CN111777684A - 冠状病毒串联表位蛋白所诱导抗体的制备方法及其应用 - Google Patents
冠状病毒串联表位蛋白所诱导抗体的制备方法及其应用 Download PDFInfo
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Abstract
本发明公开了冠状病毒串联表位蛋白所诱导抗体的制备方法及其应用，所述蛋白的核苷酸序列如SEQ：ID：1所示，其氨基酸序列如SEQ：ID：2所示。还提供了一种本发明所述冠状病毒串联表位蛋白所诱导抗体在治疗冠状病毒引起的人类呼吸道疾病药物制备过程中的应用。本发明的蛋白包含SARS‑CoV、MERS‑CoV与SARS‑CoV‑2三种冠状病毒之Spike蛋白保守区域以及串联表位。用所述串联表位蛋白免疫产蛋母鸡后，从收获蛋黄中提取卵黄抗体，将所制备的卵黄抗体制备成抗冠状病毒感染的卵黄抗体喷雾制剂，通过向口腔及鼻腔喷雾途径以阻挡冠状病毒的感染，该药物能够在体外与所述冠状病毒S蛋白具有良好的结合能力。
Description
技术领域
本发明属于生物制药技术领域，涉及一种冠状病毒串联表位蛋白所诱导抗体的制备方法及其应用，具体地说，涉及一种抗SARS-COV-2冠状病毒感染的卵黄抗体喷雾制剂和口服制剂的制备方法及其应用。
背景技术
β-冠状病毒与2003年SARS-COV病毒存在79.5％的同源序列，但与MERS-CoV和SARS-CoV有所不同，它是感染人类冠状病毒家族中第七个成员。临床症状表现为呼吸困难、四肢无力、发热、干咳等，在较严重病例中，可导致肺炎、严重急性呼吸综合征、肾衰竭，甚至死亡。2020年1月12日世界卫生组织(WHO)将此暂命名为2019新型冠状病毒(2019-nCoV)。2020年2月7日，国家卫建委将2019-nCoV引起的新型肺炎暂命名为“新型冠状病毒肺炎”，Novel Coronavirus Pneumonia(简称NCP)。2020年2月11日 WHO宣布将“新型冠状病毒感染肺炎”命名为“COVID-19”，与此同时，国际病毒分类委员会声明，将新型冠状病毒命名为“SARS-CoV-2”(Severe Acute Respiratory Syndrome Coronavirus 2)。目前2019-nCoV病毒引起的新型肺炎并无特异性治疗药物。
卵黄抗体IgY是指通过免疫母鸡，收集免疫后母鸡鸡蛋提取卵黄中IgY抗体。相对单克隆抗体和多克隆抗体，卵黄抗体具备制备简便，产量极高，成本低廉，持续性获取，研发周期短等优点。在养殖行业，IgY目前被认为是一种有望成为抗生素替代的生物制品。由于IgY不含有与补体结合的特点，国内外众多科学家将其IgY直接应用于免疫学诊断及抗体药物领域。如利用人肠病毒71型(EV71)灭活苗免疫接种母鸡，收集提取特异性的卵黄抗体，通过口服或者腹腔注射免疫后的IgY均能降低实验动物的发病率和死亡率。 Junlin Wen制备特异性IgY处理后荷毒小鼠，使其体内病毒显著减少。Kassima等发现抗弧菌特异性IgY抗体在体内外均能够发挥明显的抑菌效果，揭示IgY可作为一种抗生素替代或者口服免疫治疗药物。日本Yasuhiro Tsukamoto通过免疫雌性鸵鸟产生抗鸡传染性支气管炎病毒(IBV)的IgY，发现纯化后的IgY具有很强的中和作用，注射该IgY有助于保护受感染IBV的幼雏。我国学者Chao-Yang Fu等利用卵黄抗体技术研制抗SARS-COV 病毒IgY特异性抗体，在VERO上中和病毒效价为1:640，进一步揭示卵黄抗体技术在由冠状病毒引起的人类呼吸道疾病方面的应用前景。
IgY与病原体相互作用的确切机制尚在进一步研究中，目前比较公认以下几种机制包括凝集细菌、抑制病原体粘附，免疫调理，中和毒素。其中，抑制粘附的主要机制被认为是IgY最基本也是最重要的机制。尤其是针对肠道、呼吸道粘膜，特异性IgY抗体通过防止病原体附着在肠道和呼吸道，进而阻断与粘膜上皮细胞受体相互作用、干扰粘蛋白结合，还可以中和诸如膜蛋白、脂多糖、菌毛等因子。目前，我国上市销售的卵黄抗体产品公司有江西方信动物药业有限公司、广州市亚牧医药科技有限公司、杭州华扬奥博生物技术有限公司、南京科农生物技术研究所、香港新亚国际药业集团公司等公司。上述公司开发销售的卵黄抗体主要集中于猪类、禽类、水产类以及人类相关疾病的防治方面。湖南安邦制药有限公司主持的“卵黄抗体技术(IgY系列产品)深度开发”曾列为长沙市的重点项目之一，同时该公司引进国外IgY生物抗体技术，生产妇科用“瑞丽女人抑菌丹”第二代产品，开创了国内首家应用IgY生物制剂的绿色疗法治疗预防妇科疾病。
现有技术中，目前还没有关于抗SARS-COV-2冠状病毒感染的有效药物的相关报道。
发明内容
本发明的目的在于提供一种冠状病毒串联表位蛋白所诱导抗体的制备方法及其应用，得到可与SARS-CoV、MERS-CoV与SARS-CoV-2共3种冠状病毒发生抗原抗体反应的冠状病毒多价卵黄抗体。特别是由体外真核表达所述三种冠状病毒串联表位蛋白，该蛋白包含SARS-CoV、MERS-CoV与SARS-CoV-2三种冠状病毒之Spike蛋白保守区域以及串联表位。用所述串联表位蛋白免疫产蛋母鸡后，从收获蛋黄中提取卵黄抗体，将所制备的卵黄抗体制备成抗冠状病毒感染的卵黄抗体喷雾制剂，通过向口腔及鼻腔喷雾途径以阻挡冠状病毒的感染；也可通过口服途径以阻挡冠状病毒的感染。
其技术方案如下：
一种冠状病毒串联表位蛋白，对SARS-CoV、MERS-CoV、SARS-CoV-2病毒进行生物信息学分析，通过人工合成包含所述冠状病毒的主要免疫保护性抗原核酸序列的重组表达，将真核重组表达载体转染细胞并体外真核表达冠状病毒串联表位蛋白。其核苷酸序列如SEQ：ID：1所示，其氨基酸序列如SEQ：ID：2所示。
一种冠状病毒串联表位蛋白所诱导抗体的制备方法，包括以下步骤：
利用人工合成的冠状病毒环病毒串联表位DNA片段构建真核表达载体，然后将重组真核表达载体转化表达细胞，培养后将培养物经纯化即得冠状病毒串联表位蛋白。
进一步，所述真核表达载体为pCDNA3.1载体，优选质粒pSecTag2A-S1-6His，更优选构建并筛选pSecTag2A-S1-6His/CHO-k稳定表达细胞株。
进一步，所述表达细胞为VERO细胞，优选为HEK293T细胞。
进一步，所述培养物的纯化方法为：重组CHO-k细胞在5L、30L规格的生物反应器微载体发酵，收集细胞培养液，4℃、5000rmp条件下离心10min，取上清液于100000g、 4℃条件下离心2小时，沉淀用HNE buffer悬浮，HNE buffer的组成为25mM Tris-HCl pH 7.4，150mMNaCl，5mM EDTA；然后将悬液进行蔗糖梯度离心，蔗糖采用HNE buffer 配制，体系采用0.3g/mL蔗糖3mL，0.45g/mL蔗糖3mL，1.37g/mL蔗糖1mL，然后与4℃、 100000g条件下离心2h，收集中间层，并用折射仪测量密度；用pH6.5-7.5的PBS平衡 Sepharose6FFTM介质进行凝胶层析纯化，收集外水体积流穿峰；再采用DEAE- SepharoseFFTM介质进行离子交换层析，平衡液为pH6.5-7.5、含有0.05-0.15M氯化钠的 PBS，洗脱液为含0.2-0.5M氯化钠、pH6.5-7.5的PBS，纯化后液体即为冠状病毒串联表位蛋白溶液。
一种本发明所述冠状病毒串联表位蛋白所诱导抗体在治疗冠状病毒引起的人类呼吸道疾病药物制备过程中的应用。
进一步，所述治疗冠状病毒引起的人类呼吸道疾病药物为抗冠状病毒感染的卵黄抗体的喷雾制剂和口服制剂。
进一步，所述应用包括以下步骤：
步骤1：利用所述冠状病毒串联表位蛋白人工免疫蛋鸡；
步骤2：从收获的鸡蛋中提取卵黄抗体，经过纯化与精制后得到冠状病毒多价卵黄抗体粉末；
步骤3：按冠状病毒多价卵黄抗体粉末1％、芳樟醇0.01％、香叶醇0.01％、透明质酸 1％、磺化木质素0.3％、表面活性剂0.02％的比例溶于无菌水中，用柠檬酸调节pH值为6.5，然后0.2微米微孔滤膜过滤分装，即得抗冠状病毒感染的卵黄抗体喷雾制剂。
步骤4：按冠状病毒多价卵黄抗体粉末1％、甲基丙烯酸和丙烯酸乙酯按1:1形成的聚阴离子共聚物，该聚合物在pH>5.5时才会溶解，是优良的肠溶片包衣材料，以此制备抗冠状病毒多价卵黄抗体的口服制剂。
再进一步，步骤1中，所述人工免疫蛋鸡的具体步骤为：挑选6月龄产蛋母鸡，肌肉多点注射共1mL浓度为1mg/mL的冠状病毒串联表位蛋白溶液。共3次免疫，每次间隔 1周。末次28天后开始收集鸡蛋。
再进一步，步骤2中，所述提取卵黄抗体的步骤具体为：收集大约100个鸡蛋的蛋黄，按蛋黄液和去离子水1:7的比例加入去离子水，用玻璃棒搅拌均匀。调节蛋黄液的pH到5.00，放在4℃条件下静置过夜。待蛋黄液充分溶解后，将溶液放在7000rpm，4℃条件下离心10min,，取上清液。按照每9％(W/V)比例加入NaCl，再次调节溶液pH＝4，静置 2h。将盐析溶液按7000rpm，4℃条件下离心10min,去除上清，收集蛋白沉淀。将蛋白沉淀用适量PBS溶液溶解，4℃条件下用离子水透析过夜，之后真空冷冻干燥，即得冠状病毒多价卵黄抗体粉末。
本发明的有益效果：
本发明人工合成含有SARS-CoV、MERS-CoV、与SARS-CoV-2三种冠状病毒之Spike蛋白保守区域以及它们的串联表位的蛋白，并利用其作为抗原免疫母鸡。从收获的鸡蛋中分离卵黄抗体制备用于防止冠状病毒感染的喷雾制剂和口服制剂。本发明的喷雾制剂能够通过鼻腔、口腔喷雾并降低病毒对吸附呼吸道上皮细胞的几率，本发明的口服制剂能够通过口服避免胃酸破坏并降低病毒对肠道上皮细胞的几率，同时可通过IgY结合冠状病毒从而激发机体“抗体依赖性细胞介导的细胞毒作用(ADCC作用)，发挥免疫治疗作用。本发明的抗三种冠状病毒感染的卵黄抗体喷雾制剂、口服制剂能够在体外与所述冠状病毒S 蛋白具有良好的中和能力。
具体实施方式
下面结合具体实施例对本发明的技术方案作进一步详细地说明。
以下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)中所述的条件，或按照制造厂商所建议的条件实施。
实施例1细胞培养及细胞批库建立
VERO细胞、HEK293T细胞均来源于美国模式培养物集存库(American TypeCulture Collection，ATCC)。培养基采用DMEM培养基(Dulbecco's modified Eaglemedium)，培养基中添加100U/mL青霉素及100μg/mL链霉素，然后加入体积分数为2-10％的胎牛血清，然后在细胞工厂或细胞发酵罐进行培养，建立细胞工作种子批库，并对传代后的外源因子、致瘤性及稳定性等全面检定，细胞使用代数控制在60代以内，均符合生物制品生产介质的要求。
实施例2质粒转染及稳定高表达细胞系筛选与建库
选用实施例1建立的VERO细胞为靶细胞，优化靶细胞为HEK293T细胞。采用脂质体法将人工合成的重组真核表达载体转染VERO细胞，然后筛选稳定高表达的细胞系。具体步骤如下：1)在35mm孔中，加入重组真核表达载体1.5μg；2)加入5μL OptiMEM配置的脂质体，室温孵育45min。然后加入到被OptiMEM润洗过的宿主细胞中，孵育5h；③离心去除载体-脂质体混合物，并向细胞中加入DMEM进行培养；3)通过加压筛选表达冠状病毒串联表位蛋白的细胞株。
实施例3抗三种冠状病毒多价卵黄抗体效价测定
将所述三种冠状病毒串联表位蛋白作为包被蛋白、将真空冷冻干燥的冠状病毒多价卵黄抗体粉末稀释后作为待检抗体，通过ELISA方法测定冠状病毒多价卵黄抗体效价。具体步骤如下：1)将包被蛋白用包被缓冲液稀释至10μg/mL，抗原，每孔加100μL到酶标板内，4℃下放置过夜。2)将孔内液体液倒去，每孔加入100mL PBST洗涤液，等待3 min，倒去液体，使劲拍打，重复5次。3)每孔加入200μL封闭液(5％脱脂奶粉，PBS 溶液配)，37℃条件下放置1.5h后，按上述方法洗涤并拍干。4)用PBST溶液配制1％(W/V) 的冠状病毒多价卵黄抗体溶液，之后继续用PBST溶液进行1:100倍梯度稀释。5)将不同稀释梯度的冠状病毒多价卵黄抗体溶液加入到酶标板中，37℃孵育1h。之后洗涤拍干并加入酶标二抗，继续37℃孵育1h。6)洗涤拍干后，加入辣根过氧化物酶底物，显色后通过酶标仪读取吸光度。7)计算各稀释倍数下的P/N值，以阴性对照2.1倍所在的稀释倍数，作为冠状病毒多价卵黄抗体的滴度。
实验例4抗三种冠状病毒感染的卵黄抗体喷雾制剂和口服制剂的检定
抗三种冠状病毒卵黄抗体喷雾制剂和口服制剂的外观应均一，无异味和沉淀。通过 Lorry法测定蛋白含量1.0-2.0mg/mL范围内。通过ELISA方法检测抗体滴度应不小于1：3200。接种硫乙醇酸盐培养基、营养琼脂斜面培养基和改良马丁培养基培养14d，并以无菌生理盐水做阴性对照，培养温度为25℃、35℃。结果应无细菌生长。接种半流体和肉汤培养基，37℃初代培养21天，次代培养21天，无菌生理盐水做阴性对照，应无支原体生长。
实验例5抗三种冠状病毒感染的卵黄抗体喷雾制剂和口服制剂的过敏原检测
取0.5mL卵黄抗体喷雾制剂或者口服制剂，皮下接种体重为250～350g豚鼠，每个样品接种豚鼠5只，每只接种0.5mL，并用人血白蛋白和生理盐水作为阳性、阴性对照。注射后30分钟开始观察，持续观察3天。观察期间内应无鼻痒、喷嚏、烦躁不安、呼吸困难、休克、痉挛甚至死亡等过敏症状。
实施例6抗三种冠状病毒感染的卵黄抗体喷雾制剂和口服制剂的效力检验
为了验证本发明的抗三种冠状病毒卵黄抗体喷雾制剂和口服制剂对SARS-CoV、MERS-CoV、SARS-CoV-2冠状病毒的抑制效果，我们进行了体外抗原结合能力进行验证。
将三种冠状病毒串联表位蛋白作为包被蛋白、将真空冷冻干燥的冠状病毒多价卵黄抗体粉末稀释后作为待检抗体，通过ELISA方法测定冠状病毒多价卵黄抗体效价。具体步骤如下：1)将包被蛋白用包被缓冲液稀释至10μg/mL，抗原，每孔加100μL到酶标板内，4℃下放置过夜。2)将孔内液体液倒去，每孔加入100mL PBST洗涤液，等待3min，倒去液体，使劲拍打，重复5次。3)每孔加入200μL封闭液(5％脱脂奶粉，PBS溶液配)，37℃条件下放置1.5h后，按上述方法洗涤并拍干。4)用PBST溶液配制1％(W/V) 的冠状病毒多价卵黄抗体溶液，之后继续用PBST溶液进行1:100倍梯度稀释。5)将不同稀释梯度的冠状病毒多价卵黄抗体溶液加入到酶标板中，37℃孵育1h。之后洗涤拍干并加入酶标二抗，继续37℃孵育1h。6)洗涤拍干后，加入辣根过氧化物酶底物，显色后通过酶标仪读取吸光度。7)计算各稀释倍数下的P/N值，以阴性对照2.1倍所在的稀释倍数，作为冠状病毒多价卵黄抗体的滴度。试验结果显示本发明制备的抗多种冠状病毒感染的卵黄抗体喷雾制剂或口服制剂与SARS-CoV、MERS-CoV、SARS-CoV-2、HCoV-229E、 HCoV-NL63、HCoV-OC43、HCoV-HKU1七种冠状病毒的S蛋白的结合效价如表1中所示。
表1
| 抗原 | 效价 |
| S蛋白(SARS-CoV) | 1：6400 |
| S蛋白(MERS-CoV) | 1：3200 |
| S蛋白(SARS-CoV-2) | 1：6400 |
实验结论：本发明一种抗三种冠状病毒感染的卵黄抗体喷雾制剂和口服制剂能够在体外与所述冠状病毒S蛋白具有良好的结合能力。
以上所述，仅为本发明较佳的具体实施方式，本发明的保护范围不限于此，任何熟悉本技术领域的技术人员在本发明披露的技术范围内，可显而易见地得到的技术方案的简单变化或等效替换均落入本发明的保护范围内。
Claims (10)
1.一种冠状病毒串联表位蛋白，其特征在于：其核苷酸序列如SEQ：ID：1所示，其氨基酸序列如SEQ：ID：2所示。
2.一种权利要求1所述冠状病毒串联表位蛋白所诱导抗体的制备方法，其特征在于：包括以下步骤：
利用人工合成的冠状病毒环病毒串联表位DNA片段构建真核表达载体，然后将重组真核表达载体转化表达细胞，培养后将培养物经纯化即得冠状病毒串联表位蛋白。
3.根据权利要求2所述冠状病毒串联表位蛋白所诱导抗体的制备方法，其特征在于：所述真核表达载体为pCDNA3.1载体。
4.根据权利要求2所述冠状病毒串联表位蛋白所诱导抗体的制备方法，其特征在于：所述表达细胞为VERO细胞。
5.根据权利要求2所述冠状病毒串联表位蛋白所诱导抗体的制备方法，其特征在于：所述培养物的纯化方法为：收集细胞培养液，4℃、5000rmp条件下离心10min，取上清液于100000g、4℃条件下离心2小时，沉淀用HNE buffer悬浮，HNE buffer的组成为25 mM Tris-HCl pH 7.4，150 mM NaCl，5 mM EDTA；然后将悬液进行蔗糖梯度离心，蔗糖采用HNEbuffer配制，体系采用0.3g/mL蔗糖3mL，0.45 g/mL蔗糖3mL，1.37 g/mL蔗糖1mL，然后与4℃、100000g条件下离心2h，收集中间层，并用折射仪测量密度；用pH6.5-7.5的PBS平衡Sepharose6FFTM介质进行凝胶层析纯化，收集外水体积流穿峰；再采用DEAE-SepharoseFFTM介质进行离子交换层析，平衡液为pH6.5-7.5、含有0.05-0.15M氯化钠的PBS，洗脱液为含0.2-0.5M氯化钠、pH6.5-7.5的PBS，纯化后液体即为冠状病毒串联表位蛋白溶液。
6.一种权利要求1所述冠状病毒串联表位蛋白所诱导抗体在治疗冠状病毒引起的人类呼吸道疾病药物制备过程中的应用。
7.根据权利要求6所述的应用，其特征在于，所述治疗冠状病毒引起的人类呼吸道疾病药物为抗冠状病毒感染的卵黄抗体喷雾制剂和口服制剂。
8.根据权利要求6所述的应用，其特征在于，所述应用包括以下步骤：
步骤1、利用所述冠状病毒串联表位蛋白人工免疫蛋鸡；
步骤2、从收获的鸡蛋中提取卵黄抗体，经过纯化与精制后得到冠状病毒多价卵黄抗体粉末；
步骤3、按冠状病毒多价卵黄抗体粉末1%、芳樟醇0.01%、香叶醇0.01%、透明质酸1%、磺化木质素0.3%、表面活性剂0.02%的比例溶于无菌水中，用柠檬酸调节pH值为6.5，然后0.2微米微孔滤膜过滤分装，即得抗冠状病毒感染的卵黄抗体喷雾制剂；
步骤4、按冠状病毒多价卵黄抗体粉末1%、甲基丙烯酸和丙烯酸乙酯按1 : 1形成的聚阴离子共聚物，该聚合物在pH > 5.5时才会溶解，是优良的肠溶片包衣材料，以此制备抗冠状病毒多价卵黄抗体的口服制剂。
9.根据权利要求8所述的应用，其特征在于，步骤1中，所述人工免疫蛋鸡的具体步骤为：挑选6月龄产蛋母鸡，肌肉多点注射共1 mL浓度为 1mg/mL的冠状病毒串联表位蛋白溶液；共3次免疫，每次间隔1周；末次28天后开始收集鸡蛋。
10.根据权利要求8所述的应用，其特征在于，步骤2中，所述提取卵黄抗体的步骤具体为：收集100个鸡蛋的蛋黄，按蛋黄液和去离子水1:7的比例加入去离子水，用玻璃棒搅拌均匀；调节蛋黄液的pH到5.00，放在4℃条件下静置过夜；待蛋黄液充分溶解后，将溶液放在7000 rpm，4℃条件下离心10 min,，取上清液；按照每9%比例加入NaCl，再次调节溶液pH=4，静置2 h；将盐析溶液按7000 rpm，4℃条件下离心10 min,去除上清，收集蛋白沉淀；将蛋白沉淀用适量PBS溶液溶解，4℃条件下用离子水透析过夜，之后真空冷冻干燥，即得冠状病毒多价卵黄抗体粉末。
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