CN114470198B - 一种用于防治新冠肺炎的SARS-CoV-2卵黄中和抗体IgY喷雾剂 - Google Patents
一种用于防治新冠肺炎的SARS-CoV-2卵黄中和抗体IgY喷雾剂 Download PDFInfo
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Abstract
一种用于防治新冠肺炎的SARS‑CoV‑2卵黄中和抗体IgY喷雾剂，所述卵黄中和抗体IgY为多克隆抗体，包括新冠病毒S1蛋白受体结合域抗体或S2蛋白抗体，可阻断新冠病毒与细胞受体ACE2的结合，中和新冠病毒SARS‑CoV‑2，阻断新冠病毒的感染，抑杀病毒，预防及治疗新冠肺炎；以新冠病毒的S蛋白、S1蛋白、RBD蛋白或S2蛋白作为抗原，免疫产蛋母鸡，从鸡蛋黄中提取卵黄抗体IgY。本发明提供抗新冠病毒的卵黄中和抗体IgY制剂，可为喷雾剂、滴眼剂、口服胶囊或口服含片；作用方式安全、易大规模生产，不产生免疫排斥反应，开创安全简便、卫生有效、经济的个性化防疫方式应对疫情。
Description
技术领域
本发明属于防疫领域，具体涉及一种用于防治新冠肺炎的SARS-CoV-2卵黄中和抗体IgY喷雾剂。
背景技术
在SARS-CoV-2中，病毒表面的S蛋白介导受体识别和膜融合。在病毒感染过程中，三聚体S蛋白被切割成S1和S2亚基，S1蛋白的受体结合域(RBD)直接与靶细胞表面受体ACE2的肽酶域(peptidase domain，PD)结合，而S2负责膜融合，因此，该病毒能够特异性的识别并结合到人细胞表面受体，侵入人体细胞，造成人的感染。
研制新冠疫苗需要做I，II，III期临床试验，周期长，最快也需要3-5年时间。新冠病毒属于RNA单链病毒，容易变异，新冠病毒基因组30000多个碱基，目前已突变1000多个位点，若研制疫苗的抗原表位发生突变，则疫苗就失效了，因此，疫苗研制的速度赶不上基因突变的速度。
中和抗体能够中和病毒，使病毒失去感染性达到防治作用，若能够研制出新型冠状病毒的中和抗体，就能够中和新型冠状病毒，使病毒不能结合到细胞表面受体，阻断病毒的感染，从而达到预防和治疗新型冠状病毒感染引起的疾病作用。
目前，新型冠状病毒感染者恢复期的康复者血浆也含有新型冠状病毒的中和抗体，能够治疗新型冠状病毒感染者，但由于血浆中可能含有其它的病原体，会引起其它的传染病，又由于康复者血浆来源少，难以满足庞大病人治疗的需求。
许多科学家转而研制新冠病毒人源化中和抗体IgG，但人源化中和抗体IgG与病毒结合后有时会产生“抗体依赖的增强作用(ADE)”，协助病毒进入靶细胞，提高感染率，导致病毒致死率增加几十倍甚至几百倍。
发明内容
本发明的目的在于提供一种用于防治新冠肺炎的SARS-CoV-2卵黄中和抗体IgY喷雾剂，中和病毒，阻断新冠病毒的感染，抑杀病毒，起到预防及治疗新冠肺炎的作用，作用方式安全、容易大规模生产，不会产生免疫排斥反应，开创一种安全简便、卫生有效、经济的个性化防疫方式应对疫情。
为了达到上述目的，本发明提供如下技术方案：
提供新冠病毒卵黄中和抗体IgY在制备抗新冠病毒SARS-CoV-2制剂中的应用。
进一步，所述新冠病毒卵黄中和抗体IgY为多克隆抗体，包括新冠病毒S1蛋白受体结合域RBD的3个表位的抗体或S2蛋白抗体。
又，所述新冠病毒卵黄中和抗体IgY中，包括S1蛋白的受体结合域RBD的抗体，其重链为67-70KD，轻链为22-30KD。
又，所述新冠病毒卵黄中和抗体IgY的制备方法为：以新冠病毒的S蛋白、S1蛋白、RBD蛋白或S2蛋白免疫产蛋母鸡，每次取抗原50-250μg，对产蛋母鸡进行肌肉注射，免疫三次，每次间隔时间为7-42天；用蛋黄筛分离免疫母鸡产蛋的蛋黄，制备为蛋黄液，用1MolHCl调至pH5.1-6.0，2-8℃静置沉淀4-5小时，以8000-12000rpm转速离心20-30分钟，弃沉淀，上清加入1-2％正辛酸沉淀去除杂蛋白和卵磷脂纯化，得到所述新冠病毒卵黄中和抗体IgY，含量为2-6mg/ml，纯度40-50％。
一种抗新冠病毒SARS-CoV-2卵黄中和抗体IgY制剂，包括：所述新冠病毒卵黄中和抗体IgY、稳定剂、抑菌剂和防腐剂。
优选地，所述抑菌剂为乳酸链球菌素NISIN、苯扎溴安、溶菌酶中的一种以上；所述防腐剂为氯已定、山梨酸钾中的一种或两种。
优选地，所述新冠病毒卵黄中和抗体IgY制剂为喷雾剂、滴眼剂、口服胶囊或口服含片。
进一步，所述喷雾剂中包括：新冠病毒卵黄中和抗体IgY 0.1-0.3％、溶菌酶0.1-0.9％、乳酸链球菌素(NISIN)0.005-0.02％、甘油0.5-20％、氯已定0.01-0.05％和蒸馏水79.8-98.57％。
又，所述滴眼剂中包括：卵黄中和抗体IgY 0.1-0.3％、溶菌酶0.1-0.9％、EDTA2Na0.01-0.1％、NaCl 0.1-0.5％、苯扎溴安0.01-0.05％、乳酸链球菌素(NISIN)0.005-0.02％、甘油0.5-20％和蒸馏水72.4-96.29％。
优选地，所述口服含片中包括：卵黄中和抗体IgY 0.01-0.03％、柠檬酸1-5％、木糖醇10-30％、淀粉40-50％、山梨酸钾0.05-0.5％和蒸馏水48.99-14.97％。
又，所述口服胶囊中包括：卵黄中和抗体IgY 0.5-1.6％、乳酸链球菌素0.005-0.02％、壳聚糖0.1-0.4％、CaCl2 0.5-3％、海藻酸钠1-3％和蒸馏水97.895-91.98％。
在新冠病毒SARS-CoV-2中，病毒表面的S蛋白是糖基化蛋白，须用具有糖基化表达系统才能表达出糖基化的S蛋白，才能保持天然结构，才能有活性，才能免疫出中和抗体。大肠杆菌表达系统无糖基化系统，酵母表达系统糖基化系统不完善，昆虫表达系统也不完善，本发明选择含有完善糖基化系统的哺乳细胞表达S、S1、S2、RBD蛋白，表达出的糖基化S蛋白保持天然结构，有活性，能免疫出卵黄中和抗体。
将本发明的抗突变新冠病毒卵黄中和抗体IgY喷剂喷在鼻腔和口腔，吸入呼吸道，可中和新冠病毒SARS-CoV-2，将其雾化吸入肺内可应急治疗新冠肺炎。
本发明的抗新冠病毒SARS-CoV-2的卵黄中和抗体IgY制剂中，抗体IgY是蛋白质，浓度高，乳酸链球菌素(NISIN)的作用就象一个表面活性剂，它对细菌的营养细胞及孢子都有作用。NISIN对营养细胞的主要作用点是细胞质膜，抑制细胞壁中肽聚糖的生物合成，从而使细胞壁质膜和磷脂化合物合成受阻，并造成细胞膜内容物外泄，严重地引起细胞裂解，在万分之三到万分之五的浓度既有明显抑菌作用，对芽孢的作用是在孢子出现膨胀的起始阶段抑制其发芽。NISIN主要抑制大部分革兰氏阳性菌，特别是细菌的芽孢，还能抑制葡萄球菌属、链球菌属、小球菌属和乳杆菌属的某些菌种，抑制大部分梭菌属和芽孢杆菌属的芽孢，避免在制剂的生产过程中污染菌，防止产品腐败变质。
抗体IgY制剂在生产过程中需防止染菌，本发明中的抗体IgY是蛋白质，浓度很高，喷雾剂的生产过程中易染菌，产品易腐败变质，较常规IgY抗体的生产难度大；苯扎溴安是二甲基苄基烃铵的混合物，具有典型阳离子表面活性剂的性质，水溶液搅拌时能产生大量泡沫，性质稳定，耐光，耐热，无挥发性，可长期存放，其使用浓度为50～100mg/L，能够杀灭该IgY抗体生产过程中的污染菌；本发明口服含片中的防腐剂选用山梨酸钾，通过与微生物酶系统的巯基结合从而破坏许多酶系统，其毒性低，在酸性介质中能充分发挥防腐作用。
本发明中喷雾剂或滴眼剂在生产过程中需严格防止染菌，NISIN、溶菌酶与氯已定或苯扎溴安结合使用能有效防腐，既能杀灭真菌，又能杀灭革兰氏阳性菌和革兰氏阴性菌；氯已定又名洗必泰(Chlorhexidine)，为广谱抗菌剂，对真菌也有效，对组织无刺激性，对革兰氏阳性菌比阴性菌的抗菌作用强，溶菌酶是一种能水解细菌中黏多糖的碱性酶。溶菌酶主要通过破坏细胞壁中的N-乙酰胞壁酸和N-乙酰氨基葡萄糖之间的β-1,4糖苷键，使细胞壁不溶性黏多糖分解成可溶性糖肽，导致细胞壁破裂内容物逸出而使细菌溶解。
本发明利用新冠病毒S蛋白、S1蛋白、S2蛋白或RBD蛋白免疫产蛋母鸡，从鸡蛋黄中提取卵黄抗体IgY，然后用水稀释法结合正辛酸沉淀去除杂蛋白和卵磷脂纯化，获得的卵黄中和抗体IgY能够中和新型冠状病毒SARS-CoV-2，使病毒不能结合到细胞表面受体，阻断病毒的感染，从而达到预防和治疗新型冠状病毒感染引起的疾病作用；以RBD蛋白为抗原时，仅以RBD作为靶点，诱发中和抗体，避免了非RBD区抗体形成抗体依赖致病加重现象的可能性，对老年患者也更有利。
IgY抗体效价与免疫的抗原剂量有关，免疫抗原剂量少时，抗体的效价低，抗原剂量高时，效价高，通常为了获得效价高的抗体，一般尽可能免疫多的抗原，但抗原剂量过高时，会引起免疫抑制现象，获得的抗体效价反而少，本发明根据针对新冠病毒的试验结果，选取每次免疫200μg抗原，获得的抗体效价较高，IgY中和抗原的能力强，又能避免免疫抑制现象的产生。
在对卵黄中和抗体IgY纯化时，水的添加量、卵黄液pH值、4℃静置时间、离心速度，正辛酸的添加量、超滤都会影响抗体的含量；若产品中卵黄中和抗体IgY含量太少，使用时可能不能完全中和病毒，不能完全阻断新冠病毒的感染，预防或治疗效果降低，本发明选择合适合适的控制参数，获得的卵黄抗体IgY浓度在20μg/ml时能够中和97％的新冠病毒(SARS-CoV-2)。
本发明的卵黄中和抗体IgY喷雾剂方便携带，适用于各类人群，在坐公交、地铁、去医院、学校、开会等人群聚集场合使用，可中和新冠病毒、预防新冠肺炎；或给复工复产的人员使用，预防新冠肺炎，使复工复产尽快开始，大大减少经济损失。
本发明的卵黄中和抗体IgY口服胶囊，吞服后，可中和新冠病人消化道内的新冠病毒，阻止新冠病毒从粪便中排出，防止新冠病毒通过粪口传播；本发明的卵黄中和抗体IgY滴眼剂、以及口服的口服剂和含片，用于新型冠状病毒感染的预防和治疗，开创一种简便、安全、有效、卫生、经济的个性化防疫方式应对疫情。
由于狗、猫等宠物也会感染新冠病毒，一旦狗、猫等宠物感染新冠病毒后也会传染给人类，本发明的新冠病毒卵黄抗体IgY制剂，如口、鼻喷剂、滴眼剂、含片、口服剂、口服胶囊也可用来预防狗、猫等宠物的新冠肺炎，阻断新冠病毒通过狗、猫等宠物传染给人类。
与现有技术相比，本发明至少具有如下有益效果：
本发明以新冠病毒S蛋白、S1蛋白、S2蛋白或RBD蛋白作为抗原，免疫产蛋母鸡，从鸡蛋黄中提取卵黄抗体IgY，在功能上等价于哺乳动物血液中抗体IgG，无IgG的Fc片段，与病毒结合后不会产生抗体依赖的增强作用，安全，且无血浆中可能残留的病原体。
本发明的新冠病毒卵黄抗体IgY从抗原蛋白表达到免疫鸡生产抗体只需2个多月时间，可以快速制备突变新冠病毒卵黄抗体，当季新冠流行期间就可以进行新冠病毒的防控；同时，容易大规模生产满足全民抗疫需求，基于IgY抗体口服和外用时不具免疫原性，不会产生免疫排斥反应。
本发明的新冠病毒(SARS-CoV-2)卵黄中和抗体IgY能有效中和新冠病毒感染，经生物安全三级实验室用新冠病毒SARS-CoV-2进行新冠病毒SARS-CoV-2卵黄抗体IgY中和实验，以免疫前母鸡产的蛋中提取的不含有新冠病毒的IgY作为阴性对照，本发明的新冠病毒卵黄抗体IgY浓度在20μg/ml时中和97％的新冠病毒(SARS-CoV-2)感染，对照组无明显中和活性。
附图说明
图1为本发明实施例1中的新冠病毒(SARS-CoV-2)卵黄IgY抗体电泳图。
图2为本发明实施例6中的新冠病毒(SARS-CoV-2)卵黄IgY抗体新冠病毒中和实验结果。
具体实施方式
以下结合具体实施例对本发明作进一步说明。
实施例1制备抗新冠病毒的卵黄中和抗体IgY
1.以新冠病毒RBD蛋白免疫产蛋母鸡
以新冠病毒RBD蛋白(其氨基酸序列参见SEQ ID NO .1)作为抗原，每次取抗原200μg，加入适合免疫鸡用的佐剂，抗原：佐剂ISA 70＝30：70，乳化，体积比，对产蛋母鸡进行肌肉注，0.5ml，免疫3次，3点免疫，每次间隔时间为28天。
2.提取纯化IgY抗体
用蛋黄筛分离免疫母鸡产蛋的蛋黄，加入9倍蒸馏水搅拌混匀制备蛋黄液，用1MolHCl调pH5.2，4℃静置沉淀5小时，8000rpm离心20分钟，弃沉淀，收集上清，在上清中缓慢滴加正辛酸至1％去除杂蛋白和卵磷脂，搅拌混匀。
于4℃静置2小时，4℃8000rpm离心30分钟，收集上清液进行超滤浓缩：加入终浓度3％的硅藻土作为助滤剂，采用0.45μm混合纤维素酯膜进行真空抽滤(0.09MPa)，得抽滤液；选择5万分子截留量超滤膜，室温下进料，单段间歇操作模式对抽滤液进行超滤浓缩，得浓缩IgY，巴斯德消毒，0.22μm膜过滤除菌，即为分离纯化的新冠病毒卵黄中和抗体IgY，含量为3mg/ml，纯度50％。
3.SDS-PAGE电泳分析
对分离纯化的IgY进行SDS-PAGE电泳分析，样品制备：3％浓缩胶，8％分离胶，点样8μl，电压为80V，电泳60分钟，当指示剂到达分离胶前沿1-2cm停止电泳，考马斯亮蓝染色，脱色，拍照(如图1)。
由图1可见，所述卵黄中和抗体IgY的重链为67-70KD，轻链为22-30KD。
4.效价分析
用琼脂双向免疫扩散实验(AGP)测定IgY抗体的效价：
打孔：制备1.5％缓冲琼脂板，在中间打一孔，孔径4mm，在该孔周围距离3mm处打6个孔径3mm的小孔。
稀释抗体：采用二倍稀释法对上述纯化后的新冠病毒卵黄中和抗体IgY样品进行20、2-1、2-2、2-3、2-4、2-5、2-6、2-7、2-8连续稀释。
加样：中心孔加入新冠病毒RBD蛋白(2000μg/ml)，以不溢出孔为准，周围小孔加入纯化的IgY，按IgY的稀释倍数由小到大加入，加样后置于带盖饭盒(内铺湿纱布)，在37℃下扩散24小时。
结果观察：抗原孔与抗体孔出现沉淀线为阳性，以分离纯化的IgY能出现沉淀线的最大稀释倍数为其AGP效价。
实验结果：新冠病毒(SARS-CoV-2)卵黄IgY抗体在稀释64倍时仍能产生明显的白色抗原-抗体沉淀线，即新冠病毒(SARS-CoV-2)卵黄IgY抗体的效价为1：64。
本发明的卵黄中和抗体IgY至少在体内持续12小时，使用喷雾制剂时，早晚各喷3-4下，共消耗1ml喷剂，一天一般只需喷两次就可满足预防新冠的要求，或外出前喷雾3-4下，可预防新冠肺炎。
实施例2新冠病毒卵黄中和抗体IgY喷雾剂的制备：
各成分按照如下重量百分含量进行配比：实施例1的卵黄中和抗体IgY 0.3％，甘油10％，NISIN 0.02％，溶菌酶0.5％、氯已定0.02％，加去离子水补足100ml，用巴斯德消毒法进病毒行灭活，用0.22μm的膜过滤除菌，将溶液装入小型携带式喷雾罐中喷雾使用，用于鼻喷剂、口喷剂预防，雾化应急治疗或应急注射治疗。
实施例3防新冠肺炎的卵黄中和抗体IgY滴眼剂的制备
各成分按照如下重量百分含量进行配比：实施例1的卵黄中和抗体IgY 0.3％，溶菌酶0.5％，NISIN 0.02％，EDTA-2Na 0.05％，NaCl 0.034％，苯扎溴安0.02％，甘油5％，山梨醇5％，水溶性薄荷香精0.025％，磷酸氢二钠0.786％，磷酸二氢钠1.04％，去离子水补足余量。
用巴斯德消毒法进病毒行灭活，用0.22μm的膜过滤除菌。将溶液装入小型携带式眼药瓶滴眼使用。
实施例4防新冠肺炎的卵黄中和抗体IgY口服含片的制备
各成分按照如下重量百分含量进行配比：实施例1的卵黄中和抗体IgY 0.3％，木糖醇26％，柠檬酸5.2％，淀粉42％，山梨酸钾0.1％，ddH2O 26.4％，水溶性薄荷香精0.5％。
将除卵黄中和抗体IgY以外的原料进行微波灭菌，将经微波灭菌处理的原料按照配比搅拌混合，再经过干法造粒、压片和微波灭菌得防治新冠肺炎的卵黄中和抗体IgY口服含片，每片0.38克。
实施例5防新冠肺炎的卵黄中和抗体IgY口服胶囊的制备
各成分按照如下重量百分含量进行配比：实施例1的卵黄中和抗体IgY 1％，壳聚糖0.2％，CaCl2 2％，NISIN 0.02％，海藻酸钠2％，木糖醇5％，山梨醇5％，水溶性留兰香香精0.3％，ddH2O 84.48％。
将壳聚糖按高分子量：低分子量＝1：2的比例配制成0.2％壳聚糖包囊液，其中，高分子量指分子量在1.00-1.50×107，低分子量指分子量在2×105以下；再将CaCl2加到上述壳聚糖溶液中，调其终浓度为2％，调整pH为5.5；再按重量比海藻酸钠：IgY抗体＝3：4配制IgY与海藻酸钠混合液，并将海藻酸钠浓度稀释为2％；按30滴/分钟的速度，将IgY抗体与海藻酸钠混合液滴加到上述壳聚糖溶液中，作用15分钟，37℃干燥3h即可获得防新冠肺炎的卵黄中和抗体IgY口服胶囊。
实施例6新冠病毒SARS-CoV-2抗体中和试验
在第三方的生物安全三级实验室进行新冠病毒SARS-CoV-2抗体中和试验，具体如下：
将新冠病毒SARS-CoV-2的卵黄中和抗体IgY以20μg/ml为起始浓度，连续2倍比稀释，共计12个梯度(每个稀释度2个复孔)，即20μg/ml，10μg/ml，5μg/ml，2.5μg/ml，1.25μg/ml，0.625μg/ml，0.3125μg/ml，0.15625μg/ml，0.078125μg/ml，0.0390625μg/ml，0.01953125μg/ml，0.009765625μg/ml，作为实验组，以阴性抗体(即免疫前母鸡产的蛋中提取的不含有新冠病毒的IgY)为对照组，对照组阴性抗体以相同浓度倍比稀释；
随后，将上述实验组IgY抗体和对照组阴性抗体，分别与SARS-CoV-2病毒在37℃共孵育1h，孵育完成后加入Vero E6细胞共培养24h。细胞感染24h后弃上清，用无水甲醇-20℃固定20min；预冷PBS洗一遍，加入3％BSA室温封闭2h；加入1：400稀释的康复期病人血清作一抗，4℃过夜孵育；弃上清，预冷PBS洗三次，加入FITC标记抗人IgG抗体室温孵育1h；弃上清，预冷PBS洗一次，DAPI(4′,6-二脒基-2-苯基吲哚)室温染色10min；预冷PBS洗一次，加入100μl显微镜下观察，检测细胞中的阳性克隆数，结果如图2所示。
经细胞计数：对照孔细胞核为51000个，其中，阳性细胞数目为8500个，本发明的IgY抗体在20μg/ml浓度时中和了约97％的病毒感染，对照抗体无明显中和活性。
因此，本发明的新冠病毒SARS-CoV-2卵黄中和抗体IgY在20μg/ml浓度时可中和约97％的新型冠状病毒感染。
序列表
<110> 上海博满生物科技有限公司
<120> 一种用于防治新冠肺炎的SARS-CoV-2卵黄中和抗体IgY喷雾剂
<130> 2011205
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 254
<212> PRT
<213> 新型冠状病毒(SARS-CoV-2)
<400> 1
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Claims (1)
1.一种制备抗新型冠状病毒SARS-CoV-2卵黄中和抗体IgY的方法，其特征在于，包括以下步骤：
（1）以新型冠状病毒SARS-CoV-2 RBD蛋白免疫产蛋母鸡
以新型冠状病毒SARS-CoV-2 RBD蛋白作为抗原，所述新型冠状病毒SARS-CoV-2 RBD蛋白的氨基酸序列如SEQ ID NO .1所示，每次取抗原200μg，加入适合免疫鸡用的佐剂，抗原：佐剂ISA 70的体积比＝30：70，乳化，对产蛋母鸡进行肌肉注射，0.5ml，免疫3次，3点免疫，每次间隔时间为28天；
（2）提取纯化IgY抗体
用蛋黄筛分离免疫母鸡产蛋的蛋黄，加入9倍蒸馏水搅拌混匀制备蛋黄液，用1MolHCl调pH5.2，4℃静置沉淀5小时，8000rpm离心20分钟，弃沉淀，收集上清，在上清中缓慢滴加正辛酸至1％去除杂蛋白和卵磷脂，搅拌混匀；于4℃静置2小时，4℃8000rpm离心30分钟，收集上清液进行超滤浓缩：加入终浓度3％的硅藻土作为助滤剂，采用0.45μm混合纤维素酯膜在0.09MPa下进行真空抽滤，得抽滤液；选择5万分子截留量超滤膜，室温下进料，单段间歇操作模式对抽滤液进行超滤浓缩，得浓缩IgY；巴斯德消毒，0.22μm膜过滤除菌，即为分离纯化的新型冠状病毒SARS-CoV-2卵黄中和抗体IgY，含量为3mg/ml，纯度50％。
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