CN110201152B - 一种G3bp2多肽疫苗及应用 - Google Patents
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Abstract
本发明属于生物医药领域，具体涉及一种G3bp2多肽疫苗及其应用。本发明提供了一种皮下免疫注射的G3bp2多肽疫苗，其可以刺激小鼠体内产生大量的抗G3bp2蛋白的特异性抗体，降低血液中炎症因子水平，能够有效抑制动脉粥样硬化的发展过程，为目前动脉粥样硬化的治疗提供新的靶标和途径。此外，本发明提供的疫苗还能够降低血糖水平和总蛋红素与白蛋白比值，表明其有可能对于糖尿病和神经性疾病也具有潜在疗效。最后，本发明提供的疫苗安全性高，具有重要的临床价值。
Description
技术领域
本发明属于生物医药领域，具体涉及一种G3bp2多肽疫苗及其应用。
背景技术
G3BP(Ras-GTPase-activating protein SH3domain binding protein)是一个在真核生物进化过程中高度保守的RasGAP结合蛋白，主要定位于细胞质中。它属于RNA结合蛋白家族，在哺乳动物中至少有三种：G3bp1、G3bp2a和G3bp2b。已有许多证据表明，G3BP参与多种细胞信号途径和RNA代谢；在多种肿瘤组织和细胞中过量表达，提示G3BP有可能是一种肿瘤标志物，并且可能与肿瘤的发生发展有关。
应急颗粒(stress granules,SG)是真核细胞受到多种物理化学刺激时在细胞质中形成的颗粒，为一种适应性的保护机制。大量研究发现，应激颗粒的形成在应激反应中扮演着十分重要的作用，并且G3bp2是应激颗粒中不可或缺的组成成分。近期研究表明，G3bp2与癌症的起始，病毒感染，神经性疾病的发生密切相关，但在心血管疾病中还没有相关报道。
动脉粥样硬化是心血管疾病致残致死的首要原因，更是其主要病理性基础，严重危害人类健康和生活质量。动脉粥样硬化是一个复杂且漫长的发病过程，关于动脉粥样硬化的发病机制也有各种不同的学说，包括脂质学说，剪切力学说，炎症学说等，这些学说均有一个共同点，即均为机体对于外界各种刺激的应激反应。例如，脂质学说中的氧化应激，剪切力学说中的机械应激，炎症学说中免疫应激，最后间接或直接导致形成动脉粥样硬化。
目前，基于多肽的抗体治疗已成功用于治疗癌症，但关于动脉粥样硬化疾病的抗体治疗目前仍处于研究阶段，并且相关报道比较少，因此本发明选用G3bp2的有效抗原表位免疫ApoE-/-小鼠，研究其对动脉粥样硬化治疗的效果，为心血管疾病的治疗提供新靶标，具有重大意义。
发明内容
有鉴于此，本发明的目的之一在于提供一种G3bp2多肽疫苗，具体方案如下：
一种G3bp2多肽疫苗，所述疫苗的活性成分为G3bp2蛋白的一段特异性多肽片段，所述多肽片段的氨基酸序列包括SEQ ID NO:1所示和/或其中的片段。
进一步，所述疫苗还含有耦合剂及免疫佐剂，其中免疫所用的抗原分别为血蓝蛋白(KLH)和通过半胱氨酸和耦合剂血蓝蛋白偶联。
进一步，所述疫苗以血蓝蛋白为载体，采用化学耦联的方法，将血蓝蛋白与多肽片段联接。
进一步，所述载体与多肽片段的质量比为1-2：1-3。
优选的，所述载体与多肽片段的质量比为1：1。采用化学偶联法按1:1质量比合成以KLH为载体的KLH-G3bp2多肽疫苗。
进一步，所述疫苗的剂型为皮下注射制剂。本发明所述的多肽疫苗，通过多次重复注射到小鼠腹部背部皮下12个位置，在首次免疫注射后的第二周时收集血清，第四周时处理小鼠并收集血液，血管，心脏等重要样本。通过酶联免疫吸附实验检测出血清中具有大量G3bp2多肽片段特异性抗体。此外，体内重要脏器的病理性切片染色，脏器指数指标和血液白细胞比例检测均未发现有明显的副作用，说明本发明的多肽疫苗非常安全。
本发明的目的之二，在于提供G3bp2多肽疫苗的应用，具体方案如下：
G3bp2多肽疫苗在制备抗动脉粥样硬化药物中的应用。
G3bp2多肽疫苗在降低血液炎性因子水平中的应用。
进一步，所述炎性因子包括细胞间粘附因子-1、血管内皮细胞粘附因子-1和单核细胞趋化蛋白-1中的一种或多种。
G3bp2多肽疫苗在降低血糖水平和总蛋红素与白蛋白比值的应用。
本发明的多肽疫苗能够显著降低血液中的碱性磷酸酶浓度，葡萄糖浓度和总蛋红素与白蛋白比值，同时降低血清中血脂水平，以及炎症因子水平，并抑制动脉粥样硬化的发展。同时由于够降低血糖水平和总蛋红素与白蛋白比值，表明其有可能对于糖尿病和神经性疾病也具有潜在疗效。
一种抗动脉粥样硬化药物，所述药物包括上述G3bp2多肽疫苗和药学上可以接受的载体和助剂。
本发明的有益效果在于：
1)本发明提供了一种皮下免疫注射的G3bp2多肽疫苗，其可以刺激小鼠体内产生大量的抗G3bp2蛋白的特异性抗体，降低血液中炎症因子水平，能够有效抑制了动脉粥样硬化的发展过程，为目前动脉粥样硬化的治疗提供新的靶标和途径。
2)本发明提供的疫苗还能够降低血糖水平和总蛋红素与白蛋白比值，表明其有可能对于糖尿病和神经性疾病也具有潜在疗效。
3)本发明提供的疫苗安全性高，具有重要的临床价值。
附图说明
图1 G3bp2蛋白抗原表位预测(A和B)，皮下注射位置(C)，血清中特异性抗原滴度检测(D)。
图2免疫小鼠注射时间和后续实验流程图。
图3主动脉油红O染色(A和B)和不同位置斑块统计(C)。
图4血液中碱性磷酸酶浓度(A)，葡萄糖浓度(B)和总蛋红素与白蛋白比值(C)，以及血清中生化指标(D)和血清中炎症因子浓度(E)。
图5脏器病理性检测(A)，体重测量(B),脏器指数检测(C)和血液中不同类型白细胞比例检测(D)。
具体实施方式
以下将参照附图，对本发明的优选实施例进行详细描述。优选实施例中未注明具体条件的实验方法，通常按照常规条件，所举实施例是为了更好地对本发明的内容进行说明，但并不是本发明的内容仅限于所举实施例。所以熟悉本领域的技术人员根据上述发明内容对实施方案进行非本质的改进和调整，仍属于本发明的保护范围。
除非特别说明，本实验构建的动脉粥样硬化模型所用的ApoE-/-小鼠从8周龄开始饲喂高脂饲料至20周龄，高脂饲料脂肪比例为21％，胆固醇比例为0.15％。
除非特别说明，以下实施例中所用的试剂和材料均为分析纯级别的试剂，且可从正规渠道商合成或订购。
本实验所合成的多肽序列如SEQ ID NO:1所示，采用化学偶联法按1:1质量比合成以KLH为载体的KLH-G3bp2多肽疫苗，其中免疫小鼠的多肽纯度不低于95％。
本实验所合成的KLH和KLH-G3bp2作为免疫抗原注射免疫小鼠，合成的多肽用来包被巯基结合平板，检测血清中特异性抗体。
实施例1
免疫抗原乳浊液的配制
免疫前将1mgKLH-G3bp2或KLH溶解于1ml无菌的PBS缓冲液中，用0.2μm规格的滤头过滤除菌后备用。
抗原乳液的具体配制：准确吸取KLH-G3bp2或者KLH溶液20μl，弗氏佐剂120μl，生理盐水120μl，加入灭菌后的离心管后震荡乳化30min(每次乳化20s，然后冰上放置1min)。乳化完毕后吸取一滴液体滴在冷水面上，如果长时间保持圆珠形状并且轻轻晃动不会散开，则表明已充分乳化。前两次免疫时采用弗氏完全佐剂进行乳化，后三次采用弗氏不完全佐剂进行乳化。
实施例2
小鼠免疫和取材
小鼠高脂喂养8周后，眼眶取血，然后重复皮下免疫注射腹部背部皮下12个位置5次，在初次免疫后第4周时异氟烷麻醉后眼球取血，然后断颈处死小鼠，并收获主动脉、心脏、肝脏、脾脏、肺和肾脏样本。
实施例3
合成多肽包被巯基结合平板及免疫效果评价
1.缓冲液配制
1)包被缓冲液
分别称取碳酸钠1.59g，碳酸氢二钠2.93g，溶解至1000ml双蒸水中，混合均匀后，调节pH至9.1，存放于4℃备用。
2)洗涤缓冲液
分别称取磷酸二氢钾0.2g，十二水磷酸氢二钠0.2g，氯化钠0.8g，氯化钾0.2g，量取0.05％吐温-20 0.5ml，然后加双蒸水至1000ml。
3)封闭缓冲液
称量5g牛血清蛋白，加双蒸水至100ml，混匀分装后存放于4℃备用。
4)终止液
取双蒸水89.15ml，沿壁逐滴向其内缓慢加入98％浓硫酸10.85ml，边加边轻轻搅拌。
2.操作步骤
1)包被：用包被缓冲液稀释未偶联的多肽至20ug/ml包被巯基结合平板，100μl/孔。
2)孵育：加盖覆膜后放置于37℃培养箱中温育2h。
3)洗涤：弃去孔内液体，甩干，用洗涤液洗板3次，每孔350μl，每次5min，甩并在吸水纸上轻拍将孔内液体拍干。
4)封闭：拍干后每孔中加入100μl的封闭缓冲液，加盖覆膜后放置于37℃培养箱中温育2h或者4℃冰箱过夜。
5)洗涤：弃去封闭液，每孔加入约350μl洗涤液，摇床上200转/min震荡洗涤5min，弃去洗涤液并排干，重复洗涤5次。
6)加样：每孔加入事先以稀释液梯度稀释的血清100μl，每个样本设两个平行孔，每板设2个空白孔和2个本底对照孔，加盖覆膜后放置于37℃培养箱中温育1h。
7)洗涤：弃去孔内液体，每孔加入约350μl洗涤液，摇床上200转/min震荡洗涤5min，弃去洗涤液并排干，重复洗涤5次。
8)结合二抗：拍干后每孔加入以稀释液1:5000稀释的辣根过氧化物酶标记的山羊抗小鼠IgG 100μl，37℃孵育1h。
9)洗涤：弃去孔内液体，每孔加入约350μl洗涤液，摇床上200转/min震荡洗涤5min，弃去洗涤液并排干，重复洗涤5次。
10)显色测定：每孔加入TMB单组份显色液90μl，室温避光放置10min左右后观察显色，当出现颜色梯度后，每孔加入50μl终止液终止反应，用酶标仪在450nm波长测量各孔的光吸收值。
3.实验结果
测定结果显示，G3bp2多肽疫苗具有良好的免疫效果。
1)G3bp2多肽疫苗能有效减缓ApoE-/-小鼠动脉粥样硬化的发展。对小鼠的体重和重要脏器的重量测定结果和脏器HE染色结果均显示，小鼠体重和脏器指数并未受到多肽疫苗免疫影响，证明其安全性高。油红O染色和统计显示，免疫注射后小鼠主动脉斑块明显减少。见图3、图5。
2)血脂含量测定结果统计显示，多肽免疫后小鼠血液中胆固醇，甘油三酯和低密度脂蛋白水平显著降低。见图4。
3)ELISA试剂盒检测血清中炎症因子结果表明，多肽免疫后细胞间粘附因子-1，血管内皮细胞粘附因子-1和单核细胞趋化蛋白-1表达水平显著降低。见图4。
最后说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的宗旨和范围，其均应涵盖在本发明的权利要求范围当中。
序列表
<110>重庆大学
<120>一种G3bp2多肽疫苗及应用
<160>1
<170> Patent In Version 3.5
<210>1
<211>18
<212>PRT
<213>Artificial sequence
<400>1
Cys Ile Ile Arg Tyr Pro Asp Ser His Gln Leu Phe Val Gly Asn Leu
1 5 10 15
Pro His
Claims (9)
1.一种G3bp2多肽疫苗，其特征在于，所述疫苗的活性成分为G3bp2蛋白的一段特异性多肽片段，所述多肽片段的氨基酸序列如SEQ ID NO:1所示。
2.根据权利要求1所述的G3bp2多肽疫苗，其特征在于，所述疫苗还含有耦合剂及免疫佐剂。
3.根据权利要求1所述的G3bp2多肽疫苗，其特征在于，所述疫苗以血蓝蛋白为载体，采用化学耦联的方法，将血蓝蛋白与多肽片段联接。
4.根据权利要求3所述的G3bp2多肽疫苗，其特征在于，所述载体与多肽片段的质量比为1-2：1-3。
5.根据权利要求1所述的G3bp2多肽疫苗，其特征在于，所述疫苗的剂型为皮下注射制剂。
6.权利要求1-5任一所述的G3bp2多肽疫苗在制备抗动脉粥样硬化药物中的应用。
7.权利要求1-5任一所述的G3bp2多肽疫苗在制备降低血液炎性因子水平的药物中的应用。
8.根据权利要求7所述的应用，其特征在于，所述炎性因子为细胞间粘附因子-1、血管内皮细胞粘附因子-1和单核细胞趋化蛋白-1中的一种或多种。
9.一种抗动脉粥样硬化药物，其特征在于，所述药物包括权利要求1所述G3bp2多肽疫苗和药学上可以接受的载体和助剂。
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