CN114099639A - H1-pHSP65纳米疫苗及其制备方法和用途 - Google Patents

H1-pHSP65纳米疫苗及其制备方法和用途 Download PDF

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CN114099639A
CN114099639A CN202111416905.3A CN202111416905A CN114099639A CN 114099639 A CN114099639 A CN 114099639A CN 202111416905 A CN202111416905 A CN 202111416905A CN 114099639 A CN114099639 A CN 114099639A
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phsp65
vaccine
nano
plasmid
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杨洁
柴大飞
郑骏年
姚宏
刘玲
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Xuzhou Medical University
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Abstract

本发明涉及疫苗制备技术领域，具体公开了H1‑pHSP65纳米疫苗及其制备方法和用途，通过pHSP65质粒的构建，进一步制备获得H1‑pHSP65纳米疫苗，具体是将H1纳米载体包裹HSP65质粒DNA形成H1‑pHSP65纳米颗粒。本发明制备的H1‑pHSP65纳米颗粒能够用于制备抗肿瘤药物，能够抑制肿瘤细胞的增殖和转移，能够有效激发小鼠的肿瘤免疫反应。

Description

H1-pHSP65纳米疫苗及其制备方法和用途
技术领域
本发明属于疫苗制备技术领域，具体涉及H1-pHSP65纳米疫苗及其制备方法和用途。
背景技术
DNA疫苗在肿瘤疫苗中占有重要地位，是近年研究的热点之一。肿瘤DNA疫苗是利用基因工程技术将肿瘤相关抗原的基因负载到质粒载体上，借助载体或者人体基因表达系统，能持续引起较强的体液免疫和细胞免疫，达到预防和治疗疾病的目的。
HSP65(heat shock protein 65kD)是来源于卡介苗的热休克蛋白，是卡介苗的主要抗原之一，国际基因库登录号是M17705。卡介苗早在60年代即通过BCG膀胱内灌注用于浅表性膀胱癌的临床治疗，不仅可消灭原位癌，还可以预防肿瘤复发。但是，BCG的成分复杂，会引起一系列的毒副作用。许多肿瘤会过量表达和分泌HSP60蛋白，HSP60与肿瘤的发生、发展、转移有关，可以作为肿瘤靶标之一，但是内源性HSP60的免疫原性较差。HSP65是HSP60家族成员，与HSP60具有高度同源性(高于55％)。HSP65具有多重的T表位和B表位，具有强免疫原性，能刺激机体同时产生体液免疫和细胞免疫。
但是，核酸分子量较大、带负电等性质导致裸露DNA很难自主进入细胞，并且体内易降解，不能有效突破内皮细胞在血管腔内形成屏障，不能进入血管外的免疫细胞，从而阻碍特异性免疫反应的诱导。
纳米载体H1(PEI600-CyD-FA)是将阳离子聚合物聚乙烯亚胺PEI600通过环糊精CyD修饰和偶合叶酸FA配体修饰后得到的新型纳米复合物。H1不仅具有低毒、低免疫原性、水溶性好、生物相容性好的特点，可与带负电荷的细胞膜或质粒DNA通过静电天然吸附，具有增强细胞黏附吸收作用，促进胞间运输、缓释、基因转染效率高等多种良好特性；还能引起高水平的DC对抗原的摄取作用和更强的抗原交叉递呈作用，能显著提高抗原特异性CTL活性，增强抗原特异性的体液免疫及细胞免疫应答。由于H1具有DNA结合能力强、载体转染效率高、基因表达能力高、安全低毒等特点，适合作为基因疫苗的递呈载体。
发明内容
本发明的目的是提供了H1-pHSP65纳米疫苗及其制备方法和用途，通过热休克蛋白65/HSP65(国际基因库登录号是M17705)的基因联合H1(PEI600-CyD-FA)纳米载体构建获得H1-pHSP65纳米疫苗，制备得到的H1-pHSP65纳米疫苗能够用于制备抗肿瘤药物，能够更有效的被机体吸收，增强其免疫原性。
本发明提供了H1-pHSP65纳米疫苗的制备方法，具体包括如下步骤：
S1，pHSP65质粒的构建：根据HSP65序列设计引物，扩增得HSP65序列，以pcDNA3.1为真核表达载体构建得pHSP65质粒；
S2，H1-pHSP65纳米疫苗的制备：将质粒pHSP65转化入DH5a大肠杆菌感受态细胞，培养后提取质粒，将pHSP65质粒和H1纳米载体分别溶于PBS，静置5分钟后混合，静置10分钟后即得H1-pHSP65纳米疫苗。
进一步地，S1中，所述引物包括F引物和R引物；
所述F引物的基因序列如SEQ ID NO.1所示；
所述R引物的基因序列如SEQ ID NO.2所示。
进一步地，S2中，质粒pHSP65转化入DH5a大肠杆菌感受态细胞后培养温度为37℃。
进一步地，S2中，H1-pHSP65基因纳米疫苗是将H1纳米载体包裹HSP65质粒DNA形成H1-pHSP65纳米颗粒。
本发明还提供了由上述方法制备得到的H1-pHSP65纳米疫苗。
本发明还提供了所述的H1-pHSP65纳米疫苗在制备抗肿瘤药物中的应用。
本发明还提供了包含上述H1-pHSP65纳米疫苗的疫苗注射剂。
本发明还提供了所述的疫苗注射剂在制备抗肿瘤药物中的应用。
进一步地，所述疫苗注射剂包括但不限于用于制备抗肾癌的药物。
与现有技术相比，本发明具有以下有益效果：
1、本发明通过将H1纳米载体包裹HSP65质粒DNA形成H1-pHSP65纳米颗粒，使之更有效的被机体吸收，增强其免疫原性，制备得到的H1-pHSP65纳米疫苗能够用于制备抗肿瘤药物，能够抑制肿瘤细胞的增殖和转移，能够有效激发小鼠的肿瘤免疫反应。
2、本发明制备得到的H1-pHSP65纳米疫苗能够用于制备预防和治疗肾癌等肿瘤的药物。
附图说明
图1为本发明中质粒pcDNA3.1-HSP65示意图；
图2为本发明中琼脂糖凝胶电泳显示质粒DNA经H1纳米颗粒包裹后的电泳行为；
其中，泳道1为未包裹的质粒DNA；泳道2-5，表示同样的质粒DNA经不同量的H1纳米颗粒荷载；1-5泳道的质粒：H1的荷载比分别是1：0，1：1，1：2，1：3，1：4；
图3为本发明中质粒DNA经H1纳米颗粒包裹后的颗粒大小；
图4为本发明中制备的H1-pHSP65基因纳米疫苗对于肾癌Renca的防治作用；
图5为本发明中HE染色检测对照组及实验组荷瘤小鼠的肺脏病理切片的肿瘤转移情况；
图6为本发明中ELISPOT实验检测免疫后小鼠脾脏淋巴细胞分泌IFN-γ的表达。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明，但应当理解本发明的保护范围并不受具体实施方式的限制。下列实施例中未注明具体条件的试验方法，通常按照常规条件操作，由于不涉及发明点，故不对其步骤进行详细描述。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
本发明提供了H1-pHSP65纳米疫苗及其制备方法和用途，通过热休克蛋白65/HSP65(国际基因库登录号是M17705)的基因联合H1(PEI600-CyD-FA)纳米载体，将H1纳米载体包裹HSP65质粒DNA形成H1-pHSP65纳米颗粒，使之更有效的被机体吸收，增强其免疫原性，制备得到的H1-pHSP65纳米疫苗能够用于制备抗肿瘤药物，能够抑制肿瘤细胞的增殖和转移，能够有效激发小鼠的肿瘤免疫反应。
实施例1
热休克蛋白65/pHSP65(国际基因库登录号是M17705)
一、本实施例提供了H1-pHSP65纳米疫苗的制备方法，包括如下步骤：
S1，pHSP65质粒的构建：
根据HSP65序列设计上、下游引物，分别为F引物和R引物，以pET28a-HSP65质粒为模板进行扩增，所述F引物的核苷酸序列如SEQ ID NO.1所示，所述R引物的核苷酸序列如SEQ ID NO.2所示；
SEQ ID NO.1：
Figure BDA0003375616870000051
SEQ ID NO.2：
Figure BDA0003375616870000052
SEQ ID NO.1中双下划线表示NcoI酶切位点，SEQ ID NO.2中双下划线表示EcoRI酶切位点；
扩增反应体系为：质粒模板0.5μL、上下游引物各1μL、2x Master Mix 10μL、水7.5μL，总体积为20μL。扩增程序为：98℃3min；98℃15s、55℃5s、72℃8min，30cycles；72℃10min；4℃保存。
通过上述PCR反应获得HSP65的DNA序列、以pcDNA3.1为真核表达载体、NcoI和EcoRI为酶切位点、插入HSP65序列，构建表达质粒pcDNA3.1-HSP65并测序验证(即为pHSP65质粒)(图1)。
S2，H1-pHSP65基因纳米疫苗的制备：
将上述质粒pHSP65转化入DH5a大肠杆菌感受态细胞，37度震荡培养过夜后提取质粒，琼脂糖凝胶电泳检测纯度并用nanodrop仪器检测DNA浓度，将pHSP65质粒和H1纳米载体分别溶于PBS，静置5分钟后混合，静置10分钟后即得H1-pHSP65基因纳米疫苗(图2、图3)。经zeta sizer检测，该纳米颗粒的直径约为152nm(图3)。
二、本发明制备的H1-pHSP65纳米疫苗的功能验证
1、H1-pHSP65纳米疫苗的抗肿瘤作用
选取6-8周龄雌性BALB/c小鼠随机分组：对照组、H1-pHSP65实验组。每2周一次皮下免疫治疗，共4次。免疫结束后，收集对数生长期的Renca细胞，按5×105个细胞/只注入小鼠腹侧皮下建立小鼠皮下移植瘤模型，观察疫苗的抗肿瘤作用。荷瘤后3周处死小鼠，取血、瘤和内脏。
如图4所示，和对照组相比，H1-pHSP65免疫组的小鼠肿瘤明显较小，说明H1-pHSP65疫苗有利于抑制小鼠肿瘤细胞的增殖。
2、HE染色检测荷瘤小鼠病理切片的肿瘤肺转移情况
处死小鼠，取肺脏于10％甲醛溶液中固定，石蜡包埋，4μm厚切片，HE染色，在光学显微镜下观察并拍照(10x)。
如图5所示，对照组的肺泡中有大量的肿瘤转移灶，H1-pHSP65实验组的小鼠肺脏的肿瘤转移灶较少，说明H1-pHSP65疫苗有利于抑制小鼠肿瘤细胞的转移，减少扩散。
3、ELISPOT实验检测脾脏淋巴细胞分泌IFN-γ的功能性T细胞
使用检测试剂盒(达优mouse IFN-γ2210005，达科为)中经IFN-γ抗体预包被的96孔板，RPMI-1640培养基200mL/孔封闭5-10min；分离小鼠脾细胞，105/孔接种于ELISPOT板；加入刺激物后，37℃、5％CO2培养箱培养3d。倾倒孔内细胞和培养基，加入预冷的去离子水200μL/孔，4℃放置10min；甩去孔内液体，洗板6次，每次在吸水纸上扣干；加入生物素标记的抗体工作液，37℃孵育1h；洗板6次；加入酶标亲和素工作液，37℃孵育1h；洗板5次；弃去并扣干孔内液体；加入AEC显色液，室温避光静置5-30min；倾倒孔内液体，揭开底座，洗涤3-5遍，终止显色；室温阴凉处晾干后读板。
如图6所示，H1-pHSP65免疫可以有效激发小鼠的Th1细胞免疫反应，促进IFN-γ的表达。
尽管已描述了本发明的优选实施例，但本领域内的技术人员一旦得知了基本创造性概念，则可对这些实施例作出另外的变更和修改。所以，所附权利要求意欲解释为包括优选实施例以及落入本发明范围的所有变更和修改。
显然，本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样，倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内，则本发明也意图包含这些改动和变型在内。
序列表
<110> 徐州医科大学
<120> H1-pHSP65纳米疫苗及其制备方法和用途
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 27
<212> DNA
<213> 人工合成
<400> 1
ccaccatggc caagacaatt gcgtacg 27
<210> 2
<211> 27
<212> DNA
<213> 人工合成
<400> 2
gcagaattct cagaaatcca tgccacc 27

Claims (9)

1.H1-pHSP65纳米疫苗的制备方法，其特征在于，包括如下步骤：
S1，pHSP65质粒的构建：根据HSP65序列设计引物，以pET28a-HSP65质粒为模板扩增得HSP65序列，以pcDNA3.1为真核表达载体，NcoI和EcoRI为酶切位点、插入HSP65序列，构建得pHSP65质粒；
S2，H1-pHSP65纳米疫苗的制备：将质粒pHSP65转化入DH5a大肠杆菌感受态细胞，培养后提取质粒，将pHSP65质粒和H1纳米载体分别溶于PBS，静置5分钟后混合，静置10分钟后即得H1-pHSP65纳米疫苗。
2.根据权利要求1所述的H1-pHSP65纳米疫苗的制备方法，其特征在于，S1中，所述引物包括F引物和R引物；
所述F引物的核苷酸序列如SEQ ID NO.1所示；
所述R引物的核苷酸序列如SEQ ID NO.2所示。
3.根据权利要求1所述的H1-pHSP65纳米疫苗的制备方法，其特征在于，S2中，质粒pHSP65转化入DH5a大肠杆菌感受态细胞后培养温度为37℃。
4.根据权利要求3所述的H1-pHSP65纳米疫苗的制备方法，其特征在于，S2中，H1-pHSP65基因纳米疫苗是将H1纳米载体包裹HSP65质粒DNA形成H1-pHSP65纳米颗粒。
5.由权利要求1-4任一项所述的制备方法制备得到的H1-pHSP65纳米疫苗。
6.权利要求5所述的H1-pHSP65纳米疫苗在制备抗肿瘤药物中的应用。
7.包含权利要求5所述的H1-pHSP65纳米疫苗的疫苗注射剂。
8.权利要求7所述的疫苗注射剂在制备抗肿瘤药物中的应用。
9.权利要求8所述的疫苗注射剂在制备抗肿瘤药物中的应用，其特征在于，所述疫苗注射剂包括但不限于用于制备抗肾癌的药物。
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