CN101680016A - 携带表达人肝癌特异性抗原的肿瘤的动物模型以及利用所述动物模型分析源于树突状细胞的免疫治疗手段的预防和治疗功效的方法 - Google Patents
携带表达人肝癌特异性抗原的肿瘤的动物模型以及利用所述动物模型分析源于树突状细胞的免疫治疗手段的预防和治疗功效的方法 Download PDFInfo
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Abstract
本发明涉及利用携带表达人肝癌特异性抗原的肿瘤的动物模型分析源于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法,其包括以下步骤:(a)(a′)给正常非人动物施用待分析的树突状细胞,或者(a″)给正常非人动物施用表达人肝癌特异性抗原的癌细胞系,以在正常动物中诱发癌;(b)(b′)当在步骤(a)中进行步骤(a′)时,给动物施用表达人肝癌特异性抗原的癌细胞系来诱发癌,或者(b″)当在步骤(a)中进行步骤(a″)时,给动物施用待分析的癌树突状细胞;和(c)通过测量动物的源自所述癌细胞系的癌细胞的形成或生长来测定作为免疫治疗手段的树突状细胞对肝癌的预防和治疗功效。
Description
技术领域
本发明涉及分析源于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法,更具体涉及利用患人肝癌的动物模型分析源于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法。
背景技术
肝癌的年发病率占所有癌的4%,这对应于56万名肝癌患者,而其中的39万名位于亚洲。在韩国,每年有12000-15000人罹患肝癌,此数目仅亚于胃癌。又因其引起的死亡率仅次于肺癌和胃癌,肝癌的危害实在不容小觑。70%的肝癌由乙肝病毒引起,估计有13%的肝癌由丙肝病毒引起,其他病因占肝癌发病率的18%。
肝癌分为原发性癌和转移性癌。最为多发的原发性肝癌为肝细胞癌,通过肝门静脉扩散的转移性肝癌的发病率也同样高。
为开发肝病治疗手段的诸多努力主要着眼于肝炎领域。目前已开发并使用的肝炎治疗手段有例如干扰素和拉米夫定。与干扰素相比,拉米夫定几乎没有副作用,且便于经口施用。但据报道,具有对拉米夫定的抗药性的病毒出现率已高达几乎50%,而且其对已发展为肝癌的患者几乎不发挥治疗效力。
患肝癌的患者在早期无症状。而当当被确诊为癌时,患者的情况已经非常危险。尽管肝癌的主要治疗方法为外科手术切除,但能够进行外科手术的肝癌患者数却非常少。其他肝癌治疗方法的例子包括组织移植、系统化疗、放疗和电灼疗法。但是这些手段都表现出高复发率,且引起严重的副作用,如移植排斥反应。即便是成功的切除手术,也有25%的复发率。还知,成功的切除手术结果取得于具有2-3cm的小肿瘤大小的患者。但是即使是小肿瘤大小肝癌,手术后三年内复发的可能性仍估计大于50%。肝癌的高复发率首先是由切除手术过程中的微转移所致,其次是由源自肝硬化的新肝癌发生所致。
人们仍需要针对肝癌的无副作用及无痛的新细胞免疫疗法。目前已开发出利用树突状细胞的癌疫苗接种法,被称为主动免疫疗法。与用灭活癌细胞免疫相比,它的效果更强;与注射体外培养的患者T细胞的被动免疫相比,它的作用更持久;与直接施用细胞因子(如IL-2和IFN-α)相比,它具有更高的安全性。另外,它还对转移或复发的癌具有显著治疗效果,并且无副作用及无痛。尽管难以通过利用树突状细胞的癌疫苗接种来减小肿瘤大小,但通过引发体内的免疫应答,它在转移(例如微转移)早期或原发后治疗期对于抑制复发及明显的转移发挥显著的功效。
为临床测试利用树突状细胞的免疫疗法,有必要先在动物模型上检验其功效和安全性。然而,现今还未有人提议用于评估基于树突状细胞的针对人前列腺癌的疫苗的前列腺癌动物模型。
本申请通篇引用了多个专利和出版物,引用内容以括号表示。将这些专利和出版物通过引用整体并入本申请,以更全面描述本发明及本发明所述技术领域的状态。
发明内容
为满足本领域的上述需求,本发明人建立了表达人肝癌特异性抗原的异种癌细胞系以及利用所述细胞系的动物模型。另外,我们发现,可通过使用所述动物模型来精确分析作为免疫治疗手段的树突状细胞对肝癌的预防和治疗功效。
由此,本发明旨在提供分析基于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法。
本发明还旨在提供表达人肝癌特异性抗原的小鼠源肝癌细胞系。
本发明又旨在提供小鼠(Mus musculus)肝癌模型。
将通过以下详述及随附的权利要求和附图明晰本发明的其他目的和优点。
本发明一方面提供利用携带表达人肝癌特异性抗原的肿瘤的动物模型分析源于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法,包括以下步骤:
(a)
(a′)给正常非人动物施用待分析的树突状细胞,或者
(a″)给正常非人动物施用表达人肝癌特异性抗原的癌细胞系,以在正常动物中诱发癌;
(b)
(b′)当在步骤(a)中进行步骤(a′)时,给动物施用表达人肝癌特异性抗原的癌细胞系来诱发癌,或者
(b″)当在步骤(a)中进行步骤(a″)时,给动物施用待分析的癌树突状细胞;和
(c)通过测量所述动物的源自所述癌细胞系的癌细胞的形成或生长来测定作为免疫治疗手段的树突状细胞对肝癌的预防和治疗功效。
本发明涉及
(i)分析源于树突状细胞的免疫治疗手段对肝癌的预防功效的方法,和
(ii)分析源于树突状细胞的免疫治疗手段对肝癌的治疗功效的方法。
在这一方面,本发明的分析源于树突状细胞的免疫治疗手段对肝癌的治疗功效的方法包括以下步骤:
(a″)给正常非人动物施用表达人肝癌特异性抗原的癌细胞系,以在正常动物中诱发癌;
(b″)给动物施用待分析的癌树突状细胞;和
(c)通过测量动物中源自所述癌细胞系的癌细胞的形成或生长来分析作为免疫治疗手段的树突状细胞对肝癌的治疗功效。
本发明的用于分析源于树突状细胞的免疫治疗手段对肝癌预防功效的方法包括以下步骤:
(a′)给正常非人动物施用待分析的树突状细胞;
(b′)给动物施用表达人肝癌特异性抗原的癌细胞系,以在动物中诱发癌;和
(c)通过测量动物中源自所述癌细胞系的癌细胞的形成或生长来分析作为免疫治疗手段的树突状细胞对肝癌的预防功效。
本发明首次提供了利用动物模型分析源于人树突状细胞的免疫治疗手段对肝癌的功效的成功方案。根据传统技术,还为将动物模型用于此类分析。
在本发明中,所使用的动物包括除人之外的任何动物种类,优选哺乳动物,更优选啮齿动物,再更优选小鼠(Mus musculus),且最优选C3H/HeN小鼠。本文所述“正常动物”是指未患癌的动物。
本方法中的用于建立表达人肝癌特异性抗原的癌细胞系的抗原包括人肝癌细胞中表达的任何抗原。人肝癌特异性抗原
优选为AFP(甲胎蛋白)、GPC3(磷脂酰肌醇蛋白聚糖3)、TRP53(转化相关蛋白53)、MAGEA1(黑素瘤抗原家族A,1)、NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1),
更优选为AFP(甲胎蛋白)、GPC3(磷脂酰肌醇蛋白聚糖3)、TRP53(转化相关蛋白53)或MAGEA1(黑素瘤抗原家族A,1),
再更优选为AFP(甲胎蛋白)或GPC3(磷脂酰肌醇蛋白聚糖3),
最优选为AFP(甲胎蛋白)。
人肝癌特异性抗原可包含天然存在的全长氨基酸序列及其部分序列。
可用于本发明的抗原优选包括:
对于AFP(甲胎蛋白)而言为跨SEQ ID N0:13或14的氨基酸1-346或1-484的氨基酸序列,
对于GPC3(磷脂酰肌醇蛋白聚糖3)而言为跨SEQ ID NO:15的氨基酸1-332的氨基酸序列,
对于TRP53(转化相关蛋白53)而言为跨SEQ ID NO:16的氨基酸1-326的氨基酸序列,
对于NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1,CTAG1)而言为跨SEQ ID NO:17的氨基酸1-180的氨基酸序列,或
对于MAGEA 1(黑素瘤抗原家族A,1)而言为跨SEQ ID NO:18的氨基酸1-309的氨基酸序列。
用于在正常动物中诱发癌的癌细胞系可以源自多种动物。所述癌细胞系优选与受者动物同源或异源,更优选与受者动物同源。在优选实施方式中将小鼠用作正常动物,且将小鼠源癌细胞系用作癌细胞系。更优选将C3H/HeN小鼠用作正常动物,且将MH134小鼠源癌细胞系用作癌细胞系。
可用于本发明的癌细胞系包括肝癌细胞系、胃癌细胞系、脑癌细胞系、肺癌细胞系、乳腺癌细胞系、卵巢癌细胞系、支气管癌细胞系、鼻咽癌细胞系、喉癌细胞系、胰腺癌细胞系、膀胱癌细胞系、结肠癌细胞系以及宫颈癌细胞系。最适用于本发明的是同源的肝癌细胞系,如MH134癌细胞系。在优选实施方式中,本发明中使用的表达人肝癌特异性抗原的癌细胞系源自肝癌细胞。与此同时,还有源于小鼠的肝癌细胞系,如源于C57BL/6小鼠,C3H/HeN小鼠和BALB/c小鼠的肝癌细胞系,但是它们并不适于直接用于本发明,因为它们不表达上述肝癌特异性抗原。当使用肝癌细胞系作为癌细胞系,C3H/HeN小鼠作为受者动物时,同源的肝癌细胞系MH134最适用于本发明。
用编码人肝癌特异性抗原的核苷酸序列转化小鼠源肝癌细胞系之后,将其用于本发明。编码人肝癌特异性抗原的核苷酸序列可包含天然存在的全长核苷酸序列及其部分序列。可用于本发明的编码人肝癌特异性抗原的核苷酸序列优选包含编码以下氨基酸序列的核苷酸序列:
跨AFP(甲胎蛋白)的氨基酸1-346或1-484的氨基酸序列,
跨GPC3(磷脂酰肌醇蛋白聚糖3)的氨基酸1-332的氨基酸序列,
跨TRP53(转化相关蛋白53)的氨基酸1-326的氨基酸序列,
跨NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)的氨基酸1-180的氨基酸序列,或
跨MAGEA1(黑素瘤抗原家族A,1)的氨基酸1-309的氨基酸序列。
编码人肝癌特异性抗原的核苷酸序列更优选包含以下核苷酸序列:
对于AFP(甲胎蛋白)而言为SEQ ID NO:1的核苷酸7-1044或SEQ ID NO:2的核苷酸7-1458的核苷酸序列,
对于GPC3(磷脂酰肌醇蛋白聚糖3)而言为SEQ ID NO:3的核苷酸7-1002的核苷酸序列,
对于TRP53(转化相关蛋白53)而言为SEQ ID NO:4的核苷酸7-984的核苷酸序列,
对于NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)而言为SEQ ID NO:5的核苷酸7-546的核苷酸序列,或
对于MAGEA1(黑素瘤抗原家族A,1)而言为SEQ ID NO:6的核苷酸7-933的核苷酸序列。
编码人肝癌特异性抗原的核苷酸序列可由多种方法制备。例如,从人源肝癌细胞系(如HepG2、ZR75-1、SK-BR-3)中分离出总RNA,使用通过参考已知的编码人肝癌特异性抗原的核苷酸序列设计的引物由所述总RNA合成cDNA分子。然后将所述合成出的cDNA克隆进合适的动物细胞表达载体(如pcDNA3.1(+))中,并导入小鼠肝癌细胞(如MH134细胞系)中。从细胞中筛选出表达人肝癌特异性抗原的经转化癌细胞,并用其建立表达人肝癌特异性抗原的癌细胞系。如上所述,本发明人首次建立了表达人肝癌特异性抗原的小鼠源肝癌细胞系。所述表达人肝癌特异性抗原的癌细胞系处理被表达的人肝癌特异性抗原,并且通过I型主要组织相容性复合体在其表面呈递所述经处理的抗原分子。结果,所述癌细胞系得以被特异于人肝癌抗原的T细胞所识别。
本发明中待分析的树突状细胞可由本领域技术人员熟知的多种方法制备。例如,树突状细胞可由单核细胞、造血祖细胞或骨髓细胞获得。
以下例示了使用骨髓细胞制备树突状细胞的方法:从小鼠的股骨和胫骨中分离出骨髓细胞,并将其培养于含有适量细胞因子(如IL-4和GM-CSF)的培养基中,以使其分化为树突状细胞。用人肝癌特异性抗原刺激所获得的未成熟树突状细胞,然后在含有适量细胞因子的培养基中培养,以使树突状细胞成熟,将其作为待分析的样品。当使用缀合了CTP(细胞质转导肽)的抗原时,刺激会变得非常有效。CTP分子向细胞质而非细胞核呈递抗原,使树突状细胞更高效地通过I型主要组织相容性复合体(MHC I)在其表面产生抗原。有关CTP分子的叙述见韩国专利No.10-0608558,将其教导通过引用并入本文。
可将待分析的树突状细胞通过多种途径施用于动物,优选静脉注射或皮下注射,最优选皮下注射。可将表达人肝癌特异性抗原的癌细胞系通过多种途径施用于动物,优选静脉注射或皮下注射,最优选皮下注射(Fong,L.et al.,Dendritic cells injected via different routesinduce immunity in cancer patients.J.Immunol.166:4254.(2001))。
将步骤(a)中的树突状细胞以1×104-1×108细胞的剂量施用于动物(例如小鼠),所述剂量优选为1×105-1×107细胞,且更优选为约1×106细胞。优选以适当的时间间隔(例如一星期)进行两次所述树突状细胞注射。将步骤(a)中的癌细胞系以1×104-1×108细胞的剂量施用于动物(例如小鼠),所述剂量优选为1×105-1×107细胞,且更优选为3×105细胞。
根据本领域技术人员以往所知,一般认为,当将表达人肝癌特异性抗原的癌细胞系施用于非人动物后,它们极有可能被动物的免疫应答所排斥。而令人惊讶的是,根据本发明,将表达人肝癌特异性抗原的癌细胞系施用于非人动物后,诱发动物形成癌组织,其使本方法得以顺利实施。
上述步骤(a)中癌细胞系的施用途径和剂量也可应用于步骤(b)。
根据本发明,(i)用于在步骤(a′)中刺激树突状细胞的人肝癌特异性抗原和(ii)在步骤(b′)的癌细胞系中表达的人肝癌特异性抗原源自同一抗原。例如,当用于在步骤(a′)中刺激树突状细胞的人肝癌特异性抗原为AFP(甲胎蛋白)时,在步骤(b′)的癌细胞系中表达的人肝癌特异性抗原也为AFP。因此,由表达AFP(甲胎蛋白)的树突状细胞诱发的细胞毒性T淋巴细胞识别表达AFP的癌细胞系,导致癌细胞系溶解。
根据本发明,(i)用于在步骤(a″)中刺激树突状细胞的人肝癌特异性抗原和(ii)在步骤(b″)的癌细胞系中表达的人肝癌特异性抗原源自同一抗原。例如,当用于在步骤(a″)中刺激树突状细胞的人肝癌特异性抗原为AFP(甲胎蛋白)时,在步骤(b″)的癌细胞系中表达的人肝癌特异性抗原也为AFP。因此,由表达AFP(甲胎蛋白)的树突状细胞诱发的细胞毒性T淋巴细胞识别表达AFP的癌细胞系,导致癌细胞系溶解。
在本发明的最后一个步骤中测量了动物的癌细胞形成和生长,以确定作为免疫治疗手段的树突状细胞对肝癌的预防或治疗功效。可用肉眼或使用仪器(如卡尺)评估动物的癌细胞形成和生长。当观察到癌细胞进一步形成和生长,则可确定作为免疫治疗手段的树突状细胞对肝癌具有预防和治疗功效。
为了在临床上应用树突状细胞来预防和治疗肝癌,首先要在动物模型中确定树突状细胞的功效和安全性。本发明使可对作为免疫治疗手段的树突状细胞进行基于动物模型的评估。本发明中筛选出的树突状细胞成为针对肝癌的免疫治疗手段的有前景的候选物。
本发明另一方面提供了表达人肝癌特异性抗原的小鼠源肝癌细胞系(重组MH134细胞系),其特征在于,所述人肝癌特异性抗原为AFP(甲胎蛋白)、GPC3(磷脂酰肌醇蛋白聚糖3)、TRP53(转化相关蛋白53)、MAGEA1(黑素瘤抗原家族A,1)或NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)。
本发明人首先开发了本发明的表达人肝癌特异性抗原的小鼠源肝癌细胞系(重组MH134细胞系),用于建立肝癌动物模型。
可通过用编码AFP(甲胎蛋白)、GPC3(磷脂酰肌醇蛋白聚糖3)、TRP53(转化相关蛋白53)、MAGEA1(黑素瘤抗原家族A,1)或NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)的核苷酸序列进行转化来制备本发明的肝癌细胞系。编码人肝癌特异性抗原的核苷酸序列可包含天然存在的全长核苷酸序列及其部分序列。
优选用包含编码以下氨基酸序列的核苷酸序列的载体转化所述肝癌细胞系:
跨AFP的氨基酸1-346或1-484的氨基酸序列、
跨GPC3的氨基酸1-332的氨基酸序列、
跨TRP53的氨基酸1-326的氨基酸序列、
跨MAGEA1的氨基酸1-309的氨基酸序列、或
跨NY-ESO-1的氨基酸1-180的氨基酸序列。
本发明的表达人肝癌特异性抗原的小鼠源肝癌细胞系优选是经以下载体转化的细胞系:
pcDNA3.1(+)-标签/AFP(甲胎蛋白)、
pcDNA3.1(+)-标签/GPC3(磷脂酰肌醇蛋白聚糖3)、
pcDNA3.1(+)-标签/TRP53(转化相关蛋白53)、
pcDNA3.1(+)-标签/NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)、或
pcDNA3.1(+)-标签/MAGEA1(黑素瘤抗原家族A,1),
所述载体是通过将编码人肝癌特异性抗原的核苷酸序列整合入pcDNA3.1(+)-标签(pcDNA3.1(+)-36A)(见图2.)而制得的。
整合入图2所示的载体pcDNA3.1(+)-标签的人肝癌抗原AFP、GPC3、TRP53、NY-ESO-1和MGAEA1分别表示为:
SEQ ID NO:1的第7-1044的1038个核苷酸、
SEQ ID NO:2的第7-1458的1452个核苷酸、
SEQ ID NO:3的第7-1002的996个核苷酸、
SEQ ID NO:4的第7-984的978个核苷酸、
SEQ ID NO:5的第7-546的540个核苷酸、和
SEQ ID NO:6的第7-993的927个核苷酸。
表达人肝癌特异性抗原的癌细胞系处理被表达的人肝癌特异性抗原,并通过I型主要组织相容性复合体在其表面呈递所述经处理的抗原分子。结果,所述癌细胞系得以被特异于人肝癌抗原的T淋巴细胞识别。
本发明又一方面提供了小鼠肝癌模型,其特征在于,所述小鼠模型具有通过接种本发明的表达人肝癌特异性抗原的肝癌细胞系而形成的癌,且可通过用经所述人肝癌特异性抗体刺激的树突状细胞处理来抑制在所述小鼠模型中的形成的癌的转移或生长。
小鼠肝癌模型携带通过接种表达人肝癌特异性抗原的小鼠肝癌细胞系而形成的癌,且其可用于评估作为对肝癌的免疫治疗手段的树突状细胞。还未曾有人建议用小鼠模型评估对肝癌的预防和治疗功效。
在优选实施方式中,注射于小鼠的肝癌细胞系与小鼠同源。在优选实施方式中,将所述小鼠肝癌模型用于本发明来分析上文所述树突状细胞对肝癌的预防和治疗功效。在优选实施方式中,本发明的小鼠模型是与注射的癌细胞系同源的C3H/HeN小鼠。
附图简述
图1的凝胶图片显示编码肝脏特异性抗原AFP(甲胎蛋白)、MAGEA1(黑素瘤抗原家族A,1)、TRP53(转化相关蛋白53)、GPC3(磷脂酰肌醇蛋白聚糖3)和NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)的核苷酸序列的PCR产物。为制备重组抗原,由HepG2(人肝癌细胞系)、ZR-75-1(人乳腺癌细胞系)、SK-BR3合成cDNA分子,并用于编码肝脏特异性抗原AFP、MEGEA1、TRP53、GPC3和ESO-1的核苷酸序列的PCR扩增。泳道M、1、2、3、4、5和6分别表示
标记物、
AFP(甲胎蛋白)1/2N(1040bp)、
AFP(甲胎蛋白)2/3N(1454bp)、
GPC3(磷脂酰肌醇蛋白聚糖3)1/2N(998bp)、
TRP53(转化相关蛋白53)2/3N(980bp)、
NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)(540bp)、
MAGEA1(黑素瘤抗原家族A,1)(929bp)。
图2显示用于表达肝癌特异性抗原的重组载体的基因图谱及其部分核苷酸序列。使用由HepG2(人肝癌细胞系)、ZR-75-1(人乳腺癌细胞系)、Sk-BR3合成的cDNA分子作为模板,通过PCR扩增了编码肝脏特异性抗原AFP、MEGEA1、TRP53、GPC3和NY-ESO-1的核苷酸序列。为了进行表达,将所述扩增出的序列克隆到真核载体(pcDNA3.1-36A)或原核载体(pCTP)中。在载体的基因图谱中,36A、CMV启动子、BGH pA、fl ori和SV40ori分别代表编码36A标签的序列、巨细胞病毒启动子、牛生长激素基因的多聚腺苷酸序列、fl复制起点和SV40复制起点。抗生素代表抗生素抗性基因。将编码36A标签的序列插入到真核载体中,以辅助检测由所述载体表达的蛋白质。用于导入标签序列的引物为:
标签-XhoI/s(5′-ACCCTCGAGGTCCATGACCGGAGGTCAGCAGATGGGTCGCGACCTGTACGACGA-3′)和
标签-XbaI/as(5′-ACCTCTAGATTAGCTTCCCCATCTGTCCTTGTCGTCATCGTCGTACAGGTCGCG-S′)。
在94℃30秒-52℃30秒-72℃5分钟的温度条件下通过一个循环的PCR扩增制备了标签DNA片段。36A标签的氨基酸序列是SMTGGQQMGRDLYDDDDKDRWGS,且其核苷酸序列是TCCATG ACC GGA GGT CAG CAG ATG GGT CGC GAC CTG TACGAC GAT GAC GAC AAG GAC AGA TGG GGA AGC。将36A的核苷酸序列以XhoI-36A-Stop-XbaI插入到MCS和BGH pA之间。更多关于35A标签的叙述见韩国专利No.10-0295558。
图3显示在经转化细胞内表达的肝癌抗原的蛋白印迹分析结果。用重组的pcDNA3.1-HA-36A/AFP、pcDNA3.1-HA-36A/MAGEA1、pcDNA3.1-HA-36A/TRP53和pcDNA3.1-HA-36A/GPC3转化MH134细胞,并在抗生素G418存在的情况下进行筛选,之后进行蛋白印迹分析。使用基因特异单克隆抗体(抗-AFP抗体,H-140Santacruz;抗-MAGEA1抗体,ab3211Abcam;抗-TRP53抗体、MAB1355研发系统;抗-GPC3抗体,AF2199研发系统)作为用于分析的抗体。
图4代表显示引入到MH134细胞的肝癌抗原(AFP、P53、MAGEA1和GPC3)的表达稳定性的蛋白印迹分析结果。将建立的细胞系在无G418的条件下培养,并对1×106细胞实施蛋白印迹分析。Nc代表阴性对照、未转化的MH134细胞。
图5显示肝癌抗原(AFP、MAGEA1、GPC3、TRP53和NY-ESO-1)的SDS-PAGE分析和蛋白印迹分析结果。将编码肝癌抗原的核苷酸序列克隆到pCTP载体中,并在BL21-gold(DE3)中表达。通过12%的SDS-PAGE和蛋白印迹分析证实了所表达的重组的缀合了CTP的蛋白。泳道M、1、2、3、4、5、6、7、8、9、10、11和12分别对应于:
分子量标记、
CTP-AFP 1/2N的沉淀和上清、
CTP-AFP 2/3N的沉淀和上清、
CTP-GPC31/2N的沉淀和上清、
CTP-TRP53的沉淀和上清、
CTP-NY-ESO1的沉淀和上清、
CTP-MAGEA1的沉淀和上清、
CTP-MAGEA3的沉淀和上清。
图6a和6b显示通过用表达肝癌抗原的重组MH134和对照MH134细胞诱发的C3H/HeN小鼠中的实体瘤的相对生长率。向C3H/HeN小鼠皮下接种3×105细胞的重组的MH134或对照MH134,并在30天后观察了癌的形成及速率(图6a)。向C3H/HeN小鼠皮下接种5×105细胞的重组MH134或对照MH134,并在30天后观察了癌的形成及速率(图6b)。在接种重组的肿瘤细胞系后,每隔三天测量一次肿瘤大小。
图7显示基于DC(树突状细胞)的疫苗抑制由重组的MH134细胞系诱发的肿瘤形成的预防功效。为了探究经肝癌抗原刺激的DC的预防功效,给小鼠隔一周皮下注射两次1×106细胞/小鼠的经激发的DC。一周后,给小鼠皮下注射1×106细胞/小鼠的重组癌细胞系。此后,每隔两天测量一次肿瘤大小。
图8显示癌预防模型中经基于DC的疫苗免疫的小鼠存活率。小鼠经DC疫苗免疫,并且经重组癌细胞系攻击。对存活的小鼠计数。经注射DC疫苗的小鼠存活,甚至在对照小鼠在50天内全部死亡之后。
图9显示DC疫苗的抑制肺转移的预防功效。给小鼠隔一周施用两次经CTP-AFP刺激的DC。然后,通过尾静脉给小鼠接种重组的肝癌细胞系(MH134/AFP)。接种后20天,提取肺并进行拍照,并对形成的癌节计数。
图10显示DC疫苗在携带肿瘤的小鼠中对癌的治疗功效。给小鼠皮下注射3×105细胞/小鼠的表达人肝癌抗原的重组癌细胞系。3天后,给小鼠隔一周皮下注射两次1×106细胞/小鼠的经重组肝癌抗原CTP-MAGEA1或CTP-AFP刺激的源于骨髓的树突状细胞(Bm-DC)。注射后2天,每隔两天检测一次肿瘤的形成和大小。在注射后20天时给小鼠肿瘤拍照。
图11a显示在经DC疫苗治疗的小鼠中癌症抗原特异性细胞毒性T淋巴细胞的活性。从经DC疫苗治疗的小鼠脾脏中分离T淋巴细胞,并与经CTP抗原刺激的抗原呈递细胞(APC)以5∶1(T∶APC)的比例混合。温育5天后,测量了细胞毒性T淋巴细胞的活性。通过ELISA检验了IFN-γ和IL-4的表达水平(图11b),并用MTT测定法研究了T细胞的增殖能力(图11c)。
以下通过实施例进一步详述本发明。本领域技术人员会明晰,这些实施例仅旨在更具体阐述本发明,且随附的权利要求书中提出的本发明的保护范围不局限于这些实施例。
实施例
实施例1:表达人源肝癌抗原的小鼠细胞系的制备
实施例1-1:人源肝癌抗原的表达载体的构建
(a)人源肝癌细胞系HepG2、ZR75-1、SK-BR-3的培养
本实验中使用的HepG2、ZR75-1、SK-BR-3是表达人肝癌特异性抗原如AFP(甲胎蛋白)、TRP53(转化相关蛋白53)、GPC3(磷脂酰肌醇蛋白聚糖3)、MAGEA1(黑素瘤抗原家族A,1)、NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)的人源肝癌细胞系,从韩国细胞系研究基金会获得。将所述肝癌细胞系在含有10%FBS的RPMI-1640培养基(Gibco/BRL)中培养和维持。将培养的细胞用胰蛋白酶-EDTA处理1分钟,以获得非粘附单细胞,然后传代培养至80%的融合率。一周进行2-3次传代培养。
(b)HepG2中的AFP(甲胎蛋白)、GPC3(磷脂酰肌醇蛋白
聚糖3)、MAGEA1(黑素瘤抗原家族A,1)及SK-BR-3中的NY-ESO-1
(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)、MAGEA1(黑
素瘤抗原案族A,1)的cDNA PCR产物的制备
在收获肝癌细胞系之前,将细胞传代培养2-3次以达到60%融合率,并经胰蛋白酶处理,随后收获细胞。用Trizol(Gibco BRL)从收获的细胞中提取总RNA,并经异丙醇沉淀,并经70%乙醇洗涤纯化。为合成cDNA,将10μg总RNA和1μg的oligo(dT)12-18引物的混合物在65℃变性5分钟,并转移到冰上,并向其加入逆转录酶缓冲液、10mM DTT、1mM dNTP混合物和20单位的RNAsin。使反应物混合物在42℃预反应2分钟,然后用200单位MMLV(Molony小鼠白血病病毒)逆转录酶(Invitrogen,Inc.)在42℃进行逆转录60分钟。反应完成后,使其在70℃保持15分钟以灭活酶活性。用合成为用于扩增AFP(甲胎蛋白)、MAGEA1(黑素瘤抗原家族A,1)、GPC3(磷脂酰肌醇蛋白聚糖3)、TRP53(转化相关蛋白53)和NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)的cDNA分子的模板的cDNA分子进行PCR反应。所用的引物序列汇总于表1a和1b。
表1a
用于克隆到原核表达载体的引物
表1b
用于克隆到真核表达载体的引物
使用表1a中的引物组(Bionics,Inc.)和PCR聚合酶(SolgentCo.,Ltd.),在94℃30秒-62℃30秒-72℃50秒的温度条件进行25个循环的PCR扩增,以获得用于在原核细胞中表达的DNA片段:GPC3(磷脂酰肌醇蛋白聚糖3)(909bp)、TRP53(转化相关蛋白53)(978bp)、NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)(540bp)、AFP(甲胎蛋白)(983bp)and MAGEA1(黑素瘤抗原家族A,1)(927bp)。类似地,使用表1b中的引物组进行PCR扩增,以用于在获得用于真核细胞中表达的DNA片段:GPC3(磷脂酰肌醇蛋白聚糖3)(998bp)、TRP53(转化相关蛋白53)(980bp)、NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)(542bp)、AFP(甲胎蛋白)(1040bp)和MAGEA1(黑素瘤抗原家族A,1)(929bp)。为了能够检测到真核细胞中表达的蛋白,将CreaGen,Inc.(韩国)开发的36A标签引入扩增的核苷酸序列中。用于引入标签序列的引物为标签-XhoI/s(5′-ACCCTCGAGGTCCATGACCGGAGGTCAGCAGATGGGTCGCGACCTGTACGACGA-3′)和标签-XbaI/as(5′-ACCTCTAGATTAGCTTCCCCATCTGTCCTTGTCGTCATCGTCGTACAGGTCGCG-S′)。在94℃30秒-52℃30秒-72℃5分钟的温度条件下通过1个循环的PCR扩增制备标签DNA片段。36A标签的氨基酸序列为SMTGGQQMGRDLYDDDDKDRWGS,且其核苷酸序列为TCCATG ACC GGA GGT CAG CAG ATG GGT CGC GAC CTG TACGAC GAT GAC GAC AAG GAC AGA TGG GGA AGC。将36A的核苷酸序列以XhoI-36A-Stop-XbaI插入到MCS和BGH pA之间。更多关于35A标签的描述见韩国专利No.10-0295558。
(c)将肝癌抗原cDNA克隆到表达载体(pcDNA3.1(+)-36A标
签载体和pCTP载体)中
用KpnI/EcoRI消化每个人肝癌抗原的DNA片断,并克隆到pcDNA3.1(+)标签载体中,再经过测序确认克隆的序列(见图2和SEQ ID NO:1-SEQ ID NO:6)。为在原核细胞内获得重组肝癌抗原,使用了pCTP-Td载体。通过基因操作pTAT-HA载体(由华盛顿大学的Dr.S.Dowdy友情提供,H.Nagahara et al.,Nature Med.4:1449(1998))而构建了pCTP-Td载体。用KpnI/EcoRI消化每个人肝癌抗原的DNA片断,并克隆到pCTP在载体上,再经过测序确认克隆的序列(见图2和SEQ ID NO:7-SEQ ID NO:12)。
对克隆的核苷酸序列进行DNA测序。经证实,由克隆的序列编码的氨基酸序列与已知的AFP、MAGEA1、GPC3、TRP53和NY-ESO-1(Blast2序列搜索)的氨基酸序列具有100%的同一性。
引入到原核表达载体中的序列缺少对应于N端信号肽和与C端相邻的跨膜结构域的序列。被引入原核表达载体中的核苷酸序列编码以下氨基酸序列:
AFP的氨基酸20-346(327个氨基酸)(SEQ ID NO:19)、
GPC3的氨基酸31-331(303个氨基酸)(SEQ ID NO:21)、
TRP53的氨基酸1-326(326个氨基酸)(SEQ ID NO:22)、
NY-ESO-1的氨基酸1-180(180个氨基酸)(SEQ ID NO:23)和
MAGEA1的氨基酸1-308(308个氨基酸)(SEQ ID NO:24)。
同时,被引入真核表达载体中的核苷酸序列编码以下氨基酸序列:
AFP的氨基酸1-346(346个氨基酸)(SEQ ID NO:13)、
GPC3的氨基酸1-332(332个氨基酸)(SEQ ID NO:15)、
TRP53的氨基酸1-326(326个氨基酸)(SEQ ID NO:16)、
NY-ESO-1的氨基酸1-180(180个氨基酸)(SEQ ID NO:17)和
MAGEA1的氨基酸1-309(309个氨基酸)(SEQ ID NO:18)。
实施例1-2:表达人源肝癌抗原小鼠细胞系的建立
(a)对表达肝癌抗原的小鼠细胞系的抗原表达的分析
为制备表达肝癌抗原的细胞系,将pcDNA3.1(+)标签/肝癌抗原(AFP,SEQ ID NO:1;GPC3,SEQ ID NO:3;TRP53,SEQ ID:4;MAGEA1,SEQ ID NO:6)载体转化到小鼠肝癌细胞系MH134细胞内。
通过用限制型内切酶(Ssp I;Pvu I用于AFP)在处理37℃处理2小时来将在20μg真核细胞表达载体pcDNA3.1(+)-36A中克隆的构建体线性化。用PCR纯化试剂盒纯化经酶处理的pcDNA3.1。通过重悬浮将2×105细胞的MH134与50μl的经最终洗脱的DNA混合于550μl的1×PBS中,然后加入2μl 2M MgCl2(终浓度5mM)。在试管中加入将最终660μl DNA与MH134细胞混合物放入电穿孔杯中,并置于冰上。接下来,用BIO-RAD电击基因转移仪在280V,950μF下实施电穿孔,之后在冰上放置10分钟。将杯中的DNA和MH134细胞的混合物用黄色移液管头转移到含有10ml RPMI1640和10%FBS的50ml管中。将混合物分装到96孔微型平板内,每孔100μl。在37℃下温浴2天后,向孔中加入G418(10mg/ml),以使G418的终浓度达1mg/ml。经G418处理后观察细胞集落的形成。筛选出具有细胞集落的孔,并转移到6孔板上,之后,当细胞生长到106细胞/ml时转移到100mm皿中。增值并收获筛选出的细胞,随后通过蛋白印迹分析确认了抗原的表达模式。收获的细胞用PBS洗涤两次,在蛋白样品缓冲液中加热,并经离心除去基因组DNA分子,再通过SDS-PAGE分离上清。将呈现的蛋白条带用半干式转移点斑仪(Bio-Rad)转移到硝酸纤维素膜上,并与第一抗体、标签抗原特异性单克隆抗体和第二抗体-缀合了AP(碱性磷酸酶)的抗-鼠IgG(Sigma)反应。用含NBT/BCIP的AP反应溶液(Promega)观察条带。
(b)基于抗原表达评估细胞系的稳定性
为确定当把建立的重组细胞系注射给小鼠后是否在无抗生素(G418)的情况下保持其抗原表达能力,将细胞系在不含G418的培养基中进行传代培养并进行检测,以便检查被引入的外源序列的稳定表达。将每种细胞系(MH134/AFP、MH134/GPC3、MH134/TRP53和MH134/MAGEA1)都在无G418的情况下培养,每3天收集1×106细胞,然后利用每种抗原的特异性引物,通过RT-PCR检验了它们的抗原表达能力。
表1c
用于鉴定真核表达载体的表达的引物和用于靶基因的引物序列
MAGEA-1
寡核苷酸 | 起点 | 长度 | tm | Gc% | 任意的 | 3′ | rep | 序列 |
左侧引物 | 150 | 20 | 60.05 | 55.00 | 4.00 | 0.00 | 11.00 | GTCAACAGATCCTCCCCAGA |
右侧引物 | 387 | 20 | 59.99 | 45.00 | 5.00 | 1.00 | 12.00 | CAGCATTTCTGCCTTTGTGA |
序列大小:930
内含区大小:930
产物大小:238
AFP1/2N
寡核苷酸 | 起点 | 长度 | tm | Gc% | 任意的 | 3′ | rep | 序列 |
左侧引物 | 381 | 20 | 60.15 | 50.00 | 2.00 | 2.00 | 10.00 | ACACAAAAAGCCCACTCCAG |
右侧引物 | 595 | 20 | 59.75 | 45.00 | 5.00 | 2.00 | 11.00 | CTGCATTTTCAGCTTTGCAG |
序列大小:900
内含区大小:900
产物大小:215
TRP53(转化相关蛋白53)2/3N
寡核苷酸 | 起点 | 长度 | tm | Gc% | 任意的 | 3′ | rep | 序列 |
左侧引物 | 35 | 20 | 60.23 | 55.00 | 7.00 | 2.00 | 12.00 | CCCCTCTGAGTCAGGAAACA |
右侧引物 | 185 | 20 | 60.05 | 55.00 | 6.00 | 0.00 | 11.00 | TCATCTGGACCTGGGTCTTC |
序列大小:550
内含区大小:550
产物大小:151
GPC3(磷脂酰肌醇蛋白聚糖3)1/2N
寡核苷酸 | 起点 | 长度 | tm | Gc% | 任意的 | 3′ | rep | 序列 |
左侧引物 | 562 | 20 | 60.07 | 50.00 | 7.00 | 2.00 | 10.00 | CCTGATTCAGCCTTGGACAT |
右侧引物 | 801 | 20 | 60.01 | 55.00 | 5.00 | 1.00 | 10.00 | TCCCTGGCAGTAAGAGCAGT |
序列大小:871
内含区大小:871
产物大小:240
实施例2:用于刺激树突状细胞的重组的缀合了CTP的蛋白的纯
化及转导能力的测量
实施例2-1:重组的缀合了CTP的肝癌抗原的表达和纯化
根据Hanahan的方法,通过用带有各种肝癌抗原的cDNA(见SEQ ID NO:7和SEQ ID NO:12)的重组pCTP-Td载体转化大肠杆菌BL21Gold(DE3)感受态细胞(Stratagene)来制备转化体,并在氨苄西林-LB培养基中培养。将培养的转化体经离心、PBS洗涤和收获,再于12%的SDS-PAGE上分析肝癌抗原表达。表达后,通过Ni+-NTA树脂(Qiagen)柱纯化重组蛋白CTP-AFP、CTP-MAGEA1、CTP-TRP53、CTP-GPC3和CTP-NY-ESO-1。被分析的蛋白显示具有大出约6kDa的分子量,这归因于来自载体的非基因组序列。换言之,CTP-AFP呈现约44kDa的分子量,CTP-MAGEA1为约48kDa,CTP-GPC3为约41kDa,CTP-TRP53为约53kDa,且CTP-NY-ESO-1为约30kDa。
实施例3:动物肝癌模型的构建
实施例3-1:小鼠表达肝癌抗原细胞系引起癌形成和生长状况的
评估
我们检测了由表达人肝癌抗原的小鼠细胞系引起的癌的形成和生长。将如上制备的细胞系注射入6周龄Balb/c小鼠(Orient,Inc.,韩国)的股骨内。将表达人肝癌抗原的重组细胞系在含有10%FBS和500μg/ml G418的RPMI培养基中培养和保持。将处于最佳生长状态的细胞用PBS洗涤2-3次,用胰蛋白酶-EDTA处理以分离单细胞,并以3×105细胞/100μl和5×105细胞/100μl悬浮于PBS中。将100μl悬浮液皮下接种给小鼠。在接种细胞系后,每隔三天观察一次实体癌的形成。用卡尺测量实体癌的大小。如图6b所示,发现所有接种了肝癌细胞系的小鼠都携带实体癌。有趣的是,低剂量的细胞(如3×105细胞)能够引起肿瘤发生,且小鼠中形成的癌不被针对异源抗原的免疫应答排斥。图6b显示癌的生长速率。重组的MH134/TRP53细胞系表现出较其他细胞系低的癌生长速率,而MH134/MAGEA1细胞表现出与MH134相近的癌生长速率。可以理解为,TRP53的表达水平最高,并引起了最强的免疫应答。这些结果表明,小鼠中由表达人肝癌抗原的细胞引起的肿瘤发生不能被针对异源抗原的免疫应答阻止。因此,可以得出,本发明成功建立了肝癌小鼠模型,并且其能够用于评估基于树突状细胞的疫苗的预防和治疗功效。
实施例4:树突状细胞抗癌功效的分析
实施例4-1:基于树突状细胞的疫苗的预防功效(预防模型)
为了探究基于树突状细胞的疫苗是否可预防肝癌,用经重组的缀合了CTP的肝癌抗原刺激的树突状细胞免疫小鼠两次,并用表达肝癌特异性抗原的癌细胞系攻击,之后检测实体癌的形成及肺转移。
通过从股骨和胫骨骨髓细胞分化成树突状细胞来制备小鼠树突状细胞。解剖股骨和胫骨的两端,由此提取骨髓细胞,并将其收集到50ml管中。将收集的骨髓细胞悬浮于0.83%氯化铵溶液中,以除去红细胞,再在树突状细胞生产培养基(含10%FBS、10ng/ml小鼠重组IL-4和10ng/ml小鼠GM-CSF的RPMI-1640培养基)中培养2天。去除非粘附细胞,以只得到试管底部的粘附细胞。每隔2-3天用新鲜培养基替换培养基,以防止细胞因子的缺乏。经过6天的培养,收获未成熟的树突状细胞,并与CTP-AFP温浴。用每种抗原蛋白50μg/ml刺激未成熟的树突状细胞20小时,并向其中加入用于使DC成熟的细胞因子,如100μg/ml的IFN-γ和100μg/ml TNF-α。将1×106细胞的经抗原刺激的树突状细胞皮下注射给小鼠,以诱发针对癌的免疫应答。隔2周进行两次树突状细胞的免疫。第二次免疫一周后,用3×105细胞/小鼠的MH134/AFP皮下攻击通过经CTP-AFP刺激的树突状细胞免疫的小鼠。每三天测量一次癌的大小(长×宽)。如图7所示,用经CTP-AFP刺激的树突状细胞免疫的小鼠不携带肿瘤体。
图8显示使用树突状细胞的癌预防模型中的癌发生率。在用经CTP-AFP刺激的树突状细胞免疫的小鼠的组里,发现用MH134/AFP细胞系攻击的小鼠表现出肿瘤发生迟缓,并且在48天后仍然存活。相反,在经PBS处理的对照组中,小鼠从第25天起开始死亡,并在第42天时全部死亡。在用未激发的树突状细胞免疫的小鼠的对照组中,小鼠从第42天起开始死亡,并在第45天时全部死亡。使用经癌特异性抗原刺激的树突状细胞的癌预防模型表现出寿命延长和肿瘤发生延缓的功效。
实施例4-2:基于树突状细胞的疫苗对肝癌转移的抑制(预防模
型)
如上用基于树突状细胞的疫苗免疫小鼠两次,并通过尾静脉注射3×105细胞/小鼠的表达肝癌抗原的细胞系。MH134小鼠肝癌细胞系表现出自发转移。20天后,对小鼠实施安乐死,并评估肺转移。如图9所示,用经人肝癌抗原AFP刺激的树突状细胞免疫的小鼠表现出无肺转移。相反,用未经刺激的树突状细胞或PBS处理的小鼠表现出强烈的肺转移。可以理解为,基于树突状细胞的疫苗可引起特异于癌抗原的强烈免疫应答,并反过来抑制癌的形成和转移。
实施例4-3基于树突状细胞的疫苗的治疗功效(回归模型)
给小鼠皮下注射3×105细胞/小鼠的表达人肝癌抗原AFP的重组细胞系。三天后,隔一周给小鼠两次注射1×106细胞/小鼠的经缀合了CTP的抗原(CTP-MAGEA1和CTP-AFP)刺激的树突状细胞。在第二次施用DC之后,每隔两天观察一次癌的形成和生长1个月。如图10所示,所有使用MH134/MAGEA1和MH134/AFP细胞系建立的肝癌小鼠模型中的癌的生长被抑制。
实施例4-4:对由基于树突状细胞的疫苗引发的CTL反应的分析
使用从肺转移模型小鼠中分离的脾细胞分析了T细胞增殖和CTL活性。用颈椎脱臼法处死每只小鼠,分离出脾脏,并于RPMI中保存。使每个脾脏都通过70μm筛,并除去悬浮组织。对产物进行离心以收集细胞,之后悬浮于0.83%的氯化铵溶液以除去红细胞。使制备出的脾细胞通过尼龙羊毛层析柱以分离作为效应细胞的T淋巴细胞,并与APC(抗原呈递细胞)以5∶1的比例混合,之后培养5天。APC在实验前事先制备。另一方面,从正常小鼠中分离出脾细胞,并用3μg/ml的Con-A处理24小时。将经刺激的细胞于50μg/ml的各抗原蛋白CTP-AFP温浴24小时。在培养过程中将细胞浓度保持在1×106细胞。培养24小时后,用丝裂霉素C处理细胞40分钟,并洗涤3次以制备APC。将效应T细胞和APC的培养3天后,实施T细胞增殖测定(MTT测定),作为培养的一部分。培养4天后,测量上清中的IL-4和IFN-γ量。根据产品说明书使用R&D系统试剂盒。由于MH134细胞是悬浮细胞,用CFSE染色法检测MH134细胞的特异性溶解。将MH134细胞用作非靶细胞,并将表达抗原的稳定细胞系用作靶细胞。为区分非靶细胞和靶细胞,将细胞在洗涤后用CFSE染色。(靶细胞:加入15μl 1mM CFSE(CFSE高);非靶细胞:加入10μl 0.1mM CFSE(CFSE低))。在以相同比例混合两种经染色的细胞系后,通过用Histopaque(sigma)除去死细胞来分离出效应T细胞。将效应T细胞和靶细胞以1∶1、10∶1和20∶1的ET比例混合,并温浴6小时。之后,通过FACS分析测量了活细胞数量。
E∶T比例 | 效应细胞 | 靶混合 | RPMI-10 |
0.5∶1 | 25μl | 100μl | 175μl |
1∶1 | 50μl | 100μl | 150μl |
2∶1 | 100μl | 100μl | 100μl |
4∶1 | 200μl | 100μl | |
仅靶细胞 | 100μl | 200μl |
之后,用以下等式计算了数值。
特异性溶解的百分比=(1-仅靶细胞的比率/靶细胞比率+效应细胞)×100
如图11所示,在全部三个小鼠模型中都有效诱发了CTL。这些结果表明,施用基于经CTP-AFP刺激的树突状细胞的疫苗有效诱发特异于人肝癌抗原CTL,从而产生对癌的预防和治疗功效。
如前所述,本发明提供利用动物模型分析作为免疫治疗手段的树突状细胞对肝癌的预防和治疗功效的方法。为了在临床上应用树突状细胞对肝癌进行预防和治疗,必须先在动物模型上证实树突状细胞的功效和安全性。本发明使对作为免疫治疗手段的树突状细胞的基于动物模型的评估成为可能。本发明筛选出的基于树突状细胞的疫苗(DC疫苗)成为作为对肝癌的免疫治疗手段的有前途的候选物。
根据所述本发明的优选实施方式,本领域技术人员应该明晰其变型及改良也应落入本发明的范围之内,且本发明的范围将由随附的权利要求及其等效形式决定。
序列表
<110>Creagene
<120>携带表达人肝癌特异性抗原的肿瘤的动物模型以及利用所述动物模型分析源于树
突状细胞的免疫治疗手段的预防和治疗功效的方法
<160>24
<170>KopatentIn 1.71
<210>1
<211>1052
<212>DNA
<213>智人(Homo sapiens)
<400>1
ggtaccatga agtgggtgga atcaattttt ttaattttcc tactaaattt tactgaatcc 60
agaacactgc atagaaatga atatggaata gcttccatat tggattctta ccaatgtact 120
gcagagataa gtttagctga cctggctacc atattttttg cccagtttgt tcaagaagcc 180
acttacaagg aagtaagcaa aatggtgaaa gatgcattga ctgcaattga gaaacccact 240
ggagatgaac agtcttcagg gtgtttagaa aaccagctac ctgcctttct ggaagaactt 300
tgccatgaga aagaaatttt ggagaagtac ggacattcag actgctgcag ccaaagtgaa 360
gagggaagac ataactgttt tcttgcacac aaaaagccca ctccagcatc gatcccactt 420
ttccaagttc cagaacctgt cacaagctgt gaagcatatg aagaagacag ggagacattc 480
atgaacaaat tcatttatga gatagcaaga aggcatccct tcctgtatgc acctacaatt 540
cttctttggg ctgctcgcta tgacaaaata attccatctt gctgcaaagc tgaaaatgca 600
gttgaatgct tccaaacaaa ggcagcaaca gttacaaaag aattaagaga aagcagcttg 660
ttaaatcaac atgcatgtgc agtaatgaaa aattttggga cccgaacttt ccaagccata 720
actgttacta aactgagtca gaagtttacc aaagttaatt ttactgaaat ccagaaacta 780
gtcctggatg tggcccatgt acatgagcac tgttgcagag gagatgtgct ggattgtctg 840
caggatgggg aaaaaatcat gtcctacata tgttctcaac aagacactct gtcaaacaaa 900
ataacagaat gctgcaaact gaccacgctg gaacgtggtc aatgtataat tcatgcagaa 960
aatgatgaaa aacctgaagg tctatctcca aatctaaaca ggtttttagg agatagagat 1020
tttaaccaat tttcttcagg ggaagggaat tc 1052
<210>2
<211>1466
<212>DNA
<213>智人
<400>2
ggtaccatga agtgggtgga atcaattttt ttaattttcc tactaaattt tactgaatcc 60
agaacactgc atagaaatga atatggaata gcttccatat tggattctta ccaatgtact 120
gcagagataa gtttagctga cctggctacc atattttttg cccagtttgt tcaagaagcc 180
acttacaagg aagtaagcaa aatggtgaaa gatgcattga ctgcaattga gaaacccact 240
ggagatgaac agtcttcagg gtgtttagaa aaccagctac ctgcctttct ggaagaactt 300
tgccatgaga aagaaatttt ggagaagtac ggacattcag actgctgcag ccaaagtgaa 360
gagggaagac ataactgttt tcttgcacac aaaaagccca ctccagcatc gatcccactt 420
ttccaagttc cagaacctgt cacaagctgt gaagcatatg aagaagacag ggagacattc 480
atgaacaaat tcatttatga gatagcaaga aggcatccct tcctgtatgc acctacaatt 540
cttctttggg ctgctcgcta tgacaaaata attccatctt gctgcaaagc tgaaaatgca 600
gttgaatgct tccaaacaaa ggcagcaaca gttacaaaag aattaagaga aagcagcttg 660
ttaaatcaac atgcatgtgc agtaatgaaa aattttggga cccgaacttt ccaagccata 720
actgttacta aactgagtca gaagtttacc aaagttaatt ttactgaaat ccagaaacta 780
gtcctggatg tggcccatgt acatgagcac tgttgcagag gagatgtgct ggattgtctg 840
caggatgggg aaaaaatcat gtcctacata tgttctcaac aagacactct gtcaaacaaa 900
ataacagaat gctgcaaact gaccacgctg gaacgtggtc aatgtataat tcatgcagaa 960
aatgatgaaa aacctgaagg tctatctcca aatctaaaca ggtttttagg agatagagat 1020
tttaaccaat tttcttcagg ggaaaaaaat atcttcttgg caagttttgt tcatgaatat 1080
tcaagaagac atcctcagct tgctgtctca gtaattctaa gagttgctaa aggataccag 1140
gagttattgg agaagtgttt ccagactgaa aaccctcttg aatgccaaga taaaggagaa 1200
gaagaattac agaaatacat ccaggagagc caagcattgg caaagcgaag ctgcggcctc 1260
ttccagaaac taggagaata ttacttacaa aatgcgtttc tcgttgctta cacaaagaaa 1320
gccccccagc tgacctcgtc ggagctgatg gccatcacca gaaaaatggc agccacagca 1380
gccacttgtt gccaactcag tgaggacaaa ctattggcct gtggcgaggg agcggctgac 1440
attattatcg gacacttagg gaattc 1466
<210>3
<211>1010
<212>DNA
<213>智人
<400>3
ggtaccatgg ccgggaccgt gcgcaccgcg tgcttggtgg tggcgatgct gctcagcttg 60
gacttcccgg gacaggcgca gcccccgccg ccgccgccgg acgccacctg tcaccaagtc 120
cgctccttct tccagagact gcagcccgga ctcaagtggg tgccagaaac tcccgtgcca 180
ggatcagatt tgcaagtatg tctccctaag ggcccaacat gctgctcaag aaagatggaa 240
gaaaaatacc aactaacagc acgattgaac atggaacagc tgcttcagtc tgcaagtatg 300
gagctcaagt tcttaattat tcagaatgct gcggttttcc aagaggcctt tgaaattgtt 360
gttcgccatg ccaagaacta caccaatgcc atgttcaaga acaactaccc aagcctgact 420
ccacaagctt ttgagtttgt gggtgaattt ttcacagatg tgtctctcta catcttgggt 480
tctgacatca atgtagatga catggtcaat gaattgtttg acagcctgtt tccagtcatc 540
tatacccagc taatgaaccc aggcctgcct gattcagcct tggacatcaa tgagtgcctc 600
cgaggagcaa gacgtgacct gaaagtattt gggaatttcc ccaagcttat tatgacccag 660
gtttccaagt cactgcaagt cactaggatc ttccttcagg ctctgaatct tggaattgaa 720
gtgatcaaca caactgatca cctgaagttc agtaaggact gtggccgaat gctcaccaga 780
atgtggtact gctcttactg ccagggactg atgatggtta aaccctgtgg cggttactgc 840
aatgtggtca tgcaaggctg tatggcaggt gtggtggaga ttgacaagta ctggagagaa 900
tacattctgt cccttgaaga acttgtgaat ggcatgtaca gaatctatga catggagaac 960
gtactgcttg gtctcttttc aacaatccat gattctatcc aggggaattc 1010
<210>4
<211>992
<212>DNA
<213>智人
<400>4
ggtaccatgg aggagccgca gtcagatcct agcgtcgagc cccctctgag tcaggaaaca 60
ttttcagacc tatggaaact acttcctgaa aacaacgttc tgtccccctt gccgtcccaa 120
gcaatggatg atttgatgct gtccccggac gatattgaac aatggttcac tgaagaccca 180
ggtccagatg aagctcccag aatgccagag gctgctcccc gcgtggcccc tgcaccagca 240
gctcctacac cggcggcccc tgcaccagcc ccctcctggc ccctgtcatc ttctgtccct 300
tcccagaaaa cctaccaggg cagctacggt ttccgtctgg gcttcttgca ttctgggaca 360
gccaagtctg tgacttgcac gtactcccct gccctcaaca agatgttttg ccaactggcc 420
aagacctgcc ctgtgcagct gtgggttgat tccacacccc cgcccggcac ccgcgtccgc 480
gccatggcca tctacaagca gtcacagcac atgacggagg ttgtgaggcg ctgcccccac 540
catgagcgct gctcagatag cgatggtctg gcccctcctc agcatcttat ccgagtggaa 600
ggaaatttgc gtgtggagta tttggatgac agaaacactt ttcgacatag tgtggtggtg 660
ccctatgagc cgcctgaggt tggctctgac tgtaccacca tccactacaa ctacatgtgt 720
aacagttcct gcatgggcgg catgaaccgg aggcccatcc tcaccatcat cacactggaa 780
gactccagtg gtaatctact gggacggaac agctttgagg tgcgtgtttg tgcctgtcct 840
gggagagacc ggcgcacaga ggaagagaat ctccgcaaga aaggggagcc tcaccacgag 900
ctgcccccag ggagcactaa gcgagcactg cccaacaaca ccagctcctc tccccagcca 960
aagaagaaac cactggatgg agaagggaat tc 992
<210>5
<211>554
<212>DNA
<213>智人
<400>5
ggtaccatgc aggccgaagg ccggggcaca gggggttcga cgggcgatgc tgatggccca 60
ggaggccctg gcattcctga tggcccaggg ggcaatgctg gcggcccagg agaggcgggt 120
gccacgggcg gcagaggtcc ccggggcgca ggggcagcaa gggcctcggg gccgggagga 180
ggcgccccgc ggggtccgca tggcggcgcg gcttcagggc tgaatggatg ctgcagatgc 240
ggggccaggg ggccggagag ccgcctgctt gagttctacc tcgccatgcc tttcgcgaca 300
cccatggaag cagagttggc ccgcaggagc ctggcccagg atgccccacc gcttcccgtg 360
ccaggggtgc ttctgaagga gttcactgtg tccggcaaca tactgactat ccgactgact 420
gctgcagacc accgccaact gcagctctcc atcagctcct gtctccagca gctttccctg 480
ttgatgtgga tcacgcagtg ctttctgccc gtgtttttgg ctcagcctcc ctcagggcag 540
aggcgcggga attc 554
<210>6
<211>941
<212>DNA
<213>智人
<400>6
ggtaccatgt ctcttgagca gaggagtctg cactgcaagc ctgaggaagc ccttgaggcc 60
caacaagagg ccctgggcct ggtgtgtgtg caggctgccg cctcctcctc ctctcctctg 120
gtcctgggca ccctggagga ggtgcccact gctgggtcaa cagatcctcc ccagagtcct 180
cagggagcct ccgcctttcc cactaccatc aacttcactc gacagaggca acccagtgag 240
ggttccagca gccgtgaaga ggaggggcca agcacctctt gtatcctgga gtccttgttc 300
cgagcagtaa tcactaagaa ggtggctgat ttggttggtt ttctgctcct caaatatcga 360
gccagggagc cagtcacaaa ggcagaaatg ctggagagtg tcatcaaaaa ttacaagcac 420
tgttttcctg agatcttcgg caaagcctct gagtccttgc agctggtctt tggcattgac 480
gtgaaggaag cagaccccac cggccactcc tatgtccttg tcacctgcct aggtctctcc 540
tatgatggcc tgctgggtga taatcagatc atgcccaaga caggcttcct gataattgtc 600
ctggtcatga ttgcaatgga gggcggccat gctcctgagg aggaaatctg ggaggagctg 660
agtgtgatgg aggtgtatga tgggagggag cacagtgcct atggggagcc caggaagctg 720
ctcacccaag atttggtgca ggaaaagtac ctggagtacc ggcaggtgcc ggacagtgat 780
cccgcacgct atgagttcct gtggggtcca agggcccttg ctgaaaccag ctatgtgaaa 840
gtccttgagt atgtgatcaa ggtcagtgca agagttcgct ttttcttccc atccctgcgt 900
gaagcagctt tgagagagga ggaagaggga gtcgggaatt c 941
<210>7
<211>996
<212>DNA
<213>智人
<400>7
ggtaccacac tgcatagaaa tgaatatgga atagcttcca tattggattc ttaccaatgt 60
actgcagaga taagtttagc tgacctggct accatatttt ttgcccagtt tgttcaagaa 120
gccacttaca aggaagtaag caaaatggtg aaagatgcat tgactgcaat tgagaaaccc 180
actggagatg aacagtcttc agggtgttta gaaaaccagc tacctgcctt tctggaagaa 240
ctttgccatg agaaagaaat tttggagaag tacggacatt cagactgctg cagccaaagt 300
gaagagggaa gacataactg ttttcttgca cacaaaaagc ccactccagc atcgatccca 360
cttttccaag ttccagaacc tgtcacaagc tgtgaagcat atgaagaaga cagggagaca 420
ttcatgaaca aattcattta tgagatagca agaaggcatc ccttcctgta tgcacctaca 480
attcttcttt gggctgctcg ctatgacaaa ataattccat cttgctgcaa agctgaaaat 540
gcagttgaat gcttccaaac aaaggcagca acagttacaa aagaattaag agaaagcagc 600
ttgttaaatc aacatgcatg tgcagtaatg aaaaattttg ggacccgaac tttccaagcc 660
ataactgtta ctaaactgag tcagaagttt accaaagtta attttactga aatccagaaa 720
ctagtcctgg atgtggccca tgtacatgag cactgttgca gaggagatgt gctggattgt 780
ctgcaggatg gggaaaaaat catgtcctac atatgttctc aacaagacac tctgtcaaac 840
aaaataacag aatgctgcaa actgaccacg ctggaacgtg gtcaatgtat aattcatgca 900
gaaaatgatg aaaaacctga aggtctatct ccaaatctaa acaggttttt aggagataga 960
gattttaacc aattttcttc aggggaataa gaattc 996
<210>8
<211>1410
<212>DNA
<213>智人
<400>8
ggtaccacac tgcatagaaa tgaatatgga atagcttcca tattggattc ttaccaatgt 60
actgcagaga taagtttagc tgacctggct accatatttt ttgcccagtt tgttcaagaa 120
gccacttaca aggaagtaag caaaatggtg aaagatgcat tgactgcaat tgagaaaccc 180
actggagatg aacagtcttc agggtgttta gaaaaccagc tacctgcctt tctggaagaa 240
ctttgccatg agaaagaaat tttggagaag tacggacatt cagactgctg cagccaaagt 300
gaagagggaa gacataactg ttttcttgca cacaaaaagc ccactccagc atcgatccca 360
cttttccaag ttccagaacc tgtcacaagc tgtgaagcat atgaagaaga cagggagaca 420
ttcatgaaca aattcattta tgagatagca agaaggcatc ccttcctgta tgcacctaca 480
attcttcttt gggctgctcg ctatgacaaa ataattccat cttgctgcaa agctgaaaat 540
gcagttgaat gcttccaaac aaaggcagca acagttacaa aagaattaag agaaagcagc 600
ttgttaaatc aacatgcatg tgcagtaatg aaaaattttg ggacccgaac tttccaagcc 660
ataactgtta ctaaactgag tcagaagttt accaaagtta attttactga aatccagaaa 720
ctagtcctgg atgtggccca tgtacatgag cactgttgca gaggagatgt gctggattgt 780
ctgcaggatg gggaaaaaat catgtcctac atatgttctc aacaagacac tctgtcaaac 840
aaaataacag aatgctgcaa actgaccacg ctggaacgtg gtcaatgtat aattcatgca 900
gaaaatnatg aaaaacctga aggtctatct ccaaatctaa acaggttttt aggagataga 960
gattttaacc aattttcttc aggggaaaaa aatatcttct tggcaagttt tgttcatgaa 1020
tattcaagaa gacatcctca gcttgctgtc tcagtaattc taagagttgc taaaggatac 1080
caggagttat tggagaagtg tttccagact gaaaaccctc ttgaatgcca agataaagga 1140
gaagaagaat tacagaaata catccaggag agccaagcat tggcaaagcg aagctgcggc 1200
ctcttccaga aactaggaga atattactta caaaatgcgt ttctcgttgc ttacacaaag 1260
aaagcccccc agctgacctc gtcggagctg atggccatca ccagaaaaat ggcagccaca 1320
gcagccactt gttgccaact cagtgaggac aaactattgg cctgtggcga gggagcggct 1380
gacattatta tcggacactt ataagaattc 1410
<210>9
<211>921
<212>DNA
<213>智人
<400>9
ggtaccccgg acgccacctg tcaccaagtc cgctccttct tccagagact gcagcccgga 60
ctcaagtggg tgccagaaac tcccgtgcca ggatcagatt tgcaagtatg tctccctaag 120
ggcccaacat gctgctcaag aaagatggaa gaaaaatacc aactaacagc acgattgaac 180
atggaacagc tgcttcagtc tgcaagtatg gagctcaagt tcttaattat tcagaatgct 240
gcggttttcc aagaggcctt tgaaattgtt gttcgccatg ccaagaacta caccaatgcc 300
atgttcaaga acaactaccc aagcctgact ccacaagctt ttgagtttgt gggtgaattt 360
ttcacagatg tgtctctcta catcttgggt tctgacatca atgtagatga catggtcaat 420
gaattgtttg acagcctgtt tccagtcatc tatacccagc taatgaaccc aggcctgcct 480
gattcagcct tggacatcaa tgagtgcctc cgaggagcaa gacgtgacct gaaagtattt 540
gggaatttcc ccaagcttat tatgacccag gtttccaagt cactgcaagt cactaggatc 600
ttccttcagg ctctgaatct tggaattgaa gtgatcaaca caactgatca cctgaagttc 660
agtaaggact gtggccgaat gctcaccaga atgtggtact gctcttactg ccagggactg 720
atgatggtta aaccctgtgg cggttactgc aatgtggtca tgcaaggctg tatggcaggt 780
gtggtggaga ttgacaagta ctggagagaa tacattctgt cccttgaaga acttgtgaat 840
ggcatgtaca gaatctatga catggagaac gtactgcttg gtctcttttc aacaatccat 900
gattctatcc agtgagaatt c 921
<210>10
<211>990
<212>DNA
<213>智人
<400>10
ggtaccgagg agccgcagtc agatcctagc gtcgagcccc ctctgagtca ggaaacattt 60
tcagacctat ggaaactact tcctgaaaac aacgttctgt cccccttgcc gtcccaagca 120
atggatgatt tgatgctgtc cccggacgat attgaacaat ggttcactga agacccaggt 180
ccagatgaag ctcccagaat gccagaggct gctccccgcg tggcccctgc accagcagct 240
cctacaccgg cggcccctgc accagccccc tcctggcccc tgtcatcttc tgtcccttcc 300
cagaaaacct accagggcag ctacggtttc cgtctgggct tcttgcattc tgggacagcc 360
aagtctgtga cttgcacgta ctcccctgcc ctcaacaaga tgttttgcca actggccaag 420
acctgccctg tgcagctgtg ggttgattcc acacccccgc ccggcacccg cgtccgcgcc 480
atggccatct acaagcagtc acagcacatg acggaggttg tgaggcgctg cccccaccat 540
gagcgctgct cagatagcga tggtctggcc cctcctcagc atcttatccg agtggaagga 600
aatttgcgtg tggagtattt ggatgacaga aacacttttc gacatagtgt ggtggtgccc 660
tatgagccgc ctgaggttgg ctctgactgt accaccatcc actacaacta catgtgtaac 720
agttcctgca tgggcggcat gaaccggagg cccatcctca ccatcatcac actggaagac 780
tccagtggta atctactggg acggaacagc tttgaggtgc gtgtttgtgc ctgtcctggg 840
agagaccggc gcacagagga agagaatctc cgcaagaaag gggagcctca ccacgagctg 900
cccccaggga gcactaagcg agcactgccc aacaacacca gctcctctcc ccagccaaag 960
aagaaaccac tggatggaga atgagaattc 990
<210>11
<211>555
<212>DNA
<213>智人
<400>11
ggtaccatgc aggccgaagg ccggggcaca gggggttcga cgggcgatgc tgatggccca 60
ggaggccctg gcattcctga tggcccaggg ggcaatgctg gcggcccagg agaggcgggt 120
gccacgggcg gcagaggtcc ccggggcgca ggggcagcaa gggcctcggg gccgggagga 180
ggcgccccgc ggggtccgca tggcggcgcg gcttcagggc tgaatggatg ctgcagatgc 240
ggggccaggg ggccggagag ccgcctgctt gagttctacc tcgccatgcc tttcgcgaca 300
cccatggaag cagagttggc ccgcaggagc ctggcccagg atgccccacc gcttcccgtg 360
ccaggggtgc ttctgaagga gttcactgtg tccggcaaca tactgactat ccgactgact 420
gctgcagacc accgccaact gcagctctcc atcagctcct gtctccagca gctttccctg 480
ttgatgtgga tcacgcagtg ctttctgccc gtgtttttgg ctcagcctcc ctcagggcag 540
aggcgctaag aattc 555
<210>12
<211>939
<212>DNA
<213>智人
<400>12
ggtacctctc ttgagcagag gagtctgcac tgcaagcctg aggaagccct tgaggcccaa 60
caagaggccc tgggcctggt gtgtgtgcag gctgccgcct cctcctcctc tcctctggtc 120
ctgggcaccc tggaggaggt gcccactgct gggtcaacag atcctcccca gagtcctcag 180
ggagcctccg cctttcccac taccatcaac ttcactcgac agaggcaacc cagtgagggt 240
tccagcagcc gtgaagagga ggggccaagc acctcttgta tcctggagtc cttgttccga 300
gcagtaatca ctaagaaggt ggctgatttg gttggttttc tgctcctcaa atatcgagcc 360
agggagccag tcacaaaggc agaaatgctg gagagtgtca tcaaaaatta caagcactgt 420
tttcctgaga tcttcggcaa agcctctgag tccttgcagc tggtctttgg cattgacgtg 480
aaggaagcag accccaccgg ccactcctat gtccttgtca cctgcctagg tctctcctat 540
gatggcctgc tgggtgataa tcagatcatg cccaagacag gcttcctgat aattgtcctg 600
gtcatgattg caatggaggg cggccatgct cctgaggagg aaatctggga ggagctgagt 660
gtgatggagg tgtatgatgg gagggagcac agtgcctatg gggagcccag gaagctgctc 720
acccaagatt tggtgcagga aaagtacctg gagtaccggc aggtgccgga cagtgatccc 780
gcacgctatg agttcctgtg gggtccaagg gcccttgctg aaaccagcta tgtgaaagtc 840
cttgagtatg tgatcaaggt cagtgcaaga gttcgctttt tcttcccatc cctgcgtgaa 900
gcagctttga gagaggagga agagggagtc tgagaattc 939
<210>13
<211>346
<212>PRT
<213>智人
<400>13
Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr
1 5 10 15
Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu
20 25 30
Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr
35 40 45
Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser
50 55 60
Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp
65 70 75 80
Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu
85 90 95
Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp
100 105 110
Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His
115 120 125
Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro
130 135 140
Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn
145 150 155 160
Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro
165 170 175
Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys
180 185 190
Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr
195 200 205
Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys
210 215 220
Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val
225 230 235 240
Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln
245 250 255
Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly
260 265 270
Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile
275 280 285
Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys
290 295 300
Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp
305 310 315 320
Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335
Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu
340 345
<210>14
<211>484
<212>PRT
<213>智人
<400>14
Met Lys Trp Val Glu Ser Ile Phe Leu Ile Phe Leu Leu Asn Phe Thr
1 5 10 15
Glu Ser Arg Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu
20 25 30
Asp Ser Tyr Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr
35 40 45
Ile Phe Phe Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser
50 55 60
Lys Met Val Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp
65 70 75 80
Glu Gln Ser Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu
85 90 95
Glu Leu Cys His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp
100 105 110
Cys Cys Ser Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His
115 120 125
Lys Lys Pro Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro
130 135 140
Val Thr Ser Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn
145 150 155 160
Lys Phe Ile Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro
165 170 175
Thr Ile Leu Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys
180 185 190
Cys Lys Ala Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr
195 200 205
Val Thr Lys Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys
210 215 220
Ala Val Met Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val
225 230 235 240
Thr Lys Leu Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln
245 250 255
Lys Leu Val Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly
260 265 270
Asp Val Leu Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile
275 280 285
Cys Ser Gln Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys
290 295 300
Leu Thr Thr Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp
305 310 315 320
Glu Lys Pro Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp
325 330 335
Arg Asp Phe Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala
340 345 350
Ser Phe Val His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser
355 360 365
Val Ile Leu Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys
370 375 380
Phe Gln Thr Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu
385 390 395 400
Leu Gln Lys Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys
405 410 415
Gly Leu Phe Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu
420 425 430
Val Ala Tyr Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met
435 440 445
Ala Ile Thr Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu
450 455 460
Ser Glu Asp Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile
465 470 475 480
Ile Gly His Leu
<210>15
<211>332
<212>PRT
<213>智人
<400>15
Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu
1 5 10 15
Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp
20 25 30
Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly
35 40 45
Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val
50 55 60
Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys
65 70 75 80
Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala
85 90 95
Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln
100 105 110
Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala
115 120 125
Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe
130 135 140
Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp
145 150 155 160
Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro
165 170 175
Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu
180 185 190
Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe
195 200 205
Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln
210 215 220
Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile
225 230 235 240
Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu
245 250 255
Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys
260 265 270
Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly
275 280 285
Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu
290 295 300
Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu
305 310 315 320
Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln
325 330
<210>16
<211>326
<212>PRT
<213>智人
<400>16
Met Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln
1 5 10 15
Glu Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu
20 25 30
Ser Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp
35 40 45
Asp Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro
50 55 60
Arg Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro
65 70 75 80
Thr Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser
85 90 95
Val Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly
100 105 110
Phe Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro
115 120 125
Ala Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln
130 135 140
Leu Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met
145 150 155 160
Ala Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys
165 170 175
Pro His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln
180 185 190
His Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp
195 200 205
Arg Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu
210 215 220
Val Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser
225 230 235 240
Ser Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr
245 250 255
Leu Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val
260 265 270
Arg Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn
275 280 285
Leu Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr
290 295 300
Lys Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys
305 310 315 320
Lys Pro Leu Asp Gly Glu
325
<210>17
<211>180
<212>PRT
<213>智人
<400>17
Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp
1 5 10 15
Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly
20 25 30
Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala
35 40 45
Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro
50 55 60
His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala
65 70 75 80
Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe
85 90 95
Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp
100 105 110
Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val
115 120 125
Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln
130 135 140
Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met
145 150 155 160
Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser
165 170 175
Gly Gln Arg Arg
180
<210>18
<211>309
<212>PRT
<213>智人
<400>18
Met Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu
1 5 10 15
Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Ala
20 25 30
Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr
35 40 45
Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe
50 55 60
Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser
65 70 75 80
Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser
85 90 95
Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe
100 105 110
Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met
115 120 125
Leu Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe
130 135 140
Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys
145 150 155 160
Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly
165 170 175
Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr
180 185 190
Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His
195 200 205
Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr
210 215 220
Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr
225 230 235 240
Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp
245 250 255
Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala
260 265 270
Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala
275 280 285
Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu
290 295 300
Glu Glu Glu Gly Val
305
<210>19
<211>327
<212>PRT
<213>智人
<400>19
Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr
1 5 10 15
Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe
20 25 30
Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val
35 40 45
Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser
50 55 60
Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys
65 70 75 80
His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser
85 90 95
Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro
100 105 110
Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser
115 120 125
Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile
130 135 140
Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu
145 150 155 160
Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys Cys Lys Ala
165 170 175
Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys
180 185 190
Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Ala Val Met
195 200 205
Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu
210 215 220
Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val
225 230 235 240
Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly Asp Val Leu
245 250 255
Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln
260 265 270
Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr
275 280 285
Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Asp Glu Lys Pro
290 295 300
Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe
305 310 315 320
Asn Gln Phe Ser Ser Gly Glu
325
<210>20
<211>465
<212>PRT
<213>智人
<400>20
Thr Leu His Arg Asn Glu Tyr Gly Ile Ala Ser Ile Leu Asp Ser Tyr
1 5 10 15
Gln Cys Thr Ala Glu Ile Ser Leu Ala Asp Leu Ala Thr Ile Phe Phe
20 25 30
Ala Gln Phe Val Gln Glu Ala Thr Tyr Lys Glu Val Ser Lys Met Val
35 40 45
Lys Asp Ala Leu Thr Ala Ile Glu Lys Pro Thr Gly Asp Glu Gln Ser
50 55 60
Ser Gly Cys Leu Glu Asn Gln Leu Pro Ala Phe Leu Glu Glu Leu Cys
65 70 75 80
His Glu Lys Glu Ile Leu Glu Lys Tyr Gly His Ser Asp Cys Cys Ser
85 90 95
Gln Ser Glu Glu Gly Arg His Asn Cys Phe Leu Ala His Lys Lys Pro
100 105 110
Thr Pro Ala Ser Ile Pro Leu Phe Gln Val Pro Glu Pro Val Thr Ser
115 120 125
Cys Glu Ala Tyr Glu Glu Asp Arg Glu Thr Phe Met Asn Lys Phe Ile
130 135 140
Tyr Glu Ile Ala Arg Arg His Pro Phe Leu Tyr Ala Pro Thr Ile Leu
145 150 155 160
Leu Trp Ala Ala Arg Tyr Asp Lys Ile Ile Pro Ser Cys Cys Lys Ala
165 170 175
Glu Asn Ala Val Glu Cys Phe Gln Thr Lys Ala Ala Thr Val Thr Lys
180 185 190
Glu Leu Arg Glu Ser Ser Leu Leu Asn Gln His Ala Cys Ala Val Met
195 200 205
Lys Asn Phe Gly Thr Arg Thr Phe Gln Ala Ile Thr Val Thr Lys Leu
210 215 220
Ser Gln Lys Phe Thr Lys Val Asn Phe Thr Glu Ile Gln Lys Leu Val
225 230 235 240
Leu Asp Val Ala His Val His Glu His Cys Cys Arg Gly Asp Val Leu
245 250 255
Asp Cys Leu Gln Asp Gly Glu Lys Ile Met Ser Tyr Ile Cys Ser Gln
260 265 270
Gln Asp Thr Leu Ser Asn Lys Ile Thr Glu Cys Cys Lys Leu Thr Thr
275 280 285
Leu Glu Arg Gly Gln Cys Ile Ile His Ala Glu Asn Xaa Glu Lys Pro
290 295 300
Glu Gly Leu Ser Pro Asn Leu Asn Arg Phe Leu Gly Asp Arg Asp Phe
305 310 315 320
Asn Gln Phe Ser Ser Gly Glu Lys Asn Ile Phe Leu Ala Ser Phe Val
325 330 335
His Glu Tyr Ser Arg Arg His Pro Gln Leu Ala Val Ser Val Ile Leu
340 345 350
Arg Val Ala Lys Gly Tyr Gln Glu Leu Leu Glu Lys Cys Phe Gln Thr
355 360 365
Glu Asn Pro Leu Glu Cys Gln Asp Lys Gly Glu Glu Glu Leu Gln Lys
370 375 380
Tyr Ile Gln Glu Ser Gln Ala Leu Ala Lys Arg Ser Cys Gly Leu Phe
385 390 395 400
Gln Lys Leu Gly Glu Tyr Tyr Leu Gln Asn Ala Phe Leu Val Ala Tyr
405 410 415
Thr Lys Lys Ala Pro Gln Leu Thr Ser Ser Glu Leu Met Ala Ile Thr
420 425 430
Arg Lys Met Ala Ala Thr Ala Ala Thr Cys Cys Gln Leu Ser Glu Asp
435 440 445
Lys Leu Leu Ala Cys Gly Glu Gly Ala Ala Asp Ile Ile Ile Gly His
450 455 460
Leu
465
<210>21
<211>303
<212>PRT
<213>智人
<400>21
Pro Asp Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln
1 5 10 15
Pro Gly Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu
20 25 30
Gln Val Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu
35 40 45
Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln
50 55 60
Ser Ala Ser Met Glu Leu Lys Phe Leu IleIle Gln Asn Ala Ala Val
65 70 75 80
Phe Gln Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr
85 90 95
Asn Ala Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe
100 105 110
Glu Phe Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly
115 120 125
Ser Asp Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu
130 135 140
Phe Pro Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser
145 150 155 160
Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys
165 170 175
Val Phe Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser
180 185 190
Leu Gln Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu
195 200 205
Val Ile Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg
210 215 220
Met Leu Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met
225 230 235 240
Val Lys Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met
245 250 255
Ala Gly Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser
260 265 270
Leu Glu Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn
275 280 285
Val Leu Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Glx
290 295 300
<210>22
<211>326
<212>PRT
<213>智人
<400>22
Glu Glu Pro Gln Ser Asp Pro Ser Val Glu Pro Pro Leu Ser Gln Glu
1 5 10 15
Thr Phe Ser Asp Leu Trp Lys Leu Leu Pro Glu Asn Asn Val Leu Ser
20 25 30
Pro Leu Pro Ser Gln Ala Met Asp Asp Leu Met Leu Ser Pro Asp Asp
35 40 45
Ile Glu Gln Trp Phe Thr Glu Asp Pro Gly Pro Asp Glu Ala Pro Arg
50 55 60
Met Pro Glu Ala Ala Pro Arg Val Ala Pro Ala Pro Ala Ala Pro Thr
65 70 75 80
Pro Ala Ala Pro Ala Pro Ala Pro Ser Trp Pro Leu Ser Ser Ser Val
85 90 95
Pro Ser Gln Lys Thr Tyr Gln Gly Ser Tyr Gly Phe Arg Leu Gly Phe
100 105 110
Leu His Ser Gly Thr Ala Lys Ser Val Thr Cys Thr Tyr Ser Pro Ala
115 120 125
Leu Asn Lys Met Phe Cys Gln Leu Ala Lys Thr Cys Pro Val Gln Leu
130 135 140
Trp Val Asp Ser Thr Pro Pro Pro Gly Thr Arg Val Arg Ala Met Ala
145 150 155 160
Ile Tyr Lys Gln Ser Gln His Met Thr Glu Val Val Arg Arg Cys Pro
165 170 175
His His Glu Arg Cys Ser Asp Ser Asp Gly Leu Ala Pro Pro Gln His
180 185 190
Leu Ile Arg Val Glu Gly Asn Leu Arg Val Glu Tyr Leu Asp Asp Arg
195 200 205
Asn Thr Phe Arg His Ser Val Val Val Pro Tyr Glu Pro Pro Glu Val
210 215 220
Gly Ser Asp Cys Thr Thr Ile His Tyr Asn Tyr Met Cys Asn Ser Ser
225 230 235 240
Cys Met Gly Gly Met Asn Arg Arg Pro Ile Leu Thr Ile Ile Thr Leu
245 250 255
Glu Asp Ser Ser Gly Asn Leu Leu Gly Arg Asn Ser Phe Glu Val Arg
260 265 270
Val Cys Ala Cys Pro Gly Arg Asp Arg Arg Thr Glu Glu Glu Asn Leu
275 280 285
Arg Lys Lys Gly Glu Pro His His Glu Leu Pro Pro Gly Ser Thr Lys
290 295 300
Arg Ala Leu Pro Asn Asn Thr Ser Ser Ser Pro Gln Pro Lys Lys Lys
305 310 315 320
Pro Leu Asp Gly Glu Glx
325
<210>23
<211>180
<212>PRT
<213>智人
<400>23
Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp
1 5 10 15
Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly
20 25 30
Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala
35 40 45
Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro
50 55 60
His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala
65 70 75 80
Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe
85 90 95
Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp
100 105 110
Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val
115 120 125
Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln
130 135 140
Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met
145 150 155 160
Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser
165 170 175
Gly Gln Arg Arg
180
<210>24
<211>308
<212>PRT
<213>智人
<400>24
Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu Glu
1 5 10 15
Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Ala Ser
20 25 30
Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr Ala
35 40 45
Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe Pro
50 55 60
Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser Ser
65 70 75 80
Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser Leu
85 90 95
Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe Leu
100 105 110
Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met Leu
115 120 125
Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe Gly
130 135 140
Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys Glu
145 150 155 160
Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly Leu
165 170 175
Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr Gly
180 185 190
Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His Ala
195 200 205
Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr Asp
210 215 220
Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr Gln
225 230 235 240
Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp Ser
245 250 255
Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala Glu
260 265 270
Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala Arg
275 280 285
Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu Glu
290 295 300
Glu Glu Gly Val
305
Claims (19)
1.利用携带表达人肝癌特异性抗原的肿瘤的动物模型分析源于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法,所述方法包括以下步骤:
(a)给正常非人动物施用表达人肝癌特异性抗原的癌细胞系,以在正常动物中诱发癌;
(b)给所述动物施用待分析的癌树突状细胞;及
(c)通过测量所述动物的源自所述癌细胞系的癌细胞的形成或生长来测定作为免疫治疗手段的树突状细胞对所述癌的预防和治疗功效。
2.利用携带表达人肝癌特异性抗原的肿瘤的动物模型分析源于树突状细胞的免疫治疗手段对肝癌的预防和治疗功效的方法,所述方法包括以下步骤:
(a)给正常非人动物施用待分析的树突状细胞;
(b)给所述动物施用表达人肝癌特异性抗原的癌细胞系,以在所述动物中诱发癌;及
(c)通过测量所述动物的源自所述癌细胞系的癌细胞的形成或生长来测定作为免疫治疗手段的树突状细胞对肝癌的预防和治疗功效。
3.权利要求1或2的方法,其中所述动物是啮齿动物。
4.权利要求3的方法,其中所述啮齿动物是小鼠(Mus musculus)。
5.权利要求1或2的方法,其中所述人肝癌特异性抗原是AFP(甲胎蛋白)、MAGEA1(黑素瘤抗原家族A,1)、TRP53(转化相关蛋白53)、GPC3(磷脂酰肌醇蛋白聚糖3)或NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)。
6.权利要求5的方法,其中所述人肝癌特异性抗原是AFP(甲胎蛋白)。
7.权利要求1或2的方法,其中所述癌细胞系源于小鼠(Musmusculus)。
8.权利要求7的方法,其中所述癌细胞系与所述动物同源。
9.权利要求1或2的方法,其中所述步骤(a)或(b)中的树突状细胞或癌细胞系的施用通过皮下注射进行。
10.权利要求1或2的方法,其中所述步骤(b)或(a)中的树突状细胞或癌细胞系的施用通过皮下注射进行。
11.权利要求1或2的方法,其中所述表达人肝癌特异性抗原的癌细胞系是源于肝癌细胞的癌细胞系。
12.表达人肝癌特异性抗原的小鼠源肝癌细胞系(重组MH134细胞系),其特征在于,所述人肝癌特异性抗原是AFP(甲胎蛋白)、MAGEA1(黑素瘤抗原家族A,1)、TRP53(转化相关蛋白53)、GPC3(磷脂酰肌醇蛋白聚糖3)或NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)。
13.权利要求12的小鼠源肝癌细胞系,其中所述癌细胞系经含有编码以下氨基酸序列的核苷酸序列的载体转化:
SEQ ID NO:13的氨基酸序列、
SEQ ID NO:15的氨基酸序列、
SEQ ID NO:16的氨基酸序列、或
SEQ ID NO:18的氨基酸序列。
14.权利要求12的小鼠源肝癌细胞系,其中所述癌细胞系经含有以下核苷酸序列的载体转化:
SEQ ID NO:1的核苷酸7-1044的核苷酸序列、
SEQ ID NO:3的核苷酸7-1002的核苷酸序列、
SEQ ID NO:4的核苷酸7-984的核苷酸序列、或
SEQ ID NO:6的核苷酸7-933的核苷酸序列。
15.权利要求14的小鼠源肝癌细胞系,其中所述癌细胞系经以下载体转化:
pcDNA3.1(+)-标签/AFP(甲胎蛋白)、
pcDNA3.1(+)-标签/GPC3(磷脂酰肌醇蛋白聚糖3)、
pcDNA3.1(+)-标签/TRP53(转化相关蛋白53)、
pcDNA3.1(+)-标签/NY-ESO-1(纽约食管鳞状细胞癌1或癌/睾丸抗原1;CTAG1)、或
pcDNA3.1(+)-标签/MAGEA1(黑素瘤抗原家族A,1)。
16.权利要求12的小鼠源肝癌细胞系,其中所述癌细胞系是表达AFP(甲胎蛋白)抗原的MH134/AFP(甲胎蛋白)。
17.小鼠肝癌模型,其特征在于,所述小鼠模型具有通过接种表达人肝癌特异性抗原的权利要求12的肝癌细胞系而形成的癌,且在所述小鼠模型中形成的癌的转移或生长受用经人肝癌特异性抗原刺激的树突状细胞的治疗的抑制。
18.权利要求17的小鼠肝癌模型,其中所述肝癌细胞系与所述小鼠同源。
19.权利要求18的小鼠肝癌模型,其中所述小鼠肝癌模型用于实施权利要求1或2的方法。
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CN106604989A (zh) * | 2014-08-01 | 2017-04-26 | Jw可瑞基因株式会社 | 用于制备树突状细胞的方法、由该方法制备的树突状细胞、及其应用 |
CN107604062A (zh) * | 2017-09-01 | 2018-01-19 | 北京启辰生生物科技有限公司 | 一种用于肝癌免疫治疗的多抗原检测方法 |
CN110922492A (zh) * | 2019-12-18 | 2020-03-27 | 重庆医科大学 | 融合肽、ctp介导的诱导cml细胞免疫应答的dc疫苗及其制备方法 |
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WO2016060407A1 (ko) * | 2014-10-17 | 2016-04-21 | 동국대학교 산학협력단 | Emp2 유전자가 결여된 폐암 유발 동물모델의 제조방법 및 이의 용도 |
WO2017027063A1 (en) * | 2015-08-10 | 2017-02-16 | Yanping Kong | Method and compound for treatment of cancer using phosphorous-32 labeled dna |
CN112674028B (zh) * | 2020-12-30 | 2022-06-28 | 汉姆德(宁波)智能医疗科技有限公司 | 促诱癌剂诱发动物癌症模型建立的方法 |
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US7098306B2 (en) * | 1997-02-13 | 2006-08-29 | The Regents Of The University Of California | Method and compositions for treating hepatocellular cancer |
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CN106604989A (zh) * | 2014-08-01 | 2017-04-26 | Jw可瑞基因株式会社 | 用于制备树突状细胞的方法、由该方法制备的树突状细胞、及其应用 |
US10478479B2 (en) | 2014-08-01 | 2019-11-19 | Jw Creagene Inc. | Method for preparing dendritic cell, dendritic cell prepared thereby, and use thereof |
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CN107604062A (zh) * | 2017-09-01 | 2018-01-19 | 北京启辰生生物科技有限公司 | 一种用于肝癌免疫治疗的多抗原检测方法 |
CN110922492A (zh) * | 2019-12-18 | 2020-03-27 | 重庆医科大学 | 融合肽、ctp介导的诱导cml细胞免疫应答的dc疫苗及其制备方法 |
CN110922492B (zh) * | 2019-12-18 | 2022-02-15 | 重庆医科大学 | 融合肽、ctp介导的诱导cml细胞免疫应答的dc疫苗及其制备方法 |
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