CN102212112B - Mta1来源的抗肿瘤ctl表位肽及其应用 - Google Patents
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Abstract
本发明公开了一种MTA1来源的抗肿瘤CTL表位肽,包括九肽,所述九肽的氨基酸序列为P22:Tyr-Leu-Ile-Arg-Arg-Ile-Glu-Glu-Leu或P57:Ala-Leu-Ala-Asp-Lys-His-Ala-Thr-Leu或P109:Phe-Leu-Ser-Arg-Gln-Leu-Glu-Ser-Leu或P129:Thr-Leu-Leu-Asn-Glu-Thr-Glu-Ser-Leu或P173:Tyr-Gln-Ala-Asp-Ile-Thr-Asp-Leu-Leu 。本发明还包括上述肽在制备肿瘤治疗性多肽疫苗中的应用。本发明的优点在于采用理论和实验相结合的方法初步鉴定出肿瘤转移相关抗原的表位肽。鉴定的九肽未见文献报道,为研制基于抗原MTA1的肿瘤治疗性多肽疫苗提供理论基础,并为后续多价的抗原肽疫苗、长肽疫苗、多表位疫苗等的构建奠定基础。
Description
技术领域
本发明涉及肽,尤其是涉及MTA1来源的抗肿瘤CTL表位肽,本发明还涉及该肽在制备肿瘤治疗性多肽疫苗中的应用。
背景技术
转移是一个涉及多种原因及其产物的复杂过程,包括肿瘤细胞从原发灶脱离,侵入淋巴管、血管及周围组织,诱导血管形成,逃避宿主细胞抗肿瘤反应和在转移部位生长。因此,了解肿瘤转移所涉及的基因和基因产物是目前国内外研究的热题。
转移相关蛋白1(MTA1)是1994年Toh等人在人的乳腺癌细胞中鉴定发现的。近年来的研究发现它在多种转移性肿瘤中过表达,如乳腺癌、胃肠癌、食管癌、胰腺癌、胃癌、肝癌、非小细胞肺癌等,而在正常组织中低表达或不表达,并且其在肿瘤组织中的表达水平与其转移或浸润潜能有明显的关系。这些研究结果表明MTA1是一个非常好的肿瘤相关抗原,是肿瘤诊断和治疗的靶点。近年来大多数研究者将目光集中在MTA家族成员尤其是MTA1在多种人类肿瘤组织及肿瘤细胞中的表达情况、分子功能等方面,Li Kai等人在MTA1的抗前列腺癌血管生成方面取得了一定的研究成果。目前尚未见针对MTA1的HLA-I类限制性CTL表位肽的报道。
随着现代免疫学的发展,细胞毒性T淋巴细胞(Cytotoxic T lymphocyte,CTL)在肿瘤中发挥的重要作用越来越受到重视。如何有效激发CTL介导的特异性细胞免疫应答,发挥抗肿瘤效能,已经成为当今肿瘤治疗性多肽疫苗研制领域的一个重要课题。既往研究发现,引起CTL免疫应答反应的,并非完整的肿瘤抗原分子,而是抗原来源的特异性CTL表位(Epitope)。抗原递呈细胞摄取肿瘤抗原后,通过蛋白水解作用将其加工成为8~10个氨基酸长度的多肽片段,即CTL表位,进而与内质网腔中的主要组织相容性复合体I(MHC-I)类分子结合形成多肽-MHC复合物(peptide-MHC complex,pMHC),并最终将pMHC递呈到细胞表面供CD8+T细胞表面的T细胞受体(T cell receptor,TCR)识别,从而活化CTL细胞,引发CTL免疫应答。
发明内容
本发明的目的在于提供一种对肿瘤细胞SW620具有一定杀伤力的MTA1来源的抗肿瘤CTL表位肽,同时本发明还提供了该肽在制备肿瘤治疗性多肽疫苗中的应用。
为实现上述目的,本发明可采取下述技术方案:
本发明所述的MTA1来源的抗肿瘤CTL表位肽,包括九肽,所述九肽的氨基酸序列为
P22:Tyr-Leu-Ile-Arg-Arg-Ile-Glu-Glu-Leu 分子量为1204.49
或 P57:Ala-Leu-Ala-Asp-Lys-His-Ala-Thr-Leu 分子量为939.4
或P109:Phe-Leu-Ser-Arg-Gln-Leu-Glu-Ser-Leu 分子量为1092.1
或 P129:Thr-Leu-Leu-Asn-Glu-Thr-Glu-Ser-Leu 分子量为1019.51
或 P173:Tyr-Gln-Ala-Asp-Ile-Thr-Asp-Leu-Leu 分子量为1051.94。
本发明还包括上述肽在制备肿瘤治疗性多肽疫苗中的应用。
本发明的优点在于采用理论和实验相结合的方法初步鉴定出肿瘤转移相关抗原的表位肽。鉴定的九肽未见文献报道,为研制基于抗原MTA1的肿瘤治疗性多肽疫苗提供理论基础,并为后续多价的抗原肽疫苗、长肽疫苗、多表位疫苗等的构建奠定基础。
附图说明
图1-5是本发明表位肽的质谱分析图。
图6是本发明表位肽诱导的特异性CTL对肿瘤细胞SW620 (MTA1-阳性,HLA-A2阳性)的杀伤作用。
图7-11是本发明表位肽诱导的特异性CTL分泌IFN-γ的能力。
具体实施方式
本发明所述的MTA1来源的抗肿瘤CTL表位肽,包括九肽,所述九肽的氨基酸序列为
P22:Tyr-Leu-Ile-Arg-Arg-Ile-Glu-Glu-Leu
或 P57:Ala-Leu-Ala-Asp-Lys-His-Ala-Thr-Leu
或 P109:Phe-Leu-Ser-Arg-Gln-Leu-Glu-Ser-Leu
或 P129:Thr-Leu-Leu-Asn-Glu-Thr-Glu-Ser-Leu
或 P173:Tyr-Gln-Ala-Asp-Ile-Thr-Asp-Leu-Leu。
本发明主要采用理论与实践相结合的方法,根据抗原的一级结构,采用免疫信息学手段,运用SYFPEITHI、BIMAS、NetCTL 1.2和IEDB数据库对MTA1蛋白抗原的HLA-A*0201限制性CTL表位进行了预测分析。
筛选获得上述表位肽采用标准的Fmoc方案进行合成, 经HPLC纯化后,其纯度大于90%,质谱分析并证实其分子量符合理论值。各肽质谱鉴定图见图1-5。
上述表位肽的合成及制备:采用固相合成法。基本流程如下:首先将一个氨基被Fmoc基团保护的氨基酸连接在不溶性固相载体Wang树脂上,然后脱掉氨基的保护基,第一个氨基酸即连接至固相载体上;其次将氨基被Fmoc基团保护的第二个氨基酸的羧基用缩合剂活化,活化后的氨基酸再与已接在固相载体的第一个氨基酸的氨基反应形成肽键,此时在固相载体上就生成了一个带有保护基的二肽。重复上述的肽键形成反应,使肽链从C端向N端生长,直至达到所需要的肽链长度,最后切割得到目的肽。经HPLC纯化后,其纯度大于90%,质谱分析并证实其分子量符合理论值。(参见:①黄惟德、陈常庆著,多肽合成,科学出版社,1985年。②.N.休厄德、H.D.贾库布克著,刘克良等译,肽:化学与生物学,科学出版社,2005年)
本发明表位肽的人外周血单个核细胞(PBMCs)的分离与ELISPOT实验检测:抽取健康供者的外周血经密度梯度离心分离,获得PBMCs,添加白细胞介素2(IL-2,Peprotech公司)和人β2微球蛋白诱导分化CTL细胞,进一步在体外LDH和ELISPOT实验进行验证。方法如下:
1、PBMCs的分离与诱导:(1)以40ml PBS(PH 7.2)稀释抗凝处理后的外周血40ml;(2)离心管中加入4ml Lymphoprep 分离液(Axis-Shield 公司);(3)加8ml步骤(1)中稀释后的外周血于4ml Lymphoprep 分离液液面上;(4)20℃离心(2000rmp×20min);(5)离心后,分为四层,弃去最上层,用玻璃吸管吸取第二层即白膜层(富含PBMCs);(6)吸出的白膜层用PBS(pH 7.2)离心洗涤两次;(7)用24孔板铺板,细胞的浓度为1×10 6/ml,每孔1ml;(8)第二天每孔加入3 μg人β2微球蛋白和10 μg表位肽,同时设立PBS组(以PBS替代表位肽)作为阴性对照组; (9)第三天每孔加入50u IL-2;每2~3天换液,于七天后进行第二轮荷肽(即每孔补加50u IL-2、10 μg人β2微球蛋白和10 μg表位肽/PBS),十四天后进行第三轮荷肽。第三轮荷肽后3天,得到效应细胞CTL,然后进行LDH实验和ELISPOT实验检测待测肽的作用。
2、LDH检测:使用CytoTox 96? Non-Radioactive Cytotoxicity Assay(细胞毒性检测试剂盒,Promega公司)进行LDH试验检测细胞毒活性。步骤如下(详见试剂盒说明书):
1)设立检测板(100μl/孔)
(1)设立实验组:以肿瘤细胞EC-9706(ATCC公司)作为靶细胞(最优靶细胞数:5000)按不同效靶比10:1 、20:1、40:1加入上述效应细胞CTL ;
(2)设立效应细胞自发释放组;
(3)设立靶细胞自发释放组;
(4)设立靶细胞最大释放组;
(5)设立体积校正对照组;
(6)设立背景对照组。
2)细胞裂解及收获上清
(1)37℃、5%CO2孵育检测板(5h);
(2)靶细胞最大释放组和体积校正对照组中加入裂解液,每100μl培养基中加入10μl裂解液(10×),收获上清前45min加入裂解液;
(3)250g离心4min,收获上清。
3)LDH检测
(1)转移50μl上清至另一孔板;
(2)50μl/孔迅速添加稀释的底物混合液,室温避光孵育30min;
(3)添加50μl终止溶液;
(4)将孔中含有的气泡去除,一小时内检测490nm吸收值OD。
细胞杀伤率计算公式如下:
杀伤率(%) =[(OD实验组-OD效应细胞自发释放组-OD靶细胞自发释放组)/( OD靶细胞最大释放组-OD靶细胞自发释放组)]×100%
(注:所有实验组、效应细胞自发释放组、靶细胞自发释放组的吸收值均应减去背景平均吸收值;靶细胞最大释放组吸收值应减去体积校正对照组的平均吸收值)
结果见图6,由图6可知,表位肽诱导的特异性CTL对肿瘤细胞SW620(HLA-A*0201-阳性,MTA1-阳性)均显示出一定的杀伤率。
3、 ELISPOT实验:具体实验步骤如下:(1)封闭:取出所需板条,用含5% FCS RPMI 1640培养基的培养基封闭,200μL /孔,室温下(25℃)静置5~10 min后将其扣出(FCS可以封闭所包被抗体的FCS受体以降低非特异性反应);(2)细胞上板:诱导的CTL作为效应细胞(1×105/孔),荷肽的T2A2细胞作为刺激细胞(1×105/孔)铺板。100 μL/孔。细胞在孔中的分布要尽量均匀(加入细胞之后,不要再震动或者拍击ELISPOT板);● 正对照:细胞浓度为1×105/孔、加入10 μL PHA,该浓度能有效刺激 IFN-γ的分泌;● 背景负对照:加入100 μL的含5%FCS的RPMI 1640培养基;(3) 加完所有的样品之后,盖上板盖,放入CO2培养箱,37 ℃培养18h;(4)裂解细胞:倾倒孔内的细胞及培养基,200 μL/孔加入冰冷的去离子水,4 ℃冰浴反应10 min(低渗法裂解细胞);(5)洗涤:倾倒孔内的液体,1×的Washing Buffer,200 μL/孔,洗涤5~7次,每次停留30~60 s,最后一次,在吸水纸上扣干;(6)加入检测抗体孵育:每孔加入100 μL稀释好的生物素标记检测抗体,37 ℃孵育1h;(7)洗涤:倾倒孔内的液体,1×的Washing Buffer,200 μL/孔,洗涤5次,每次停留时间为30~60 s,最后一次,在吸水纸上扣干;(8)酶联亲和素孵育:将稀释好的酶联亲和素工作液加入到实验孔,100 μL/孔, 37 ℃孵育1h;(9)洗涤:倾倒孔内的液体,1×的Washing Buffer,200uL/孔,洗涤5次,每次停留时间为30~60 s,最后一次,在吸水纸上扣干;(10)显色:解冻已配好的AEC显色液。每孔加入100 μL的显色液,室温静置15-45min(在20 -25°C,显色25min较合适),注意避光;(11)终止显色:倾倒孔内液体,揭开板底座,以去离子水洗涤3~5遍,终止显色过程。将板倒扣在吸水纸上,拍干细小的水珠,之后取下保护层,放在通风的地方,室温静置10-30min,让膜自然晾干;用 ELISPOT 图象分析仪计数96孔板中每孔形成的斑点数。
体外ELISPOT实验结果显示:候选肽P22、P57、P109、P129、P173均能够在6名健康供者的外周血中诱导获得CTL,且获得的CTL经刺激后分泌较高量的IFN-γ(如图7-11)。
本发明鉴定出的肿瘤转移相关抗原的表位肽为研制基于抗原MTA1的肿瘤治疗性多肽疫苗提供了理论基础,并为后续多价的抗原肽疫苗、长肽疫苗、多表位疫苗等的构建奠定了基础。
Claims (2)
1.一种MTA1来源的抗肿瘤CTL表位肽,包括九肽,其特征在于:所述九肽的氨基酸序列为P129:Thr-Leu-Leu-Asn-Glu-Thr-Glu-Ser-Leu。
2.根据权利要求1所述的MTA1来源的抗肿瘤CTL表位肽在制备高表达该抗原的肿瘤细胞治疗性多肽疫苗中的应用。
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Current developments with peptide-based human tumor vaccines;Khazaie K.等;《Current Opinion in Oncology》;20091231;第21卷;524-530 * |
Geng Li 等.Identification of Metastasis Associated Antigen 1 (MTA1) by Serological Screening of Prostate Cancer cDNA Libraries.《The Open Biochemistry Journal》.2008,(第2期),100-107. |
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