CN111388661A - 基于tipe-2的新冠病毒负调疫苗及其制备方法和应用 - Google Patents
基于tipe-2的新冠病毒负调疫苗及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种基于TIPE‑2的新冠病毒负调疫苗及其制备方法和应用,该负调疫苗是以阳离子多肽和新冠病毒S蛋白CD8+T细胞表位肽的复合物为基因载体包裹TIPE‑2基因重组表达载体自组装而成;将该负调疫苗转染树突状细胞并与T细胞共培养,能够有效降低新冠病毒S蛋白刺激下T细胞的增殖能力、细胞因子分泌能力和表面分子的活化分子表达率,表明该负调疫苗能够有效诱导新冠病毒S蛋白特异性的T细胞耐受,可用于制备诱导新冠病毒S蛋白特异性T细胞耐受的药物,在抗新冠病毒导致的组织损伤治疗领域具有良好的开发应用前景。
Description
技术领域
本发明涉及一种负调疫苗,特别涉及一种基于TIPE-2的负调疫苗,还涉及该负调疫苗的制备方法和应用。
背景技术
新型冠状病毒肺炎(Corona Virus Disease 2019,COVID-19)是由一种新的β冠状病毒-新型冠状病毒(2019novel corona virus,2019-nCoV)引起,以血管紧张素转化酶2(Angiotensin converting enzyme 2,ACE-2)作为受体侵入细胞导致肺损伤,病情加重与继发引起的全身性炎症反应密切相关,重症患者会出现急性呼吸窘迫综合征(Acuterespiratory distress syndrome,ARDS)和感染性休克,最终出现多器官功能衰竭。目前大多数药物如巴利替尼、瑞德西韦、氯喹等尚处于临床试验阶段,尚无特效药治疗。COVID-19传染性强,可引起多种严重的并发症,对全球公共安全造成了极大威胁。研究其病原学及发病机制,进而开展对治疗药物和疫苗的临床研究,对及时控制病毒传播,防止病情恶化进展,减少并发症及病死率具有重大意义。
冠状病毒的基因组大小从26到32千碱基(Kb)不等,拥有RNA病毒最大的基因组。所有冠状病毒在基因组的组织和表达上都有相似之处,其中16个非结构蛋白由5'端的开放阅读框架(ORF)1a/b编码,紧随其后的是结构蛋白刺突蛋白(S)、包膜蛋白(E)、膜蛋白(M)和核衣壳蛋白(N),这些蛋白在3'端由其他ORF编码。其中S蛋白介导病毒与宿主细胞表面受体的附着以及与宿主细胞膜之间的融合,促进病毒进入宿主细胞。SARS冠状病毒引起的细胞因子风暴主要涉及白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-12(IL-12)、γ-干扰素(IFN-γ)、趋化因子IP-10(Interferon-inducible protein-10,IP-10)以及人单核细胞趋化蛋-1(Membrane cofactorprotein-1,MCP-1)等,COVID-19患者的病理结果提示CD8+T细胞中含有高浓度的细胞毒性颗粒,CD4+和CD8+T淋巴细胞减少,但同时也被过度激活。T淋巴细胞介导的损伤可能是导致患者病情恶化的重要因素。诱导免疫耐受是治疗移植排斥、自身免疫性疾病和过敏性疾病的重要方式。由于T细胞的活化在机体免疫系统的活化中扮有重要角色,因此在诱导免疫耐受的各种策略中,诱导T细胞耐受是一种比较理想的策略。研究表明,T细胞的活化不仅需要抗原肽与主要组织相容性复合体(MHC)的复合刺激,而且需要B7家族成员提供关键的共刺激信号。
胖瘤坏死因子α诱导蛋白8样分子2(tumor necrosis fator-α-inducedprotein8-like2,TIPE-2)最早在小鼠实验性自身免疫性脑脊髓炎动物模型中发现,主要表达于胸腺、淋巴结等组织及巨噬细胞、淋巴细胞等免疫细胞,在免疫反应中发挥重要的负向调节作用。研究发现TIPE-2在系统性红斑狼疮、慢性乙肝病毒感染及哮喘中表达均有改变,并通过诱导巨噬细胞极化减轻炎症反应。TLRs是一类重要的跨膜病原体识别受体,激活固有免疫反应。同时TLRs作为“桥梁”连接固有免疫系统和获得性免疫系统,参与维持机体免疫功能的动态稳定。深入研究发现TIPE-2通过负向调控TLR维持免疫自稳。TIPE-2有抑制T细胞活化的作用,是一个有力的T细胞活化负调节分子。
由于细胞膜的屏障作用,生物大分子不能自由进入细胞。近年来,一些具有细胞膜穿透能力的小分子多肽相继被发现,其可有效携带外源性大分子进入细胞,并对宿主细胞没有显著毒副作用。研究发现,KKKFAFAKKKFAFA为带正电荷的短肽,可通过静电作用与带负电荷的质粒DNA聚合形成致密颗粒,显著增强质粒DNA在体内外的转染效率。
发明内容
有鉴于此,本发明的目的之一在于提供一种基于TIPE-2的负调疫苗,能够有效诱导TIPE-2特异性T细胞耐受;目的之二在于提供所述基于TIPE-2的负调疫苗的制备方法;目的之三在于提供所述基于TIPE-2的负调疫苗在医药方面的应用。
为达到上述目的,本发明采用如下技术方案:
基于TIPE-2的负调疫苗,其特征在于:该疫苗为nCoV1表位肽、nCoV2表位肽、nCoV3表位肽与TIPE-2基因重组表达载体的的复合物;
该负调疫苗是以阳离子多肽KKKFAFAKKKFAFA和新冠病毒S蛋白CD8+T细胞表位肽的复合物为基因载体包裹TIPE-2基因重组表达载体自组装而成。
所述nCoV1表位肽由阳离子多肽KKKFAFAKKKFAFA、新冠病毒S蛋白CD8+T细胞表位nCoV 278-286:GSDVNCNGY组成
所述nCoV2表位肽由阳离子多肽KKKFAFAKKKFAFA、新冠病毒S蛋白CD8+T细胞表位nCoV 243-251:LVDGVSRLY组成
所述nCoV3表位肽由阳离子多肽KKKFAFAKKKFAFA、新冠病毒S蛋白CD8+T细胞表位nCoV 428-436:ATKIQNLLY组成
在所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽中,阳离子多肽序列均位于氨基端,CD8+T细胞表位序列均位于羧基端。
所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的摩尔比为1:1:1。
所述TIPE-2基因重组表达载体是将TIPE-2全长cDNA插入真核表达载体pEGFP中而得到。
步骤c的具体方法为:于室温、涡旋条件下,向含有浓度为500μg/ml的pEGFP/TIPE-2和浓度为1mg/ml的(NH4)2SO4,的水溶液中,缓慢滴加等体积的浓度为200μg/ml的阳离子多肽KKKFAFAKKKFAFA和新冠病毒S蛋白CD8+T细胞表位肽的复合物溶液,滴加完毕后,室温下涡旋45分钟,再静置20分钟,即得基于TIPE-2的负调疫苗。
所述基于TIPE-2的负调疫苗在制备诱导新冠病毒S蛋白特异性的T细胞耐受的药物及抗新冠病毒导致的组织损伤治疗领域。
本发明的有益效果在于:阳离子多肽KKKFAFAKKKFAFA与新冠病毒S蛋白CD8+T细胞表位肽的复合物带正电荷,可以与带负电荷的TIPE-2基因重组表达载体通过静电作用聚合形成与天然病毒大小相近的致密颗粒,该颗粒具有阳离子多肽KKKFAFAKKKFAFA的穿膜活性,易于被抗原递呈细胞摄取,体内转染效率高;使TIPE-2基因重组表达载体在抗原递呈细胞内表达TIPE-2,从而有效诱导新冠病毒S蛋白特异性T细胞耐受;研究结果显示,本发明负调疫苗转染的树突状细胞与同种T细胞共培养,能够有效降低新冠病毒S蛋白刺激下T细胞的增殖能力、细胞因子分泌能力和表面分子的活化分子表达率,表明本发明的负调疫苗能够有效诱导特异性T细胞耐受,可用于制备诱导T细胞耐受的药物,在抗新冠病毒导致的组织损伤治疗领域具有良好的开发应用前景。
附图说明
为了使本发明的目的、技术方案和优点更加清楚,下面将结合附图对本发明作进一步的详细描述,其中:
图1为本发明疫苗的结构示意图;
图2为TIPE-2基因的琼脂糖凝胶电泳鉴定图;
图3为TIPE-2重组真核表达质粒双酶切产物电泳鉴定图;
图4为本发明疫苗的透射电镜图;
图5为特异性T细胞增殖能力检测图;
图6为特异性T细胞分泌细胞因子能力检测图;
图7为特异性T细胞表面分子的活化分子表达率检测图。
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的描述。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,例如分子克隆实验指南(第三版,J.萨姆布鲁克等著,黄培堂等译,科学出版社,2002年)中所述的条件,或按照制造厂商所建议的条件。
一、基于TIPE-2的新冠病毒负调疫苗的制备
1、nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的设计与合成
nCoV1表位肽的氨基酸序列如SEQ ID No.1所示,nCoV2表位肽的氨基酸序列如SEQID No.2所示,nCoV3表位肽的氨基酸序列如SEQ ID No.3所示;同时,以卵清蛋白(OVA)表位肽作为对照肽(由表位OVA257-264、穿膜序列HIV-Tat49-57、内质网滞留信号序列KDEL和接头序列组成,其氨基酸序列如SEQ ID No.4所示。
多肽的合成在ABI 431A型固相多肽合成仪(美国PE公司)上进行。方法采用标准芴甲氧羰基(Fmoc)方案,精氨酸采用两次偶联。起始选用0.125mmol对羟甲基苯氧甲基聚苯乙烯树脂(HMP树脂),按照多肽序列使肽链从羧基端逐个向氨基端延伸,每种氨基酸的用量为0.5mmol,与树脂的摩尔比为4:1。各种氨基酸的α-氨基为Fmoc保护,其余侧链保护基团分别为:Lys(Boc)、Ser(tBu)、Glu(OtBu)、Arg(Pmc)、His(Trt)、Thr(tBu)和Tyr(tBu)。第一个氨基酸连接到树脂上用4-二甲氨基吡啶(DMAP),氨基酸的活化用1-羟基苯并三唑(HOBt)和二环己基碳二亚胺(DCC),偶联后用体积分数为20%的哌啶水溶液去除Fmoc保护基。多肽合成后,将树脂-粗肽产品在冰浴条件下混合于10mL切割液A(由结晶苯酚0.75g、1,2-乙二硫醇即EDT 0.25mL、苯甲硫醚0.5mL、去离子水0.25mL和三氟乙酸即TFA 10mL组成)中,待切割液温度上升至室温后,搅拌反应2小时,使肽链从树脂上裂解下来,同时去除多种保护基团。将反应混合液经G4玻砂漏斗过滤以去除树脂,先后用1mL TFA、5~10mL二氯甲烷反复冲洗反应瓶、树脂和漏斗。将滤液在常温低压下蒸发至1~2mL,加入50mL预冷乙醚沉淀多肽,4℃放置过夜,G6玻砂漏斗过滤,真空抽干,即得多肽粗品,-20℃保存备用。
将多肽粗品用二甲基亚砜(DMSO)溶解制成浓度为20mg/mL的溶液,经孔径为0.45μm的微孔滤膜过滤后,在AKTA explorer 100型中压液相色谱仪(瑞典Amerssham Bioscienc公司)上用SOURCE凝胶柱进行纯化。流动相A由体积百分含量为10%的乙醇和体积百分含量为0.1%的TFA组成,流动相B由体积百分含量为90%的乙醇和体积百分含量为0.1%的TFA组成;洗脱梯度为:先用流动相A 1.5个柱体积洗脱,再用流动相A和流动相B的混合液(流动相B占混合液的体积分数在8个柱体积内由0%逐渐增加至80%)洗脱,再用流动相A和流动相B的混合液(流动相B占混合液的体积分数在0.5个柱体积内由80%逐渐增加至100%)洗脱,在主峰处收集多肽溶液,冷冻干燥,即得多肽纯品,用DMSO溶解,-20℃保存备用。
将多肽纯品用Delta 600型高压液相色谱仪(美国Waters公司)鉴定纯度,采用Symmetry ShieldTM C18柱,流动相由体积百分含量为10%~60%的乙腈和体积百分含量为0.1%的TFA组成,梯度洗脱,流速为1mL/min。结果显示,合成多肽的纯度均达到90%以上。同时,将多肽纯品用API 2000LC/MS型电喷雾离子化质谱仪测定分子量。结果显示,合成多肽的分子量均与理论值相符。
2、TIPE-2基因重组表达载体的构建
(1)TIPE-2全长编码基因的克隆
根据GenBank登录号为NM_024575.5的TIPE-2基因序列,设计并合成上下游引物,序列如SEQ ID No.5和SEQ ID No.6所示。PCR引物以扩增TIPE-2全长编码基因;以人胎盘cDNA文库为模板进行PCR,PCR条件为:94℃预变性3分钟,然后94℃变性30秒、56℃退火30秒,72℃延伸30秒,共30个循环,最后72℃延伸5分钟;PCR产物经琼脂糖凝胶电泳鉴定、凝胶回收试剂盒切胶回收纯化后,与载体pGEM-T连接,连接产物转化大肠杆菌JM109感受态细胞,用含有Amp/IPTG/X-GAL的培养基进行蓝白斑筛选,挑取白斑培养,提取质粒,委托上海生工公司测定基因序列,将序列正确的阳性克隆质粒命名为pGEM-T/TIPE-2;
PCR产物的琼脂糖凝胶电泳鉴定图如图2所示,其中M泳道为DNA分子量标准,1泳道为PCR产物,可见1泳道在约500bp处呈单一特异性条带,与预期结果相符;质粒的测序结果显示插入基因序列与TIPE-2全长编码基因序列一致。
(2)TIPE-2基因重组真核表达载体的制备
根据TIPE-2全长编码基因序列和真核表达载体pEGFP的多克隆位点,设计并合成PCR引物,序列如SEQ ID No.7和SEQ ID No.8所示;以扩增包含TIPE-2全长编码基因、5’端BglⅡ酶切位点和3’端BsmⅠ酶切位点的cDNA片段;以pGEM-T/TIPE-2为模板进行PCR,PCR条件与步骤(1)相同;PCR产物经琼脂糖凝胶电泳鉴定、凝胶回收试剂盒切胶回收纯化后,用限制性内切酶BglⅡ和BsmⅠ进行双酶切,双酶切产物经凝胶回收试剂盒切胶回收纯化后,与同样经BglⅡ和BsmⅠ双酶切的pcDNA3.0在T4 DNA连接酶的作用下进行连接,连接产物转化大肠杆菌TOP10感受态细胞,用含有Amp/IPTG/X-GAL的培养基进行蓝白斑筛选,挑取白斑培养,提取质粒,用BglⅡ和BsmⅠ进行双酶切鉴定,并委托上海生工公司测定基因序列,将序列和阅读框架正确的阳性克隆质粒命名为pEGFP/TIPE-2;
重组真核表达质粒双酶切产物的琼脂糖凝胶电泳鉴定图如图3所示,其中M泳道为DNA分子量标准,1泳道为双酶切产物,可见1泳道在约2500bp和500bp处出现两条电泳带,与预期结果相符;质粒的测序结果显示插入基因序列无突变,阅读框架正确,无移码。
3、nCoV1表位肽、nCoV2表位肽、nCoV3表位肽与TIPE-2基因重组表达载体的复合物的制备
将pcDNA3.0/TIPE-2用浓度为1mg/ml的(NH4)2SO4,的水溶液中溶解制成浓度为500μg/mL的溶液,取该溶液100μL,在混旋状态下以5μL/min的速度滴加总浓度为1000μg/mL的多肽溶液(由摩尔比为1:1:1的nCoV1表位肽、nCoV2表位肽、nCoV3表位肽组成)100μL,滴加完毕后继续混旋45分钟,再静置20分钟,即得nCoV1表位肽、nCoV2表位肽、nCoV3表位肽与TIPE-2基因重组表达载体的复合物,本发明所述基于TIPE-2的新冠病毒负调疫苗,结构示意图如图1所示。
透射电镜分析:将新制复合物滴于200目铜网上,吸附3分钟,用吸水纸吸干,晾干30秒,以质量体积百分浓度为1%的醋酸铀水溶液负染30秒,用吸水纸吸干,晾干30秒,80kV透射电镜观察。结果如图4所示,所得复合物呈大小均一的近圆形颗粒,绝大多数颗粒长径小于25nm。
二、基于TIPE-2的负调疫苗诱导T细胞耐受能力的检测
1、分选树突状细胞
取健康志愿者外周血浓缩白细胞,用淋巴细胞分离液密度梯度离心,获得外周血单个核细胞(PBMC),将PBMC用完全RPMI1640培养基重悬后接种于6孔板,在温度为37℃、CO2气体体积分数为5%的条件下培养2小时,吸弃培养上清液,洗涤培养板2次去除非贴壁细胞,获得贴壁的单核细胞,每孔加入完全DMEM培养基3.3ml,并加入浓度为50ng/mL的粒细胞-巨噬细胞集落刺激因子(GM-CSF)和浓度为20ng/mL的白介素4(IL-4),在温度为37℃、CO2气体体积分数为5%的条件下培养,每隔48小时补充完全培养基、GM-CSF和IL24,培养第5天收获细胞,即得树突状细胞。
2、基于TIPE-2的负调疫苗转染树突状细胞
将树突状细胞于6孔板内单层培养至覆盖面积约30%,换入无血清DMEM培养基2ml,再加入基于TIPE-2的负调疫苗100μl,孵育4小时,换入完全DMEM培养基培养48小时,收集细胞,用PBS洗涤并重悬,即得基于TIPE-2的负调疫苗转染的树突状细胞。
3、T细胞增殖能力检测
取健康志愿者外周血浓缩白细胞,用淋巴细胞分离液密度梯度离心获得PBMC,将PBMC用完全RPMI1640培养基重悬后接种于6孔板,在温度为37℃、CO2气体体积分数为5%的条件下培养2小时,收集悬浮细胞,磁珠分选CD8+T细胞(纯度大于95%)作为同种反应细胞。取基于TIPE-2的负调疫苗转染树突状细胞,用丝列霉素处理2小时作为刺激细胞,用完全RPMI1640培养基调整细胞浓度为1×106个/ml,接种至96孔板,每孔100μl,同时设对照组(未经负调疫苗转染的树突状细胞),每组设3个复孔,每孔再加入1×105个同种CD8+T细胞,在温度为37℃、CO2气体体积分数为5%的条件下共培养96小时,再每孔加入浓度为1×103μCi/L的3H-胸腺嘧啶核苷(3H-TdR)溶液0.1ml,继续培养12小时,用PBS漂洗细胞3次,甲醛固定10分钟,再用温度4℃预冷的体积分数为5%的三氯醋酸溶液漂洗3次,每孔加入0.5ml浓度为0.3mol/L的NaOH溶液,60℃水浴反应30分钟,冷却至室温,转移入闪烁瓶中,加入5ml闪烁液,用液体闪烁计数器计数每分钟的闪烁次数(cpm),测定DPM值(反映细胞DNA合成速率),每组重复测定3次。
结果见图4,与基于TIPE-2的负调疫苗转染树突状细胞共培养的CD8+T细胞的cpm值为34000±3700,相应对照组的cpm值为52000±5300;表明本发明负调疫苗转染树突状细胞能够有效降低CD8+T细胞的增殖能力。
2、T细胞分泌细胞因子能力检测
取健康志愿者外周血浓缩白细胞,用淋巴细胞分离液密度梯度离心获得PBMC,将PBMC用完全RPMI1640培养基重悬后接种于6孔板,在温度为37℃、CO2气体体积分数为5%的条件下培养2小时,收集悬浮细胞,磁珠分选CD8+T细胞(纯度大于95%)作为同种反应细胞。取基于TIPE-2的负调疫苗转染树突状细胞,用丝列霉素处理2小时作为刺激细胞,用完全RPMI1640培养基调整细胞浓度为1×106个/ml,接种至96孔板,每孔100μl,同时设对照组(未经负调疫苗转染的树突状细胞),每组设3个复孔,每孔再加入1×105个同种CD8+T细胞,在温度为37℃、CO2气体体积分数为5%的条件下共培养96小时,用ELISA法检测白介素2(IL-2)和干扰素-γ(IFN-γ)的含量。
结果见图5,与基于TIPE-2的负调疫苗转染树突状细胞共培养的的CD8+T细胞分泌IL-2和IFN-γ的量分别为45.3±4.1pg/ml和41.3±3.7pg/ml,相应对照组分泌IL-2和IFN-γ的量分别为106.5±8.5pg/ml和91.5±7.9pg/ml;表明本发明负调疫苗转染树突状细胞能够有效降低CD8+T细胞分泌细胞因子的能力。
3、T细胞表面分子的活化分子表达率检测
取健康志愿者外周血浓缩白细胞,用淋巴细胞分离液密度梯度离心获得PBMC,将PBMC用完全RPMI1640培养基重悬后接种于6孔板,在温度为37℃、CO2气体体积分数为5%的条件下培养2小时,收集悬浮细胞,磁珠分选CD8+T细胞(纯度大于95%)作为同种反应细胞。取基于TIPE-2的负调疫苗转染树突状细胞,用丝列霉素处理2小时作为刺激细胞,用完全RPMI1640培养基调整细胞浓度为1×106个/ml,接种至96孔板,每孔100μl,同时设对照组(未经负调疫苗转染的树突状细胞),每组设3个复孔,每孔再加入1×105个同种CD8+T细胞,在温度为37℃、CO2气体体积分数为5%的条件下共培养96小时,用CD25或CD69抗体标记细胞,用流式细胞仪检测。
结果见图6,与基于TIPE-2的负调疫苗转染树突状细胞共培养的CD8+T细胞的CD25和CD69的表达率分别为7.1±1.2%和8.1±2.1%,相应对照组CD25和CD69的表达率分别为12.3±2.1%和17.2±2.2%;表明本发明负调疫苗转染树突状细胞能够有效降低CD8+T细胞表面分子的活化分子表达率。
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,尽管通过参照本发明的优选实施例已经对本发明进行了描述,但本领域的普通技术人员应当理解,可以在形式上和细节上对其作出各种各样的改变,而不偏离所附权利要求书所限定的本发明的精神和范围。
序列表
<110> 重庆医科大学附属永川医院
<120> 基于TIPE-2的新冠病毒负调疫苗及其制备方法和应用
<141> 2020-03-26
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Claims (5)
1.基于TIPE-2的负调疫苗,其特征在于:该疫苗为nCoV1表位肽、nCoV2表位肽、nCoV3表位肽与TIPE-2基因重组表达载体的复合物;
该负调疫苗是以阳离子多肽KKKFAFAKKKFAFA和新冠病毒S蛋白CD8+T细胞表位肽的复合物为基因载体包裹TIPE-2基因重组表达载体自组装而成;
所述nCoV1表位肽由阳离子多肽KKKFAFAKKKFAFA、新冠病毒S蛋白CD8+T细胞表位nCoV278-286:GSDVNCNGY组成;
所述nCoV2表位肽由阳离子多肽KKKFAFAKKKFAFA、新冠病毒S蛋白CD8+T细胞表位nCoV243-251:LVDGVSRLY组成;
所述nCoV3表位肽由阳离子多肽KKKFAFAKKKFAFA、新冠病毒S蛋白CD8+T细胞表位nCoV428-436:ATKIQNLLY组成;
在所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽中,阳离子多肽序列均位于氨基端,CD8+T细胞表位序列均位于羧基端。
2.根据权利要求1所述基于TIPE-2的负调疫苗,其特征在于:所述nCoV1表位肽、nCoV2表位肽、nCoV3表位肽的摩尔比为1:1:1。
3.根据权利要求1所述基于TIPE-2的负调疫苗,其特征在于:所述TIPE-2基因重组表达载体是将TIPE-2全长cDNA插入真核表达载体pEGFP中而得到。
4.根据权利要求1所述基于TIPE-2的负调疫苗的制备方法,其特征在于:于室温、涡旋条件下,向含有浓度为500μg/ml的pEGFP/TIPE-2和浓度为1mg/ml的(NH4)2SO4,的水溶液中,缓慢滴加等体积的浓度为200μg/ml的阳离子多肽KKKFAFAKKKFAFA和新冠病毒S蛋白CD8+T细胞表位肽的复合物溶液,滴加完毕后,室温下涡旋45分钟,再静置20分钟,即得基于TIPE-2的负调疫苗。
5.根据权利要求1所述基于TIPE-2的负调疫苗在制备诱导新冠病毒S蛋白特异性的T细胞耐受的药物及抗新冠病毒导致的组织损伤治疗领域。
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