CN112279912A - 新型冠状病毒禽蛋抗体SARS-CoV-2-IgY制剂及其制备方法与应用 - Google Patents
新型冠状病毒禽蛋抗体SARS-CoV-2-IgY制剂及其制备方法与应用 Download PDFInfo
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Abstract
本发明公开了新型冠状病毒禽蛋抗体SARS‑CoV‑2‑IgY制剂及其制备方法与应用,是将新冠病毒(SARS‑CoV‑2)的S蛋白(刺突蛋白)加不完全佛氏佐剂注射到雌禽的体内,再从禽所生的蛋中提取,纯化得到能与新冠病毒S蛋白结合的禽蛋抗体(SARS‑CoV‑2‑IgY),再将抗体蛋白经膜过滤、除菌、冷冻或喷雾干燥,制成喷雾剂或者口服制剂(口服液,片剂,胶囊等而成),也可加入到牛奶,豆奶等饮品中作为增强免疫的功能性食品。是一种安全、可靠、经济简便、易于推广的生物制剂,是防治新型冠状病毒感染的创新策略。
Description
技术领域
本发明涉及新型冠状病毒(SARS-CoV-2)治疗与预防制剂的开发,特别是新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)禽蛋抗体制剂及其制备方法与应用。
背景技术
冠状病毒(CoVs)属巢病毒目冠状病毒科正冠状病毒亚科,是一组高度多样化、有包膜的单链正义RNA病毒。根据病毒的血清学和基因组特点该亚科分为α、β、γ、δ四个属。自2002年以来,β冠状病毒(CoV)已经导致了三次人畜共患疾病的爆发,SARS-CoV于2002-2003年,MERS-CoV 于2012年及 SARS-CoV-2 于 2019爆发,2019新型冠状病毒(SARS-CoV-2)是2019年在人体中发现的冠状病毒新毒株。该病毒已确认存在人传人现象,且潜伏期具有传染性,其主要传播途径为空气传播与接触传播,中间宿主尚不明确。
刺突蛋白 S、包膜 E、膜糖蛋白 M 和核衣壳 N是冠状病毒完成病毒组装和感染必不可少的四种编码结构蛋白。研究发现,S 蛋白位于SARS-CoV-2病毒表面,是病毒的融合蛋白,负责病毒与宿主细胞受体结合,介导 SARS-CoV-2 感染的第一步SARS-CoV-2入侵人体细胞的受体为血管紧张素转换酶2(ACE2)。SARS-CoV-2病毒与ACE2结合后引发炎症反应是COVID-19患者主要的病理过程。发烧、干咳和疲劳是COVID-19发作时的常见症状。大多数患者在胸部CT扫描中出现双侧毛玻璃样阴影改变和淋巴细胞减少。有基础性疾病、年龄大的患者感染后,更容易发生重症和死亡。由于ACE2广泛丰富表达于心血管系统、肠道、肾脏和肺脏中,在病毒感染后机体免疫系统被过度激活,IL-6、TNF-α、TNF-γ等多种细胞因子显著升高,引发细胞因子风暴,导致出现急性呼吸窘迫综合症(ARDS)或伴多器官功能障碍,尽管插管可改善状况,死亡率仍然高达45%。因而免疫系统介导的炎症细胞因子风暴现象,是COVID-19患者发展至重型或危重型的致命因素。
由于SARS-CoV-2病毒传染源尚未明确、并存在多种变种,到目前为止尚无特效的药物治疗方法,作为防治和阻止病毒感染疫情蔓延的主要手段,疫苗的研究在也紧锣密鼓地进行,在近百种SARS-CoV-2候选疫苗中,已有数种疫苗进入临床试验,其最终人群免疫中和抗体的产生与持续的情况还需更广泛的使用证明。在病毒抗体被动免疫治疗方面,有学者发现,利用体外纯化的重组人类ACE2蛋白“中和”SARS-CoV-2病毒,可削减其感染能力1000-5000倍。近日有报道,EUSA Pharma公司IL-6抗体Siltuximab使21例COVID-19患者病情改善和稳定,疾病控制率达76%; Kiniksa公司表示GM-CSFRα抗体Mavrilimumab使 6例患者病情缓解,药物耐受性良好。这些治疗性抗体为抗击SARS-CoV-2感染带来了新的希望。
血浆疗法的技术原理主要是利用康复者血浆中针对病毒的特异性抗体,迅速识别并捕捉病毒,激活体内的补体系统清除病毒。血浆治疗不仅在近年国流行的传染性疾病SARS、MERS、埃博拉和H1N1 流感等重大疫情取得较好的疗效,近期研究数据也显示,将恢复期SARS-CoV-2感染者含有高滴度中和抗体的血浆输注给重症患者,可在短期内恢复器官功能、病毒核酸检测转阴及增加特异性抗体浓度。然而,鉴于恢复期血浆短缺,血浆需求量与供给量差距悬殊,以及供者血浆安全性等因素,获取合格的恢复期血浆仍存在较大困难。
从免疫的禽类卵黄中提取分离得到的卵黄免疫球蛋白Immunoglobulin Y (IgY))是被动免疫所需抗体的极好来源。IgY以单体的形式存在,且与哺乳动物的IgG在分子量、结构和生化功能上存在差异。相比于IgG,IgY理化性质稳定,耐热:在60-70 ℃稳定,4℃稳定5年,室温下稳定6个月、易于收集、含量高,每个鸡蛋含特异性抗体1mg, 相当于一只家兔全血的含量,不激活补体。耐酸,耐酶,可以口服。IgY早已用于预防和治疗禽类、畜类的感染性疾病,并且IgY在人类疾病诊治的应用研究已成为热点。如抗幽门螺杆菌、志贺大肠杆菌、霍乱弧菌等细菌感染。抗人类轮状病毒、流感病毒、SARS病毒,手足口病等病毒感染。
IgY被认为是安全无毒的,已被美国FDA列为安全食品。病毒感染引起的过度免疫反应性炎症风暴被认为与激活补体C5a有关,IgY不激活补体,所以相对于其它抗体来说更加安全。
为此,我们提出研发一种针对SARS-CoV-2病毒较快速,经济,方便,高效,安全的应急性禽类(鸡)蛋抗体的对策,作为血清抗体的替代品用于SARS-CoV-2病毒感染性疾病的预防与治疗。
发明内容
本发明的目的是要提供一种安全、高效、稳定,经济简便、易于推广的新型冠状病毒(SARS-CoV-2)家禽蛋抗体制剂及其制备方法与应用。
本发明目的通过下述技术方案来实现:
新型冠状病毒(SARS-CoV-2)家禽蛋抗体制剂,是将新冠病毒(SARS-CoV-2)的S蛋白(刺突蛋白)加不完全佛氏佐剂注射到雌禽的体内,再从禽所生的蛋中提取,纯化得到能与新冠病毒S蛋白结合的禽蛋抗体(SARS-CoV-2-IgY),再将抗体蛋白经膜过滤、除菌、冷冻或喷雾干燥,制成喷雾剂或者口服制剂(口服液,片剂,胶囊等而成),也可加入到牛奶,豆奶等饮品中作为增强免疫的功能性食品。
将抗新型冠状病毒禽蛋总IgY用磁珠抗体纯化方法将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体纯化到99%以上的纯度再溶入无菌注射液中,制成注射剂作为肌肉及静脉给药制剂。
所述制剂的使用方法是:
鼻咽部喷雾或口服:每天2-3次,喷雾给药每次1~2 mg抗体,口服给药每次10~20 mg抗体,或者在临时需要时(开会发言,运动,高危人群,高危环境:在疫区旅行,医院病房等)可多次临时使用达到预防效果。
功能性食品:牛奶,豆奶等饮品1~2 mg/100ml,一天一100ml;
注射剂:肌注,或静脉滴注每天1~2次,1~2 mg/次。
新型冠状病毒家禽蛋抗体(SARS-CoV-2-IgY)制剂的制备方法,包括如下步骤:
1.禽类的免疫:
取新型冠状病毒(SARS-CoV-2)S蛋白与不完全佛氏佐剂混匀乳化,肌肉注射到成年雌禽体内,每周一次,共4次,14天后出现IgY特异抗体,收集家禽蛋,取样检测禽蛋抗体含量(ELISA)4℃冰箱保存,根据需要雌禽可每隔30天重复免疫一次。
2.抗体的纯化与制备:
用75%的酒精消毒禽蛋外壳,去壳,分离取出蛋黄,用PEG或硫酸葡萄糖或自然胶(藻酸钠、角叉菜胶)或乙醇沉淀法纯化抗体蛋白质,再将纯化后的抗进行膜过滤,除菌,冷冻(喷雾)干燥,制成喷雾剂或者口服制剂(口服液,片剂,胶囊等而成)。将抗新型冠状病毒禽蛋总IgY用磁珠抗体纯化方法将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体纯化到99%以上的纯度再溶入无菌注射液中,制成注射剂作为肌肉及静脉给药制剂;将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体加入到牛奶,豆奶等饮品中作为增强免疫的功能性食品,4℃冰箱贮存。
所述的禽包括鸡、鸭、鹅、鹌鹑、鸽等。
本发明的优点在于:
采用新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)制剂预防与治疗新型冠状病毒感染性疾病(肺炎),有如下优势:1. 雌禽产生有效免疫所需抗原量小,抗体产量大,来源丰富.一个鸡蛋所含的特异性抗体约为1mg,相当于一只家兔全血所含的抗体量;2.IgY稳定,耐高温,在60~70 ℃稳定,4℃稳定5年,室温下稳定6个月;3.耐酸,耐酶,可以口服;4.不激活补体,不易引起新型冠状病毒感染后期发生的致命性炎症风暴,在安全性方面优于其它来源的新型冠状病毒治疗性抗体;5.雌禽易于饲养,禽蛋产量大,产品成本低,治疗费用低。新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)制剂是一种安全、可靠、经济简便、易于推广的生物制剂,是防治新型冠状病毒感染的创新策略。6. 通过接种疫苗产生病毒中和性抗体的时间与效能尚无定论,或存在个体差异,因此,研发低度高效的被动免疫抗体疗法很有必要。
附图说明
图1为新型冠状病毒S蛋白IgY Western blot 鉴定分析图;
图2为新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY对易感细胞中新型冠状病毒(SARS-CoV-2)的抑制作用对比图。
具体实施方式
通过下面给出的本发明的具体实施例,可以进一步清楚地了解本发明的方法,但它们不是对本发明的限定。
实施例1:
新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)制剂的制备包括如下步骤:
1.禽类的免疫:
取新型冠状病毒(SARS-CoV-2)S蛋白与不完全佛氏佐剂混匀乳化,肌肉注射到成年雌禽体内,每周一次,共4次,14天后出现IgY特异抗体,收集家禽蛋,取样检测禽蛋抗体含量(ELISA)4℃冰箱保存,根据需要雌禽可每隔30天重复免疫一次。
2.抗体的纯化与制备:
用75%的酒精消毒禽蛋外壳,去壳,分离取出蛋黄,用PEG或硫酸葡萄糖或自然胶(藻酸钠、角叉菜胶)或乙醇沉淀法纯化抗体蛋白质,再将纯化后的抗进行膜过滤,除菌,冷冻(喷雾)干燥,制成喷雾剂或者口服制剂(口服液,片剂,胶囊等而成);将抗新型冠状病毒禽蛋总IgY用磁珠抗体纯化方法将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体纯化到99%以上的纯度再溶入无菌注射液中,制成注射剂作为肌肉及静脉给药制剂;将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体加入到牛奶,豆奶等饮品中作为增强免疫的功能性食品,4℃冰箱贮存。
实施例2:
新型冠状病毒禽抗体(SARS-CoV-2-IgY)的活性分析
1. 蛋白免疫印迹法(Western Blot)检测SARS-CoV-2-IgY的特异性。
将SARS-CoV-2-S抗原(病毒S蛋白)用聚丙烯酰胺凝胶电泳分离,用鸡卵黄中提取纯化的抗SARS-CoV-2-IgY (稀释比例:1:10000)作为一抗;用封闭液以 1:10000 的比例稀释 HRP 标记的羊抗鸡 IgY作为二抗。对X 线胶片进行拍照或图像扫描,结果见图1,图中:在180KD标记上有一明显条带,说明新型冠状病毒抗体能识别并与新型冠状病毒S蛋白结合。
2. 新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)体外抗病毒 qPCR实验
将25ul新型冠状病毒(100PFU)与等体积不同浓度梯度的 IgY 或 SARS-CoV-2-IgY 孵育10分钟后,加入1×104vero 细胞(非洲绿猴肾细胞系)中,在37度培养箱培养4小时后,将细胞培养液换成完全培养基。48小时后,收集细胞并提取总 RNA,逆转录后检测各样品中新冠状病毒基因组的拷贝数。结果不同浓度的新型冠状病毒禽蛋抗体能够明显抑制新型冠状病毒的(SARS-CoV-2)在vero细胞中的复制,各剂量组与免疫前对照组比较差异明显 P< 0.001且量效关系明显,其半数抑制率IC50为0.8 µg/ml结果见图2。
3.新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)体内抗病毒动物实验
将K18 hACE2(血管紧张素﹣转换酶)转基因雄性6周龄小鼠安置在 P3实验室无病原
体设施中,分组为SARS-CoV-2感染对照组(A组)与SARS-CoV-2感染禽蛋抗体(SARS-CoV-2-
IgY)处理组(B组),每组动物8只,在第0天,A组每鼠于鼻腔部喷雾未经免疫的0.1%的禽蛋抗
体蛋白对照溶液,0.2ml/小鼠1次,B组每鼠于鼻腔部喷雾0.1%新型冠状病毒禽蛋抗体
0.2ml/小鼠1次,30分钟内各组小鼠鼻内感染3×105 298 PFU的SARS299 CoV-2,24小时后
再给药一次,48小时后处死小鼠,小鼠鼻腔取分泌物用PCR方法测定病毒进行组间比较分
析,结果A组8只小鼠新型冠状病毒检测均为阳性,B组8只小鼠检测结果均为阴性,结果见表
1
表1新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)体内抗病毒动物实验结果
分组 | 动物数 | 新冠状病毒感染率(%) | 对新冠状病毒感染的保护率(%) |
A | 8 | 100 | 0 |
B | 8 | 0 | 100 |
注:A 组与B组比较P< 0.001;阴性标准:病毒载量<40拷贝数/ml。
Claims (8)
1.新型冠状病毒禽蛋抗体SARS-CoV-2-IgY制剂,其特征是:用新型冠状病毒(SARS-CoV-2)S蛋白与不完全佛氏佐剂混匀乳化,肌肉注射到成年雌禽体内,每周一次,共4次,从该禽所生的蛋中提取出新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY),抗将纯化后的抗进行膜过滤,除菌,喷雾干燥,制成喷雾剂。
2.新型冠状病毒禽蛋抗体SARS-CoV-2-IgY制剂,其特征是:用新型冠状病毒(SARS-CoV-2)S蛋白与不完全佛氏佐剂混匀乳化,肌肉注射到成年雌禽体内,每周一次,共4次,从该禽所生的蛋中提取出新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY),抗将纯化后的抗进行膜过滤,除菌,冷冻干燥,制成口服制剂或注射剂。
3.新型冠状病毒禽蛋抗体SARS-CoV-2-IgY制剂,其特征是:用新型冠状病毒(SARS-CoV-2)S蛋白与不完全佛氏佐剂混匀乳化,肌肉注射到成年雌禽体内,每周一次,共4次,从该禽所生的蛋中提取出新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY),抗将纯化后的抗进行膜过滤,除菌,冷冻干燥,将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体加入到牛奶,豆奶饮品中作为增强免疫的功能性食品。
4.根据权利要求1所述的制剂,其特征是:所述口服制剂为口服液、片剂或胶囊。
5.权利要求1-4之一所述的制剂,其特征是:将新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY)用于预防与治疗新型冠状病毒(SARS-CoV-2)感染性疾病(SARS-CoV-2)。
6.权利要求1-4之一所述制剂的制备方法,其特征是:包括如下步骤:
(1)雌禽免疫:
用新型冠状病毒(SARS-CoV-2)S蛋白与不完全佛氏佐剂混匀乳化,肌肉注射到成年雌禽体内,每周一次,共4次,从该禽所生的蛋中提取出新型冠状病毒禽蛋抗体(SARS-CoV-2-IgY);
(2)抗体的纯化与制备:
用75%的酒精消毒禽A所下蛋的外壳,去壳,分离取出蛋黄,用PEG、硫酸葡萄糖、自然胶或乙醇沉淀法纯化抗体蛋白质,将纯化后的抗体蛋白进行膜过滤,干燥,制成喷雾剂,口服液,胶囊及片剂,将抗新型冠状病毒禽蛋总IgY用磁珠抗体纯化方法将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体纯化到99%以上的纯度再溶入无菌注射液中,制成注射剂作为肌肉及静脉给药制剂;将新型冠状病毒(SARS-CoV-2)家禽蛋特异性抗体加入到牛奶,豆奶等饮品中作为增强免疫的功能性食品,4℃冰箱贮存。
7.如权利要求6所述制剂的制备方法,其特征是:步骤(1)所述的对雌禽实施免疫4次,雌禽可每隔30天重复免疫一次。
8.如权利要求6所述制剂的制备方法,其特征是:步骤(2)所述的自然胶为藻酸钠或角叉菜胶。
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