CN112724247A - 一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用 - Google Patents
一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用 Download PDFInfo
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Abstract
本发明公开一种SARS‑CoV‑2冠状病毒IgA抗体，一种SARS‑CoV‑2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用，包括下列技术措施：1）根据SARS‑CoV‑2冠状病毒侵入人体后自身产生的所有抗体全长序列信息与未感染SARS‑CoV‑2冠状病毒者体内抗体序列进行比对分析；2）并进行了原核密码子优化，并将pET32a—IgA质粒转化至E.coli BL21(DE3)感受态中，在E.coliBL21(DE3)菌株中高效表达并纯化，最后获得纯化的IgA蛋白。3）鉴定人群是否感染SARS‑CoV‑2冠状病毒或者检测人血清中是否含有SARS‑CoV‑2冠状。具有灵敏度高，特异性好以及纯度高等特点,本发明的ELISA试剂盒灵敏度较高，特异性较强，操作简单，检测时间短,临床实用性强。
Description
技术领域
本发明涉及基因工程技术领域，具体一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用。
背景技术
冠状病毒(Coronaviruses, Co Vs)属冠状病毒科(Coronaviridae), 其下包括4属： Alpha Coronavirus(a—CoV)、Beta Coronavirus(f3—CoV)、Gamma Corona virus(y—CoV)、 Delta Coronavirus(o—CoV)。目前为止，已知的人类冠状病毒共有六种。其中两种冠状病毒传染率高、病死率高，分别是导致2003年于中国广东省暴发SARS、2012年于沙特暴发MERS的病原体严重急性呼吸综合征冠状病毒(SARS—CoV)和中东呼吸综合征冠状病毒(MERS—CoV),它们均属于B冠状病毒。MERS症状通常包括发热、咳嗽和呼吸急促，甚至发展为肺炎，病死率约为34.4%。SARS症状通常包括发热、畏寒和身体疼痛，甚至发展为肺炎，病死率约为9.6%。
发明内容
为了解决上述问题，本发明公开了一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用。
本发明的技术方案为：一种SARS-CoV-2冠状病毒IgA抗体， IgA抗体蛋白其序列号为
MNWVRQAPGEGLEWLSYISTSSNNIFYADSVKGRFTVSRDNAKNSLYLQMNSLRDEDTAVYYCAGHTGSNWFDYWGQGTLVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY*。
一种SARS-CoV-2冠状病毒IgA抗体及其在ELISA检测试剂盒中的应用，包括下列技术措施：
1）根据SARS-CoV-2冠状病毒侵入人体后自身产生的所有抗体全长序列信息与未感染SARS-CoV-2冠状病毒者体内抗体序列进行比对分析，比对出抗SARS-CoV-2冠状病毒特异性抗体IgA全长序列；
2）并进行了原核密码子优化，优化后的序列为SEQ ID N0.2所示，嵌入pET32a质粒中、合成了pET32a-IgA原核表达质粒，并将pET32a—IgA质粒转化至E.coli BL21 (DE3)感受态中，在E.coliBL21(DE3)菌株中高效表达并纯化，最后获得纯化的IgA蛋白。
3）鉴定人群是否感染SARS-CoV-2冠状病毒或者检测人血清中是否含有SARS-CoV-2冠状。
本发明的有益之处：1、本发明使用的抗体为抗SARS-CoV-2冠状病毒特异性抗体IgA，优化密码子后，嵌入到原核载体pet32a的重组抗原pET32a—IgA,该抗体为可溶性抗体并且经诱导表达纯化后，具有灵敏度高，特异性好以及纯度高等特点。同时，pET32a—IgA具有免疫原性，因此是可作为检测试剂盒的包被抗体。
2、本发明制备的ELISA抗原检测试剂盒能够准确检测样品中是否含有SARS-CoV-2冠状病毒抗原，本发明的ELISA试剂盒灵敏度较高，特异性较强，操作简单，检测时间短。该试剂盒用样品0D630nm/阳性对照0D630nm (S/P) 来判定样品是否为阳性，最终确定：当待检样品的S/P大于或等于0.25,则判为SARS-CoV-2冠状病毒抗原阳性；当S/P<0.25,则判为SARS-CoV-2冠状病毒抗原阴性。本发明的ELISA试剂盒灵敏度较高，特异性较强，操作简单，检测时间短。
3、抗SARS-CoV-2冠状病毒特异性抗体IgA全长蛋白表达于上清且表达量高以及临床实用性强。
具体实施方式
为了加深对本发明的理解，下面详细描述本发明的具体实施方式，该实施例仅用于解释本发明，并不构成对本发明的保护范围的限定。
实施例1：SARS-CoV-2冠状病毒IgA抗体蛋白的获得：
1.重组质粒的获得
对抗SARS-CoV-2冠状病毒特异性抗体IgA全长序列进行PCR靶向扩增，同时将扩增的IgA完整序列嵌入pET32a载体中，获得重组质粒pET32a-IgA。将重组质粒pET32a—IgA转化至E.coliBL21 (DE3) 感受态中，获得重组菌pET32a-IgA。
2.重组蛋白的表达与纯化
挑取单菌落接种于5ml含50µg/ml氨苄的LB液体培养基中，37℃，200r/min振荡培养8小时,再按1%的比例将菌液接种于400ml含50µg/ml氨苄的LB液体培养基中，37℃，200r/min振荡培养至菌液OD 600值为0.5时，加入IPTG至终浓度为0.8mmol/L, 诱导表达3小时，离心收集菌体,收集的菌体，用原培养峈1/10体积的BindingBuffer重悬菌体，在低温高压破碎仪中破碎菌体，重复破碎3次，菌体裂解液lOOOOr/min,离心15分钟，收集上清。用0.22µm滤器过滤收集的上清，2℃保存备用。
亲和层析纯化具体步骤为：首先，用5个柱体积的Binding Buffer平衡层析柱，然后上样，上样结束后再用10个柱体积的Binding Buffer平衡层析柱,然后，用ElutionBuffer进行洗脱，观察仪器屏幕上出现蛋白峰时开始收集，直至峰结束后停止收集，整个过程保持液体流速为1.O ml/min, 收集的蛋白用TE缓冲液透析36小时，每隔12小时换1次透析液，透析完成后收集抗体蛋白，并进行SDS- PAGE分析，同时经Image J软件计算分析蛋白纯度，所得蛋白纯度约为95%, 蛋白的氨基酸序列为SEQ ID NO.l所示；超微量核酸蛋白检测仪测定蛋白浓度，所得蛋白浓度约为l.88 mg/mL。
3.重组蛋白pET32a—IgA的免疫原性检测
为了进一步验证所得的IgA全长蛋白的免疫原性，进行Western-blot检测以表明IgA全长蛋白具有生物学活性。
实施例2：SARS-CoV-2冠状病毒IgA抗体蛋白构建ELISA检测试剂盒
1.阳性对照的制备
采集SARS-CoV-2冠状病毒感染中后期患者血液，待血液充分凝固后，4000r/min离心10分钟，分离血清，60 ℃灭活30分钟，加入终浓度为0.01%的硫柳汞钠，置气-70 ℃以下保存备用。将制备的血清用保护剂稀释10倍，0.22μm滤器过滤除菌，即为阳性对照。
2.阴性对照的制备
采集未感染过SARS-CoV-2冠状病毒的健康人血液，待血液充分凝固后，4000r/min离心10分钟，分离血清，60 ℃灭活30分钟，加入终浓度为0.01%的硫柳汞钠，置气-70 ℃以下保存备用。将制备的血清用保护剂稀释10倍，0.22μm滤器过滤除菌，即为阴性对照。
3.酶标板的准备
通过方阵法确定抗体的包被浓度为1:5000, 在2℃包被14小时。弃去包被液，加入1%牛血清白蛋白(BSA) 作为封闭液37℃封闭2小时。弃去封闭液，自然风干后抽真空包装成成品试剂盒酶标板。
4.试剂盒其他组分的配备
包被液：碳酸钠(Na2C03) 1. 59g、碳酸氢钠(NaHC03) 2.93g, 加注射用水至1000ml, 调pH值至9.6, 置2-8℃保存备用。
封闭液：牛血清白蛋白(BSA) 5 . Og、庶糖10.0g、氯化钠(NaCl) 8. 5g、Tween-200.5ml、硫柳汞钠0.10g, 加注射用水至1000ml, 置2-8℃保存备用。
保护剂：取氯化钾(KCl) 0.2g、氯化钠(NaCl) 8.Og、磷酸二氢钾(KH2P04) 0.27g、十二水磷酸氢二钠(Na2HP04·12H20) 1.42g、牛血清白蛋白(BSA) 5.Og、硫柳汞钠0.lg、吐温20(Tween-20) 0.5ml、庶糖2.0g, 加注射用水至1000ml, 置2-8°C保存备用。
样品稀释液：氯化钠(NaCl) 8.Og、十二水磷酸氢二钠(Na2HP04·12H20)2.9g、磷酸二氢钾(KH2P04)0.2g、氯化钾(KCl) 0.2g、硫柳汞钠O.lg,加注射用水至1000ml。
20倍浓缩洗涤液：吐温20 (Tween-20) 10.0ml, 氯化钠(NaCl) 160.0g 、十二水磷酸氢二钠(Na2HP04·12H20)58.0g、磷酸二氢钾(KH2P04)4.Og 、氯化钾(KCl) 4.Og, 加注射用水至1000ml。
底物显色液：拧檬酸盐—磷酸盐缓冲液(pH 5. 0) 配制0.lmol/L柠檬酸盐(C6H8O7•H2O) 和0.2mol/L磷酸盐(Na2HP04·12H20)按6.1ml : 6.4ml混合配制而成,TMB贮存液配制TMB溶于二甲基亚枫(DMSO) , 终浓度为32mmol/L,取TMB贮存液用柠檬酸盐—磷酸盐缓冲液作1:20倍稀释，再加入终浓度7.5mmol/L聚乙二醇(PEG) 、lOOmmol/L葡萄糖和2.94mmol/L H2O2，完全溶解后，避光保存，置2-8℃保存备用。
终止液：2.5ml氢氟酸(HF) 加到900ml去离子水中，定容至1000ml, 分装，lOml/瓶，28℃保存备用。
实施例3：SARS-CoV-2冠状病毒IgA抗体蛋白制备的ELISA检测试剂盒的使用方法：
样品处理：取全血，待血液凝固后4000转/分钟离心10分钟，收集上清。也可采集血液，待凝固后自然析出血清，要求血清清亮，无溶血。
洗涤液配制：使用前，将浓缩的洗涤液从试剂盒中取出，平衡至室温(20℃) ,并摇动，使沉淀溶解（最好在37℃水浴中加热5分钟），然后用蒸馏水作20倍稀释，混匀，稀释好的洗涤液在2℃可以存放7 日。
样品初步稀释：将待检血清样品在血清稀释板中按1:40的比例稀释（例如：在血清稀释板中先加入195μ1样品稀释液，再加5μ1待检血清），阳性对照和阴性对照在血清稀释板中按1:4倍稀释（如：180μ1样品稀释液中加60μ1阳性对照或阴性对照），不同的样品要注意换吸头,样品在稀释过程中要充分混匀。
取适量的抗体包被板，每孔加入200μ1 洗涤液，洗涤1 次，拍干。再将稀释好的待检血清、阳性对照和阴性对照各取100µl加至抗体包被板中，待检血清设1孔，阳性对照和阴性对照各设2孔，置37℃温育30分钟。
2.弃去孔中液体，匈孔加入200μl洗涤液，重复洗涤5次，最后一次拍干。
3.每孔加入100μ1 酶标记物，置37℃温育30分钟。
4.弃去孔中液体，洗涤方法同步骤2。
5.每孔加入100µ1底物显色液，置20-25℃避光显色10分钟。
6.每孔加入50µ1终止液，10分钟内读取0D630nm值。
试验成立条件是：阳性对照0D630nm平均值与阴性对照0D630nm平均值之差≥0.8。S为样品0D630nm值，P为阳性对照0D630nm平均值，N为阴性对照0D630nm平均值。若S/P值≥0.25,样品判定为阳性；若S/P值<0.25,样品判定为阴性。
Claims (2)
1.一种SARS-CoV-2冠状病毒IgA抗体，其特征在于：IgA抗体蛋白其序列号为MNWVRQAPGEGLEWLSYISTSSNNIFYADSVKGRFTVSRDNAKNSLYLQMNSLRDEDTAVYYCAGHTGSNWFDYWGQGTLVTVSSASPTSPKVFPLSLCSTQPDGNVVIACLVQGFFPQEPLSVTWSESGQGVTARNFPPSQDASGDLYTTSSQLTLPATQCLAGKSVTCHVKHYTNPSQDVTVPCPVPSTPPTPSPSTPPTPSPSCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTCY*。
2.根据权利要求1所述的一种SARS-CoV-2冠状病毒IgA抗体在ELISA检测试剂盒中的应用，其特征在于，包括下列技术措施：
1）根据SARS-CoV-2冠状病毒侵入人体后自身产生的所有抗体全长序列信息与未感染SARS-CoV-2冠状病毒者体内抗体序列进行比对分析，比对出抗SARS-CoV-2冠状病毒特异性抗体IgA全长序列；
2）并进行了原核密码子优化，优化后的序列为SEQ ID N0.2所示，嵌入pET32a质粒中、合成了pET32a-IgA原核表达质粒，并将pET32a—IgA质粒转化至E.coli BL21 (DE3)感受态中，在E.coliBL21(DE3)菌株中高效表达并纯化，最后获得纯化的IgA蛋白；
3）鉴定人群是否感染SARS-CoV-2冠状病毒或者检测人血清中是否含有SARS-CoV-2冠状病毒抗原，IgA蛋白作为包被抗体，建立ELISA抗原检测方法。
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| US11999777B2 (en) | 2020-06-03 | 2024-06-04 | Regeneron Pharmaceuticals, Inc. | Methods for treating or preventing SARS-CoV-2 infections and COVID-19 with anti-SARS-CoV-2 spike glycoprotein antibodies |
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| US11999777B2 (en) | 2020-06-03 | 2024-06-04 | Regeneron Pharmaceuticals, Inc. | Methods for treating or preventing SARS-CoV-2 infections and COVID-19 with anti-SARS-CoV-2 spike glycoprotein antibodies |
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