgacccaccaccccatctctgaccatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagatcacactgacctggcagcgggatggcgaggaccaaactcaggacaccgagcttgtggagaccagaccagcaggagatagaaccttccagaagtgggcagctgtggtggtgccttctggagaagagcagagatacacatgccatgtacagcatgaggggctgccgaagcccctcaccctgagatgggagccatcttcccagtccaccatccccatcgtgggcattgttgctggcctggctgtcctagcagttgtggtcatcggagctgtggtcgctactgtgatgtgtaggaggaagagctcaggtggaaaaggagggagctactctcaggctgcgtccagcgacagtgcccagggctctgatgtgtctctcacagcttga
将上述得到的重组质粒转化感受态细胞，挑取单克隆，进行测序，选择测序结果正确的克隆，进行后续实验；
2)以天根质粒大提试剂盒(购自于天根生化科技(北京)有限公司)进行质粒的提取，方法按照厂家说明书操作。
3)慢病毒包装：复苏293T细胞，传两代后用于慢病毒包装；细胞转染：(T175培养瓶)细胞密度为2×107个/瓶；取一支15mL离心管(标记为A)，将包装质粒4K(17μg)、6K(34μg)和目的质粒(51μg)加入1mLjetPRIME配套的Buffer中，轻轻地混匀。将jetPRIME(标记为B)缓慢滴加入A离心管中，加入同时匀速晃动A管，得到C溶液，之后静置10min；将T175中旧的培养基倒出，将18mL DMEM(不含抗生素和血清)加入C中，加入T175培养瓶中，置于37℃，5％CO2培养箱中培养。转染4～6h后，吸去含有转染复合物的培养基，更换为37℃预热的新鲜培养基。培养48h和72h并收集。
4)慢病毒转染3T3细胞：使用DMEM配制含有7％FBS的全细胞培养基，记为DMEM。使用DMEM把3T3细胞浓度调整到5×105/mL，加入6孔板，每孔1mL，37℃培养箱孵育过夜。弃去6孔板中DMEM培养基，使用1mLAIM-V-P混匀病毒液加入对应各孔中。培养3天后转入T75培养瓶中，加入嘌呤霉素进行筛选；筛选6天后，得到稳定表达HLA-A2601、HLA-A0201和HLA-B1501的3T3细胞，标记为3T3-HLA；
5.杀伤实验——LDH法
1)收集靶细胞3T3和3T3-HLA，1000rpm离心5min；
2)以PBS洗一遍，1000rpm离心5min；
3)加入抗原溶液，9号和16号抗原为一组；31号和45号为一组，7号、8号和23号为一组，每组中每种抗原浓度为50ng/mL，体积为3mL，进行有效新抗原组合的负载，37℃负载4h；
4)1000rpm离心5min，除去多余溶液，并以PBS洗2次；
5)以1640+2％FBS重悬后，计数调整至8×104cells/mL，分至96孔板中(U型底)，50μL/孔，备用；
6)收集CTL细胞，1000rpm离心5min；
7)以PBS洗一遍，1000rpm离心5min；
8)以1640+2％FBS重悬细胞计数后，分至96孔板中(U型底)，50μL/孔，设置效靶比(CTL：3T3-HLA或CTL：3T3-HLA负载抗原组合)为40:1、20:1、10:1、5:1、2.5:1和1.25:1；37℃5％CO2共孵育20h后，进行LDH检测，LDH检测的试剂盒为Cytotoxicity LDHAssay
厂家：Dojindo货号为CK12。
结果如图3、图4和图5所示，经过抗原刺激培养的CTL，对负载抗原多肽的靶细胞比只负载HLA的靶细胞具有更高的杀伤效率，说明，培养的CTL，可以识别此抗原，且具有特异的杀伤作用，可以作为输卵管癌治疗的首选抗原。
6.流式分型
1)收集CTL，1000rpm离心5min；
2)1×106cells/管，加入CD3、CD4、CD8和CD56的抗体，室温避光30min；
3)PBS洗两次，1000rpm离心5min；
4)PBS重悬，上机检测。
以筛选到有效新抗原，进行CTL的培养，流式分析细胞表型，结果如图6所示，NKT(CD56+CD3+)比例为29.31％，50.93％为CD8+T细胞；NK会产生非特异性的杀伤，NK比例低说明非特异性的杀伤小，特异性杀伤作用显著。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京鼎成肽源生物技术有限公司
<120> 一种输卵管癌靶标抗原、输卵管癌靶标抗原刺激培养的CTL细胞及其应用
<160> 53
<170> SIPOSequenceListing 1.0
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Ala Leu Ala Arg Ala Val Ser Ala Val
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<213> 人工序列(Artificial Sequence)
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Glu Lys Ser Glu Ala Gly Leu Ala Gly Ala Leu
1 5 10
<210> 3
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<213> 人工序列(Artificial Sequence)
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Gly Leu Ala Gly Ala Leu Ala Arg Ala Val
1 5 10
<210> 4
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Gly Ala Leu Ala Arg Ala Val Ser Ala Val
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Leu Ala Arg Ala Val Ser Ala Val Lys Asn Met
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Ser Val Ala Gly Asn Cys Arg Met Tyr
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Arg Leu Ser Val Ala Gly Asn Cys Arg Met Tyr
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Arg Tyr Gln Cys Leu Ala Leu Lys Gly Phe
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Tyr His Lys Glu Ile Glu Val Asp
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<210> 10
<211> 10
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Asn Leu His Asp Tyr Gly Val Arg Glu Leu
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<210> 11
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Leu Asp Asn Asn Asn Leu His Asp Tyr Gly Val
1 5 10
<210> 12
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
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Lys Asn Leu Tyr Phe Leu Leu Ile Gln Ile
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<212> PRT
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Thr Val Trp Gly Asn Met Leu Ile Val Val
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<210> 14
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
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Val Thr Val Trp Gly Asn Met Leu Ile Val
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<210> 15
<211> 10
<212> PRT
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Asn Ala Thr Val Tyr Gly Thr Gly Val Tyr
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Thr Val Tyr Gly Thr Gly Val Tyr Phe
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Ile Arg Ser Pro Gln Thr His Ile Leu Leu Val
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<210> 18
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Thr Ser Ile Arg Ser Pro Gln Thr His Ile Leu
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<210> 19
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Gln Thr His Ile Leu Leu Val Gly Gly
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Asp Leu Phe Glu Gly Ser Asn Glu Gly Ser Leu
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Trp Glu Met Ser Ser Leu Phe Arg Glu Leu
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Tyr Ser Val Ile Arg Gly Glu Val
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Gln Cys Leu Ala Leu Lys Gly Phe
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Phe Ser Leu Pro Glu Glu Tyr Val
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<213> 人工序列(Artificial Sequence)
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His Arg Ile Pro Cys Arg Asp Met Ser Pro
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<210> 26
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<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 26
Asp Met Ser Pro Thr Leu Ile Arg Thr
1 5
<210> 27
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Trp Val Trp Val Thr Ser Thr Gly Arg
1 5
<210> 28
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 28
Glu Ala Gly Leu Ala Gly Ala Leu
1 5
<210> 29
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
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Leu Ala Arg Ala Val Ser Ala Val Lys Asn Met
1 5 10
<210> 30
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<213> 人工序列(Artificial Sequence)
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Ser Glu Ala Gly Leu Ala Gly Ala Leu
1 5
<210> 31
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Val Asp Val Glu Glu Met Cys Leu Leu Thr Val
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<210> 32
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<213> 人工序列(Artificial Sequence)
<400> 32
Phe Val Asn Met Trp Ile Glu Arg Thr Ile Tyr
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<210> 33
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Gly Thr Gly Pro Pro Gly Gly Ala Leu Tyr
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<210> 34
<211> 9
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<213> 人工序列(Artificial Sequence)
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Ile Arg Ser Pro Gln Thr His Ile Leu
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<210> 35
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<213> 人工序列(Artificial Sequence)
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Cys Gln Pro Ala Cys Cys Met Pro Val Ser
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Val Cys Gln Pro Ala Cys Cys Met Pro
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<213> 人工序列(Artificial Sequence)
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Leu Ser Val Ala Gly Asn Cys Arg Met Tyr
1 5 10
<210> 38
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<213> 人工序列(Artificial Sequence)
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Leu His Asp Tyr Gly Val Arg Glu Leu
1 5
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Thr Val Trp Gly Asn Met Leu Ile Val
1 5
<210> 40
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Ser Leu Ser Ser Gly Ser Ser Gly Ala
1 5
<210> 41
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Ala Arg Ala Pro Val Ala Ala Ile Ile
1 5
<210> 42
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Gly Gly Ser Ala Leu Phe Ser Glu Tyr
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Asp His Glu Glu Asn Val Ala Leu Glu
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<213> 人工序列(Artificial Sequence)
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Gln Asp His Glu Glu Asn Val Ala Leu Glu Ala
1 5 10
<210> 45
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<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 45
Leu Leu Gly Arg Thr Ala Phe Cys
1 5
<210> 46
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 46
Leu Val Trp Glu Met Ser Ser Leu Phe
1 5
<210> 47
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 47
Trp Glu Met Ser Ser Leu Phe Arg Glu Leu
1 5 10
<210> 48
<211> 10
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 48
Arg Thr Tyr Arg Tyr Phe Tyr Leu Phe Ile
1 5 10
<210> 49
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<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 49
Thr Ser Thr Gln Ala Ser Pro Asp Gln Leu Leu
1 5 10
<210> 50
<211> 11
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 50
Leu Tyr Thr Trp Glu Met Phe Gln Asp Pro Val
1 5 10
<210> 51
<211> 1098
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 51
atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggccct ggccctgacc 60
cagacctggg cgggctccca ctccatgagg tatttctaca cctccgtgtc ccggcccggc 120
cgcggggagc cccgcttcat cgccgtgggc tacgtggacg acacgcagtt cgtgcggttc 180
gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggg 240
ccggagtatt gggaccggaa cacacggaat gtgaaggccc actcacagac tgaccgagcg 300
aacctgggga ccctgcgcgg ctactacaac cagagcgagg acggttctca caccatccag 360
aggatgtatg gctgcgacgt ggggccggac gggcgcttcc tccgcgggta ccagcaggac 420
gcttacgacg gcaaggatta catcgccctg aacgaggacc tgcgctcttg gaccgcggcg 480
gacatggcgg ctcagatcac ccagcgcaag tgggagacgg cccatgaggc ggagcagtgg 540
agagcctacc tggagggccg gtgcgtggag tggctccgca gatacctgga gaacgggaag 600
gagacgctgc agcgcacgga cgcccccaag acgcatatga ctcaccacgc tgtctctgac 660
catgaggcca ccctgaggtg ctgggccctg agcttctacc ctgcggagat cacactgacc 720
tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 780
ggggatggga ccttccagaa gtgggcgtct gtggtggtgc cttctggaca ggagcagaga 840
tacacctgcc atgtgcagca tgagggtctg cccaagcccc tcaccctgag atgggagccg 900
tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ctttggagct 960
gtgatcgctg gagctgtggt cgctgctgtg atgtggagga ggaagagctc agatagaaaa 1020
ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tatgtctctc 1080
acagcttgta aagtgtga 1098
<210> 52
<211> 1098
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 52
atggccgtca tggcgccccg aaccctcgtc ctgctactct cgggggctct ggccctgacc 60
cagacctggg cgggctctca ctccatgagg tatttcttca catccgtgtc ccggcccggc 120
cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacgcagtt cgtgcggttc 180
gacagcgacg ccgcgagcca gaggatggag ccgcgggcgc cgtggataga gcaggagggt 240
ccggagtatt gggacgggga gacacggaaa gtgaaggccc actcacagac tcaccgagtg 300
gacctgggga ccctgcgcgg ctactacaac cagagcgagg ccggttctca caccgtccag 360
aggatgtatg gctgcgacgt ggggtcggac tggcgcttcc tccgcgggta ccaccagtac 420
gcctacgacg gcaaggatta catcgccctg aaagaggacc tgcgctcttg gaccgcggcg 480
gacatggcag ctcagaccac caagcacaag tgggaggcgg cccatgtggc ggagcagttg 540
agagcctacc tggagggcac gtgcgtggag tggctccgca gatacctgga gaacgggaag 600
gagacgctgc agcgcacgga cgcccccaaa acgcatatga ctcaccacgc tgtctctgac 660
catgaagcca ccctgaggtg ctgggccctg agcttctacc ctgcggagat cacactgacc 720
tggcagcggg atggggagga ccagacccag gacacggagc tcgtggagac caggcctgca 780
ggggatggaa ccttccagaa gtgggcggct gtggtggtgc cttctggaca ggagcagaga 840
tacacctgcc atgtgcagca tgagggtttg cccaagcccc tcaccctgag atgggagccg 900
tcttcccagc ccaccatccc catcgtgggc atcattgctg gcctggttct ctttggagct 960
gtgatcactg gagctgtggt cgctgctgtg atgtggagga ggaagagctc agatagaaaa 1020
ggagggagct actctcaggc tgcaagcagt gacagtgccc agggctctga tgtgtctctc 1080
acagcttgta aagtgtga 1098
<210> 53
<211> 1089
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 53
atgcgggtca cggcgccccg aaccgtcctc ctgctgctct cgggagccct ggccctgacc 60
gagacctggg ccggctccca ctccatgagg tatttctaca ccgccatgtc ccggcccggc 120
cgcggggagc cccgcttcat cgcagtgggc tacgtggacg acacccagtt cgtgaggttc 180
gacagcgacg ccgcgagtcc gaggatggcg ccccgggcgc catggataga gcaggagggg 240
ccggagtatt gggaccggga gacacagatc tccaagacca acacacagac ttaccgagag 300
agcctgcgga acctgcgcgg ctactacaac cagagcgagg ccgggtctca caccctccag 360
aggatgtacg gctgcgacgt ggggccggac gggcgcctcc tccgcgggca tgaccagtcc 420
gcctacgacg gcaaggatta catcgccctg aacgaggacc tgagctcctg gaccgcggcg 480
gacacggcgg ctcagatcac ccagcgcaag tgggaggcgg cccgtgaggc ggagcagtgg 540
agagcctacc tggagggcct gtgcgtggag tggctccgca gatacctgga gaacgggaag 600
gagacgctgc agcgcgcgga ccccccaaag acacatgtga cccaccaccc catctctgac 660
catgaggcca ccctgaggtg ctgggccctg ggcttctacc ctgcggagat cacactgacc 720
tggcagcggg atggcgagga ccaaactcag gacaccgagc ttgtggagac cagaccagca 780
ggagatagaa ccttccagaa gtgggcagct gtggtggtgc cttctggaga agagcagaga 840
tacacatgcc atgtacagca tgaggggctg ccgaagcccc tcaccctgag atgggagcca 900
tcttcccagt ccaccatccc catcgtgggc attgttgctg gcctggctgt cctagcagtt 960
gtggtcatcg gagctgtggt cgctactgtg atgtgtagga ggaagagctc aggtggaaaa 1020
ggagggagct actctcaggc tgcgtccagc gacagtgccc agggctctga tgtgtctctc 1080
acagcttga 1089
Claims (10)
1.一种输卵管癌靶标抗原，其特征在于，所述输卵管癌靶标抗原的氨基酸序列如SEQID No.23所示。
2.权利要求1所述的输卵管癌靶标抗原在制备治疗输卵管癌的药物中的应用。
3.权利要求1中所述的输卵管癌靶标抗原在制备输卵管癌CTL细胞中的应用。
4.一种CTL细胞，其特征在于，所述CTL细胞为权利要求1中所述的输卵管癌靶标抗原刺激培养的CTL细胞。
5.权利要求4所述的CTL细胞的制备方法，包括以下步骤：
1)将CTL细胞浓度调整至2～3×105cells/mL；
2)将调整细胞浓度后的CTL细胞、IL-2和权利要求1中所述的输卵管癌靶标抗原混合获得培养体系；
3)将所述培养体系进行培养获得输卵管癌靶标抗原刺激培养的CTL细胞。
6.根据权利要求5所述的制备方法，其特征在于，IL-2在培养体系中的终浓度为150～250U/mL。
7.根据权利要求5或6所述的制备方法，其特征在于，输卵管癌靶标抗原在培养体系中的终浓度为40～60ng/mL。
8.根据权利要求5所述的制备方法，其特征在于，步骤3)中所述培养的温度为36～38℃，所述培养的时间为22～26h。
9.根据权利要求5所述的制备方法，其特征在于，步骤1)中所述的CTL细胞是通过PBMC细胞与APC细胞共培养获得。
10.权利要求4所述的CTL细胞、权利要求5～9任意一项所述的制备方法制备获得的CTL细胞在制备治疗输卵管癌药物中的应用。
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