CN112691188A - 一种纳米化肿瘤疫苗的制备方法 - Google Patents
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Abstract
本发明涉及一种由Flagellin及细胞因子和/或细胞因子诱导剂纳米化后与肿瘤抗原体外组合制备肿瘤疫苗的方法。本发明的肿瘤疫苗具有如下特征：(1)Flagellin及细胞因子和/或细胞因子诱导剂纳米化后具有靶向、缓释的特点；(2)Flagellin和细胞因子和/或细胞因子诱导剂分别作为T细胞激活的第二和第三信号，能够较为强烈地激活非特异性免疫；(3)Flagellin能够增强APC的抗原递程能力，增强特异性免疫；(4)广泛适用于各种类型肿瘤的预防复发、预防转移及治疗，具有良好的应用前景。
Description
技术领域
本发明属于生物医药领域，具体涉及一种纳米化肿瘤疫苗的制备方法及应用。
背景技术
自1893年癌症免疫疗法之父William B.Coley的开创性工作支持肿瘤表达特异性抗原，使其在提供充分免疫刺激的情况下具有自然的免疫原性以来，激活机体自身免疫系统来抑制肿瘤的发生发展，消灭体内的肿瘤细胞成为肿瘤治疗的研究热点。免疫检查点阻断(immune checkpoint blockade，CPB)类药物及过继性细胞转移(adoptive celltransfer，ACT)疗法在肿瘤的治疗中占据了非常重要的位置，但是CPB药物适用范围窄，ACT中代表性的CAR-T疗法也存在成本高、靶点选择性高及体外培养操作复杂等缺点，这都极大了地限制了在肿瘤治疗中的应用。肿瘤疫苗是一种通过主动免疫来增强特异性T细胞反应的疫苗，活性成分主要包括肿瘤抗原、免疫佐剂、载体、剂型。肿瘤抗原一般可分为肿瘤相关抗原和肿瘤特异性抗原。
免疫佐剂能够非特异性地激活免疫细胞。主要分为第一激活信号佐剂；第二激活信号/共刺激信号佐剂；第三激活信号佐剂；改善肿瘤免疫抑制微环境佐剂五种。Flagellin属于第二激活信号/共刺激信号佐剂，能够与细胞膜上的TLR5受体结合，促使TLR5二聚体化，从而激活MyD88,MyD88招募下游活化因子(IRAK1/IRAK4)后使IKK磷酸化，IKK激活后催化IκB磷酸化，IκB磷酸化后于NF-κB解离，NF-κB暴露出核定位序列并转运至细胞核内，与相应的DNA结合，启动基因转录并释放出TNF-ɑ,IL-6,IL-12等细胞因子。
大多数抗原递程细胞(antigen-presenting cell,APC)表面存在大量的TLR5受体，Flagellin能够激活并促使这些APC增殖及成熟，增其抗原摄取能力，增强向引流淋巴结迁移的能力，完成对T辅助细胞招募、激活、促分化，最终启动获得性免疫。从而使机体的免疫系统发挥出更强大的功能，杀伤及清除肿瘤细胞。
随着人们对纳米材料的认识与的发展，纳米技术已经广泛的应用到医药研究领域，已有很多基于不同组织或病灶部位生理环境差异而设计的具有靶向、缓释等作用的纳米化药物。已有学者发现部分纳米材料能够在特定pH条件下发挥质子海绵效应，破坏细胞膜，促进物质的胞质运输，这种材料在抗原胞质输送及运输至淋巴结有很大的潜力。除此之外，利用纳米材料包裹抗原或佐剂等蛋白类易被机体降解的蛋白、多肽等，延长该类物质在体内的代谢的半衰期或起到缓释作用。因此纳米化肿瘤疫苗是非常可行且具有优势的。
发明内容
1.本发明的目的是提供一种纳米化肿瘤疫苗及其应用
2.本发明提供的纳米化肿瘤疫苗的制备方法，具体包括以下步骤：
(1)利用分子生物学手段表达纯化得到的鞭毛蛋白Flagellin和/或细胞因子和/或细胞因子诱导剂用纳米材料包裹；
(2)将所述纳米制剂与肿瘤抗原体外组合得到肿瘤疫苗。
3.通过分子生物学常规手段，构建鼠伤寒沙门氏菌的鞭毛蛋白Flagellin的表达载体，将构建成功的表达载体利用化学和/或物理方法导入表达宿主中，可以是原核表达宿主，也可以是真核表达宿主。
4.发酵获得鞭毛蛋白Flagellin后根据蛋白所带标签的种类及蛋白自身的理化性质，选择合适的分离纯化条件，得到电泳纯的鞭毛蛋白Flagellin，并利用已有体外活性检测方法检测鞭毛蛋白Flagellin的活性。
5.上述步骤1至4中同样的方法构建表达并纯化各类激活T细胞和/或与抗原递程细胞结合的细胞因子和/或细胞因子诱导剂。
6.肿瘤抗原可以是肿瘤细胞通过反复冻融，X-ray辐照，顺铂等放疗性化学药品处理等常用手段灭活的肿瘤细胞；也可以是通过常规分子生物学手段合成的多肽等类型的肿瘤抗原。
7.利用带正电的和/或与肿瘤抗原特异性结合的纳米材料，探索合适的条件将鞭毛蛋白Flagellin；鞭毛蛋白Flagellin和一种或几种细胞因子；鞭毛蛋白Flagellin和一种或几种细胞因子诱导剂；鞭毛蛋白Flagellin和一种或几种细胞因子和细胞因子诱导剂用合适的纳米材料包裹。
8.利用所选纳米材料与各类肿瘤抗原通过其物理吸附或者化学结合等分子间相互作用力，将佐剂与肿瘤抗原进行组合，产生不同的肿瘤疫苗组合物。
9.本发明提供的肿瘤疫苗将佐剂与肿瘤抗原有效地组合起来，并且由于佐剂纳米化后体内半衰期延长，纳米化制剂也可以发挥靶向作用，从而更强地激活机体免疫系统，清除肿瘤。
附图说明
图1为纳米化肿瘤疫苗构建示意图。
图2为Flagellin表达情况的Western blotting，其中Line1:Marker；Line2:发酵第1天Flagellin表达量；Line3:发酵第2天Flagellin表达量；Line4:发酵第3天Flagellin表达量；Line5:发酵第4天Flagellin表达量；Line6:发酵第5天Flagellin表达量。
图3为Flagellin纯化结果的SDS-PAGE图，其中Line1:Marker；Line2:发酵原液；Line3:上样流穿液；Line4:50mM咪唑阶段性洗脱收集第1管洗脱液；Line5:50mM咪唑阶段性洗脱收集第2管洗脱液；Line6:50mM咪唑阶段性洗脱收集第3管洗脱液；Line7:50mM咪唑阶段性洗脱收集第4管洗脱液；Line8:50mM咪唑阶段性洗脱收集第5管洗脱液；Line9:50mM咪唑阶段性洗脱收集第6管洗脱液。
图4为纯化后Flagellin活性检测结果图。
具体实施方式
除本发明另作说明外，否则本申请中所使用的名词、术语、短语具有本领域内通常的含义。另外，除本发明另有规定外，否则本申请中所使用的试剂、仪器等均为本领域内的常规试剂或仪器，并且他们可在市场上自由获得。
实施例
实施例1 Flagellin在真核细胞中的表达
构建好的293-F-Flagellin表达细胞株于液氮罐中取出后复苏，复苏48h后计数，密度达1×106–3×106个/mL后传代，传代后细胞数在0.2×106–0.5×106个/mL，于37℃、5％CO2、125rpm摇床中培养分别于传代后第1、2、3、4、5天取细胞悬浮液500μL,1000-1500rpm，4℃离心5min，取上清，上清8000rpm，4℃离心10min取上清，Western blotting检测上清中Flagellin的表达量。结果如图1所示。
实施例2 Flagellin的纯化
发酵第5天收蛋白，具体操作：发酵液400G，4℃离心5min后取上清，上清8000rpm，4℃离心10min，依次过0.8μm，0.45μm以及0.22μm的水系滤膜，10kDa TFF超滤浓缩，Ni柱纯化。Elution Buffer(20mM PB+50mM咪唑)阶段性洗脱。纯化结果如图2所示。
实施例3 Flagellin体外活性检测
Flagellin能够刺激THP-1细胞释放TNF-α，因此可以ELISA检测TNF-α的含量反应Flagellin的活性。具体操作：将生长状态良好的THP-1细胞铺入24孔板中，待其汇合度达到80-90％时加入不同浓度Flagellin及对照蛋白，于37℃、5％CO2静置培养箱中培养48h，取上清用ELISA试剂盒检测上清液中TNF-α的表达量。结果如图3所示。
结果表明我们表达纯化所得的Flagellin有活性。
Claims (10)
1.一种纳米化肿瘤疫苗的制备方法，具体包括以下步骤：
(1)利用分子生物学手段表达纯化得到的鞭毛蛋白Flagellin和/或细胞因子和/或细胞因子诱导剂用纳米材料包裹；
(2)将所述纳米制剂与肿瘤抗原体外组合得到肿瘤疫苗。
2.如权利要求1所述肿瘤疫苗，其特征在于：鞭毛蛋白Flagellin来源于鼠伤寒沙门氏菌。
3.如权利要求1或2中任意所述的肿瘤疫苗，其特征在于：鞭毛蛋白Flagellin可通过生物分子学手段获得。
4.如权利要求1所述肿瘤疫苗，其特征在于：所用细胞因子和/或细胞因子诱导剂包括各类激活T细胞和/或与抗原递程细胞结合的细胞因子和/或细胞因子诱导剂。
5.如权利要求1或4中任意所述的肿瘤疫苗，其特征在于：所用细胞因子和/或细胞因子诱导剂为IL2,IL15,INF-γ,TNF-α,GM-CSF等其他细胞因子和/或细胞因子诱导剂。
6.如权利要求1至4中任意所述的肿瘤疫苗，其特征在于：鞭毛蛋白Flagellin可以单独纳米化，也可以和一种或者几种细胞因子和/或细胞因子诱导剂共同纳米化。
7.如权利要求1或5中任意所述的肿瘤疫苗，其特征在于：纳米化材料为带正电的和/或与肿瘤抗原特异性结合的物质。
8.如权利要求1所述肿瘤疫苗，其特征在于：肿瘤抗原包括灭活肿瘤细胞，肿瘤细胞裂解物及利用分子生物学手段设计生产的肿瘤抗原。
9.如权利要求1或7中任意所述的肿瘤疫苗，其特征在于：可通过各种物理化学方式灭活肿瘤细胞。
10.如权利要求1至7中任意所述的肿瘤疫苗的制备方法和应用。
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