CN110922492B - 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 - Google Patents
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 Download PDFInfo
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- CN110922492B CN110922492B CN201911308578.2A CN201911308578A CN110922492B CN 110922492 B CN110922492 B CN 110922492B CN 201911308578 A CN201911308578 A CN 201911308578A CN 110922492 B CN110922492 B CN 110922492B
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Abstract
本发明公开了一种融合肽，所述融合肽的氨基酸序列如SEQ ID No:1或2所示。还公开了一种分离的多核苷酸，编码前述的融合肽。还公开了CTP介导的诱导CML细胞免疫应答的DC疫苗，为通过将前述的融合肽与成熟的树突状细胞共孵育得到。还公开了该疫苗的制备方法，包括如下步骤：1)合成前述的融合肽序列；2)培养成熟的树突状细胞；3)将融合肽与成熟的树突状细胞共孵育、洗涤，即得到DC疫苗。本发明的树突状细胞疫苗免疫小鼠后，刺激小鼠体内特异性细胞免疫应答，诱导产生细胞毒性T淋巴细胞，并杀伤慢粒细胞系BaF3‑P210，证实了CTP介导形成DC疫苗和DC疫苗诱导免疫效能的可行性。
Description
技术领域
本发明涉及肿瘤治疗学和免疫学技术领域，具体涉及一种融合肽、CTP介导的诱导CML细胞免疫应答的DC疫苗及其制备方法。
背景技术
慢性髓系白血病(Chronic Myeloid Leukemia,CML)是一组来源于骨髓造血干细胞的恶性增殖性肿瘤。其主要致病机制是，来源于9号染色体上的abl基因和22号染色体上的bcr基因，断裂易位融合形成bcr-abl融合基因。该基因可编码产生具有强烈酪氨酸激酶活性的BCR-ABL融合蛋白，从而促进疾病的发生和发展。目前，针对BCR-ABL融合蛋白的酪氨酸激酶活性所开发出的酪氨酸激酶抑制剂如伊马替尼，达沙替尼和尼洛替尼的临床应用，使得慢粒的治疗取得了里程碑式的进展，但因不能清除CML恶性克隆，从而导致复发和耐药的发生。
过继性细胞免疫疗法(Adoptive cellular immunotherapy,ACI)，如肿瘤浸润淋巴细胞(Tumor Infiltrating Lymphocyte,TIL)、淋巴因子激活的杀伤细胞(LymphokineActivated Killer,LAK)、自然杀伤细胞(Natural Killer,NK)、树突状细胞(DendriticCell,DC)等已先后应用于临床，其治疗效果良好且不良反应较少。
抗CML免疫疗法因为能够产生特异性清除慢性粒细胞白血病细胞而受到广泛的关注。bcr-abl融合基因编码的BCR-ABL融合蛋白是CML特异性的细胞遗传学标志。有研究报道，其抗原性限定在BCR-ABL融合位点内，并由MHC一类分子递呈至细胞表面，这段氨基酸序列在正常细胞中不存在，因此是非常理想的细胞免疫治疗靶标。这为慢性粒细胞白血病的免疫治疗的依据。已有研究证实，BCR-ABL抗原特异性细胞毒性T淋巴细胞(Cytotoxic Tlymphocyte，CTL)应答在抗CML免疫中发挥关键性作用，通过BCR-ABL抗原负载DC(树突状细胞)，将外源性抗原递呈给CD8+ T淋巴细胞，诱导形成特异性CTL应答已成为CML治疗新策略，但由于外源性抗原经DC递呈效率不高，导致治疗效果尚不显著。
树突状细胞作为目前所知功能最为强大的抗原提呈细胞，胞内还存在着交叉抗原提呈途径。其可在交叉提呈外源性肿瘤抗原后，刺激机体产生特异性细胞免疫应答。交叉抗原提呈效率直接决定了免疫杀伤效应。而现有的抗原负载***均不能充分激发DC疫苗交叉提呈抗原的潜力，是长期困扰免疫学家和临床肿瘤工作者的一大难题，也是提高抗CML免疫治疗效率的瓶颈问题。
交叉抗原提呈理论认为，外源性抗原经胞吞依赖途径被DC摄取后形成胞饮体，外源性抗原必须从胞饮体“渗漏”进入胞质才能被DC视为内源性抗原，再进行加工处理、进入MHC-I类分子交叉提呈途径。换言之，为激活DC的交叉提呈潜能，必须使外源性抗原进行胞质定位而“内源化”。当前，基于交叉抗原提呈理论已研发了多种新型抗原负载工具，如细胞穿透肽、脂质体和纳米颗粒等，但由于以上途径仍然通过胞吞进入DC递送抗原，因“抗原从胞饮体渗漏进入胞质的渗漏率”低，限制了DC交叉抗原提呈潜力的发挥，因而无法强效激活CTL应答。因此，若能使外源性抗原以“胞吞-非依赖性途径”直接穿透DC胞膜并定位于胞质中，则可有效解决“因外源性抗原内源化效率低而阻碍交叉提呈”这一难题。
胞质转导肽(Cytoplasmic transduction peptide，CTP)是一种新近报导的可携带肽段穿透胞膜并专性定位于胞质的转导肽，以“胞吞-非依赖性”机制发挥转导功能。我们前期实验证实，CTP在各种常规方法难以转染的小鼠前髓、前B淋巴细胞以及各种人血液***原始细胞均显示出高效的转导活性和胞质定位偏性。由于其不影响胞核，因此对细胞无明显毒副作用。
发明内容
本发明的目的是提供一种融合肽、CTP介导的诱导CML细胞免疫应答的DC疫苗及其制备方法。
本发明实现其目的采用的技术方案如下：
一种融合肽，所述融合肽的氨基酸序列如SEQ ID No:1或2所示。
一种分离的多核苷酸，所述多核苷酸编码上述的融合肽。
一种CTP介导的诱导CML细胞免疫应答的DC疫苗，所述DC疫苗为通过将上述的融合肽与成熟的树突状细胞共孵育得到。
上述DC疫苗的制备方法，包括如下步骤：
1)合成权利要求1所述的融合肽序列；
2)培养成熟的树突状细胞；
3)将融合肽与成熟的树突状细胞共孵育、洗涤，即得到DC疫苗。
所述步骤2)培养成熟的树突状细胞的具体方法为：提取小鼠骨髓细胞，裂解其中的红细胞，用含粒细胞-巨噬细胞集落刺激因子、白细胞介素-4、以及10％胎牛血清的1640培养基培养7天，在第八天时加入肿瘤坏死因子-α刺激树突状细胞成熟。
本发明的有益效果是：设计与CTP融合的来源于BCR-ABL融合位点的抗原肽段，抗原来源于慢粒细胞胞质中BCR-ABL癌蛋白融合区域的9个氨基酸序列，可以经由MHC一类分子递呈至慢性粒细胞白血病细胞表面。合成的融合肽段在CTP的介导下，直接穿透DC胞膜并定位于胞质内，通过激活“外源性抗原的MHC-I类分子交叉提呈途径”而诱导CML的特异性CTL应答利用胞质转导肽(CTP)高效的胞质定位潜能。实验证明本发明的树突状细胞疫苗免疫小鼠后，刺激小鼠体内特异性细胞免疫应答，诱导产生细胞毒性T淋巴细胞，并杀伤慢粒细胞系BaF3-P210，证实了CTP介导形成DC疫苗和DC疫苗诱导免疫效能的可行性。本发明中的DC疫苗设计策略，对各类型由于各种融合基因引起的白血病的免疫治疗亦有重要的指导和参考意义。
附图说明
图1是免疫荧光检测CTP融合肽段在DC胞质定位图。
图2是激光共聚焦显微镜观察CTP融合肽段DC胞质细胞器亚定位荧光图。
图3是ELISA检测共培养上清液中IL-2水平结果。
图4是流式细胞术检测DC疫苗体内刺激后T淋巴细胞表CD69,CD137,CD107a分子的表达结果。
图5是乳酸脱氢酶释放实验检测细胞溶解效率结果。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例1 CTP介导的慢粒特异性树突状细胞疫苗的制备
一、根据CTP的肽段序列和来源于BCR-ABL癌蛋白融合位点的序列，设计合成2条新的融合肽段以及这两个肽段加了HA标签的肽段作为实验组(同时设置5个对照组)；为了后续定位实验的需要，在已有的序列中加入HA标签蛋白，肽段名称和具体氨基酸序列见表1。
表1
表1中的SEQ ID No:1～9肽段与成熟树突状细胞共培养后得到的负载肽段的树突状细胞分别命名为：DC/CTP-GF、DC/CTP-SS、DC/CTP-HA-GF、DC/CTP-HA-SS、DC/GF、DC/SS、DC/HA-GF、DC/HA-SS、DC/CTP。
二、树突状细胞培养方法：提取小鼠骨髓细胞，裂解其中的红细胞，用含10ng/L粒细胞-巨噬细胞集落刺激因子(R&D Systems，USA)，5ng/L白细胞介素-4(即IL-4，R&DSystems，USA)以及10％胎牛血清的1640培养基(Gibco，USA)培养7天；在第八天时加入5ng/L肿瘤坏死因子-α(即TNF-α，R&D Systems，USA)刺激树突状细胞成熟。
三、合成肽段细胞胞质定位观察
(1)将表1中的肽段分别与成熟树突状细胞共培养30分钟后，经由磷酸缓冲盐溶液(PBS)洗涤五遍；
(2)离心后弃大部分上清，用残留的上清液重悬细胞沉淀，将细胞均匀涂抹于清洁载玻片上；
(3)甲醇-20℃固定过夜；
(4)固定完成后，洗涤残留在载玻片上的甲醇；
(5)透化：使用1％的Triton-100透化15min，后用PBS洗涤三遍；
(6)封闭：使用山羊血清，4℃封闭1小时；
(7)孵育一抗：将来源于兔HA-Tag单克隆抗体使用山羊血清按1﹕1000的比例进行稀释，直接加到载玻片上，4℃孵育过夜；
(8)过夜后洗涤，用1％Triton-100按照1：800的比例稀释Cy3标记的羊抗兔单克隆抗体，稀释后的抗体按照每张玻片200μl的量加在玻片上，37℃水浴箱里孵育1小时；
(9)洗涤玻片，4',6-二脒基-2-苯基吲哚(DAPI)按照1：1000的比例用磷酸缓冲盐溶液稀释后染核；
(10)应用磷酸缓冲盐溶液将玻片洗涤三遍，干燥后用甘油封片，待观察；结果如图1所示，CTP融合肽段在胞质中具有良好的定位效果。
四、合成肽段细胞器亚定位观察：
(1)将表1中的肽段分别与成熟树突状细胞共培养30分钟后，经由磷酸缓冲盐溶液洗涤五遍；
(2)加入内质网红色荧光探针(碧云天，上海)，具体染色步骤按照制造商说明书进行；
(3)按照说明书完成内质网红色荧光探针染色后，将细胞用磷酸缓冲盐溶液重悬，均匀涂抹于载玻片上，晾干后甲醇固定，再用内质网红色荧光探针进行复染，具体操作步骤按照制造商说明书进行；
结果如图2所示：大部分肽段定位于胞质中的内质网上。
五、抗原负载方法
将合成的CTP融合肽段以10μmol/L的终浓度与成熟的树突状细胞共孵育，过夜后用磷酸缓冲盐溶液洗涤三遍，即得到特异性抗原负载后的树突状细胞，即是CTP融合肽段负载的DC疫苗。
六、交叉抗原提呈效率检测
将步骤五中所得特异性抗原负载后的树突状细胞与CD8+ T淋巴细胞分选磁珠(试剂盒购自美天旎，德国)分选出的初始CD8+ T淋巴细胞共培养20小时，后收集上清，应用ELISA法(试剂盒购自R&D Systems，USA)检测上清液中白细胞介素-2(IL-2)的分泌量，具体操作按照试剂盒操作手册进行。结果如图3所示：CTP融合肽段实验组中检测到的IL-2分泌量明显更高，说明CTP融合肽段负载的DC疫苗能更有效的引发交叉抗原提呈反应。
实施例2CTP融合肽段负载的DC疫苗的应用
一、小鼠免疫策略
将3×106个成熟树突状细胞与10μmol/L CTP融合肽段共孵育过夜，洗涤后注射入小鼠腹股沟皮下***处，记为第一天；7天后，再行第二次免疫；又过七天后，收集小鼠的脾脏和外周血淋巴细胞。
二、小鼠脾脏淋巴细胞分离
收集小鼠的脾脏，将其在研钵中制备成单细胞悬液，并用滤网过滤；将小鼠脾脏淋巴细胞分离液(来源于天津市灏洋生物制品科技有限责任公司)与制备好的单细胞悬液按照1﹕1的比例加入到离心管中，下层为脾脏淋巴细胞分离液，上层为单细胞悬液，要求两层界限清晰，不能混合；按照2000r/min的转速离心，后吸取白膜层，洗涤后得到的即为分离所得脾脏淋巴细胞。
外周血淋巴细胞分离步骤同上，所用淋巴细胞分离液为小鼠外周血淋巴细胞分离液(购自天津灏洋公司)。
结果显示：得到了厚度尚可的白膜层，即淋巴细胞数量可以满足后续的实验。
三、CTL细胞扩增及磁珠分选
以丝裂霉素C处理后的靶细胞BaF3-P210(本实验室构建保藏)作为滋养细胞，按照3﹕1的效靶比进行培养，第一天时共加入含50μmol/L 2-巯基乙醇的RPMI 1640完全T淋巴细胞培养基。1天后加入小鼠白介素-2(IL-2)至终浓度10μmol/L。培养至第3天和第6天时，分别去除1ml培养基，添加含10μmol/L IL-2的新鲜安全RPMI 1640培养基。孵育七天后，收集效应CTL细胞。收集效应CTL细胞时，采用德国美天旎CD8+ T淋巴细胞磁珠分选试剂盒，具体步骤按照说明书进行。
四、T淋巴细胞活化(CD69、CD137表达检测)
将步骤三中分选所得CTL细胞与靶细胞BaF3-P210按照3﹕1的比例进行共孵育，直接加入APC标记的小鼠抗CD69单克隆抗体(购自eBioscience公司，USA)，在12h后收集细胞进行洗涤，标记上FITC标记的小鼠抗CD8单克隆抗体(购自eBioscience，USA)，洗涤后进行流式检测。
CD137表达量检测和CD69操作相同，应用APC标记的小鼠抗CD137单克隆抗体(购自eBioscience，USA)进行检测，但是是在24h后进行检测。
结果如图4所示：CTP融合肽段实验组免疫所得的T淋巴细胞上的CD69和CD137的表达量明显高于对照组，说明实验组中T淋巴细胞具有更好的活性。
五、T淋巴细胞脱颗粒效应
将步骤三中分选所得CTL细胞与靶细胞BaF3-P210按照3﹕1的比例进行共孵育，直接加入APC标记的小鼠抗CD107a单克隆抗体(购自eBioscience，USA)，在24h后收集细胞进行洗涤，标记上FITC标记的小鼠抗CD8单克隆抗体(购自eBioscience，USA)，洗涤后进行流式检测；
结果：CTP融合肽段实验组中T淋巴细胞表达CD107a的量更高，说明此组中的T淋巴细胞脱颗粒效应更加明显，能释放足够的细胞毒成分。
六、诱导的CTL细胞对靶细胞的细胞毒作用
将步骤三中所分选的CTL细胞与靶细胞BaF3-P210按照不同的效靶比在96孔板中共孵育，每孔含靶细胞1×104个，终体积为200μl，设置三个复孔。培养4小时后，收集上清液，采用乳酸脱氢酶释放试剂盒(Promega，USA)检测效应CTL细胞对靶细胞的杀伤作用。
结果如图5所示：CTP融合肽段实验组中细胞溶解效率更加明显，说明CTP融合肽段负载的DC疫苗，能诱导出CML特异性细胞免疫应答，从而杀伤慢粒细胞。
序列表
<110> 重庆医科大学
<120> 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
<160> 9
<210> 1
<211> 21
<212> PRT
<213> 人工序列
<223> CTP-GF
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<223> CTP-SS
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Arg Arg Arg Arg Lys
20
<210> 3
<211> 30
<212> PRT
<213> 人工序列
<223> CTP-HA-GF
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Tyr Ala Gly Gly Arg Arg Ala Arg Arg Arg Arg Arg Arg Lys
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<212> PRT
<213> 人工序列
<223> CTP-HA-SS
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Ser Ser Lys Ala Leu Gln Arg Pro Val Tyr Pro Tyr Asp Val Pro Asp
1 5 10 15
Tyr Ala Gly Gly Arg Arg Ala Arg Arg Arg Arg Arg Arg Lys
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<213> 人工序列
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Ser Ser Lys Ala Leu Gln Arg Pro Val
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Ala Leu
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Claims (3)
1.一种 CTP介导的诱导CML细胞免疫应答的DC疫苗，其特征在于：所述DC疫苗为通过将如SEQ ID No:1或2所示的融合肽与成熟的树突状细胞共孵育得到的负载所述融合肽的树突状细胞。
2.权利要求1所述的DC疫苗的制备方法，其特征在于，包括如下步骤：
1）合成如SEQ ID No:1或2所示的融合肽序列；
2）培养成熟的树突状细胞；
3）将融合肽与成熟的树突状细胞共孵育、洗涤，即得到DC疫苗。
3.如权利要求2所述的制备方法，其特征在于，所述步骤2）培养成熟的树突状细胞的具体方法为：提取小鼠骨髓细胞，裂解其中的红细胞，用含粒细胞-巨噬细胞集落刺激因子、白细胞介素-4、以及10%胎牛血清的1640培养基培养7天，在第八天时加入肿瘤坏死因子-α刺激树突状细胞成熟。
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