CN113813378A - 蛋黄igy多克隆抗体中和，抑制新冠病毒covid-19的作用 - Google Patents
蛋黄igy多克隆抗体中和，抑制新冠病毒covid-19的作用 Download PDFInfo
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Abstract
新冠状病毒COVID‑19，N蛋白免疫鸡在鸡蛋黄有IGY多克隆抗体，试验证明这种IGY有中和，抑制新冠状病毒COVID‑19作用，这种来自天然食品作为预防和治疗新冠状病毒，人们容易接受，而且价格便宜操作简单，是一种理想的预防和治疗新冠状病毒COVID‑19的药物或保健食品。
Description
技术领域：医学免疫治疗领域。
背景技术：使用抗原免疫鸡可产生抗体转移和积累于蛋黄中，从蛋黄提取的抗体主要成分是IGY。
IGY理化性质：
1，在65℃一个小时活性不变，可以进行巴氏消毒消灭病原体又可以维持活性
2，在酸性环境PH3-11环境中仍有有比较高的活性。
3，IGY在体外模拟试验对胃蛋白酶，胰蛋白酶处理后仍有活性。
上述内容IgY抗体可作为药用或保健品口服
IgY功能：
重多文献报导IGY对疾病防治和诊断功能的报导。值得提出的著者杜洪桥综述文章，【卵黄抗体IGY在临床医学中的应用】附件一，抗病毒治疗，胡国柱等以灭活SAS-CoV免疫产蛋母鸡用水稀释法获得IGY，研究发现该抗体提取液稀释1∶512时扔可以中和SARS-CoY保护50％的VEROE6的细胞不被SARS-CoV感染。它与SARS患者恢复期血清中和SARS-CoV的效价相同。这说明抗SARS-CoV IGY多克隆抗体可阻断S蛋白与细胞受体结合，抑制SARS-CoV感染VEAOE6细胞。
实验员苗成等：利用大肠癌细胞免疫产蛋母鸡获得IGY，取10只裸鼠在背部注射(种植)大肠癌细胞，分两组，试验组5只鼠口服IGY抗体，另5只为对照组，结果试验组5只裸鼠3只没有肿瘤生长，2只肿瘤生长R1-2mm.对照组在肿瘤细胞注射部位全部生长肿瘤R3-5mm.附件二，说明IGY有抑制肿瘤细胞生长作用。
发明内容：
一、制备N蛋白
1 2019-nCoV
从NCBI上查找到2019-nCov基因序列，GenBank号为NC_045512，序列为1260bp，如下所示，送去南京金斯瑞公司合成，经过密码子优化，通过限制性内切酶EcoRI和XhoI构建至pET-28a表达载体上，记为N蛋白。
2019-nCoV原始序列(1260bp)：
N蛋白氨基酸序列：
2转化大肠杆菌感受态细胞BL21
合成后是质粒干粉4ug。加入40uL TE Buffer溶解为100ng/uL。转化至大肠杆菌感受态细胞BL21，涂平板培养过夜。挑取单菌落摇菌，菌液鉴定正确后即可进行蛋白表达。
3 N蛋白的表达和纯化
3.1 N蛋白的表达
取鉴定正确的菌液在小瓶中进行活化，过夜培养，第二天转入大瓶摇菌，生长处于对数期，OD600为0.6～0.8时，加入终浓度为0.01mM的IPTG诱导表达目的蛋白，诱导过夜培养，离心9000rpm，10min，收集菌体，-20度冰箱保存备用。
3.2 N蛋白的纯化
称取菌体，按1∶10加入破菌液进行超声破碎，同时实验，确定N蛋白表达在上清。超声破碎后离心收集上清，过镍柱纯化，梯度浓度咪唑缓冲液洗脱目的蛋白，根据SDS-PAGE凝胶电泳胶图，选取洗脱纯度和浓度都较高的洗脱样品，透析出去咪唑，测浓度后保存备用。
N蛋白氨基酸序列：
2转化大肠杆菌感受态细胞BL21
合成后是质粒干粉4ug。加入40uL TE Buffer溶解为100ng/uL。转化至大肠杆菌感受态细胞BL21，涂平板培养过夜。挑取单菌落摇菌，菌液鉴定正确后即可进行蛋白表达。
3 N蛋白的表达和纯化
3.1 N蛋白的表达
取鉴定正确的菌液在小瓶中进行活化，过夜培养，第二天转入大瓶摇菌，生长处于对数期，OD600为0.6～0.8时，加入终浓度为0.01mM的IPTG诱导表达目的蛋白，诱导过夜培养，离心9000rpm，10min，收集菌体，-20度冰箱保存备用。
3.2 N蛋白的纯化
称取菌体，按1∶10加入破菌液进行超声破碎，同时实验，确定N蛋白表达在上清。超声破碎后离心收集上清，过镍柱纯化，梯度浓度咪唑缓冲液洗脱目的蛋白，根据SDS-PAGE凝胶电泳胶图，选取洗脱纯度和浓度都较高的洗脱样品，透析出去咪唑，测浓度后保存备用。
二、N蛋白免疫及纯化
取N蛋白免疫产蛋母鸡。
每只母鸡N蛋白2mg加完全佐剂加大肠杆菌不耐热蛋白混合，注射鸡的翅膀皮下，10-15天进行第二次加不完全佐剂免疫，50-60天取蛋纯化。纯化方法使用水纯化法，获得IGY抗体每个蛋产率100mg左右
三、亲和力鉴定
1，ELISA方法：检测N蛋白免疫IGY活性8ng/ml，1，6ng/ml。附件三。ELSA检测报告。
2，新冠状病毒COVID-19抗体检测胶体金方法，原理是人体感染新冠状病毒COVID-19血液中出现IgM，IgG抗体，使用N蛋白标记金，硝酸纤维膜包被IgM，IgG抗抗体。人体被新冠状病毒covid-19感染的IgM，IgG与N蛋白有亲和力与N蛋白结合也标上金颗粒，层析作用达到硝酸纤维膜上与IgM，IgG抗抗体结合出现红线为阳性。证明N蛋白与新冠状病毒covid-19有同源性亲和力。。附件四，
四，综合上述N蛋白免疫禽类获得卵黄IGY多克隆抗体，服用有预防，治疗新冠状病毒COVID-19的作用。
Claims (4)
1.新冠状病毒COVID-19，N蛋白免疫产蛋母鸡，获得卵黄IGY多克隆抗体有中和，抑制新冠状病毒COVID-19作用，能够预防治疗新冠状病毒COVID-19。
2.根据权利1，N蛋白作为免疫原，灭活新冠状病毒COVID-19，和从新冠状病毒COVID-19提取或重组同源性所有的蛋白质如S蛋白都属于免疫原。
3.根据权利1，鸡属于禽类，凡是禽类都可以免疫产生卵黄IGY。
4.免疫佐剂大肠杆菌不耐热蛋白有强化免疫的作用。
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| US20030185856A1 (en) * | 2001-01-05 | 2003-10-02 | Nam-Hyung Lee | Method for the production of the egg containing anti-pathogenic bacteria specific antbodies(igy) and the yogurt and ice cream containing the igy |
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Application publication date: 20211221 |