CN115453113A - 一种广谱性通用型新型冠状病毒双抗原夹心elisa抗体检测试剂盒及其应用 - Google Patents
一种广谱性通用型新型冠状病毒双抗原夹心elisa抗体检测试剂盒及其应用 Download PDFInfo
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- CN115453113A CN115453113A CN202210382555.1A CN202210382555A CN115453113A CN 115453113 A CN115453113 A CN 115453113A CN 202210382555 A CN202210382555 A CN 202210382555A CN 115453113 A CN115453113 A CN 115453113A
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Abstract
本发明公开了一种广谱性通用型新型冠状病毒双抗原夹心ELISA抗体检测试剂盒及其应用，属于生物检测技术领域。为了对新型冠状病毒抗体检测提供一种通用型检测技术。本发明涉及一种新型冠状病毒抗体检测试剂盒，含包被重组新型冠状病毒RBD蛋白的支持介质，酶标记重组蛋白RBD，样品稀释液，阴性抗体与阳性抗体对照，浓缩洗液，显色液与终止液，样品稀释板以及封口膜组分。试剂盒可检测人及动物感染或免疫后机体内产生的新型冠状病毒特异性抗体，不受检测对象种属限制，对不同类型的抗体，包括IgM和IgG、对病毒感染的早期与中晚期都可进行检测，用于人及动物疫苗接种后的免疫效果评价，用于病毒宿主与多种动物的流行病学监测。
Description
技术领域
本发明属于生物检测技术领域，具体涉及一种广谱性通用型新型冠状病毒双抗原夹心 ELISA抗体检测试剂盒及其应用。
背景技术
科学家们发现家猫、水貂等多种动物对新冠病毒具有很强的易感性。且报道了多起家养宠物猫、养殖场水貂、动物园猫科动物以及野生白尾鹿感染阳性的事例。因此在控制新型冠状病毒的措施中迫切需要对动物进行流行病学监测，因此也迫切需要建立一种不受动物种属限制的通用型抗体检测方法。
新型冠状病毒为RNA病毒，基因组具有易突变性，至目前已出现了多种变异病毒株， 现在亟需针对大部分毒株提供一种具有较为理想检测效果的方法。
发明内容
本发明的目的是为了对新型冠状病毒抗体检测提供一种通用型检测技术，且对不同流行毒株的抗体具有都具有很好的检测效果。
本发明提供了一种新型冠状病毒双抗原夹心ELISA抗体检测试剂盒，所述试剂盒由以下部分组成：包被重组新型冠状病毒RBD蛋白的支持介质、酶标试剂、阴性对照、阳性对照、浓缩洗液、显色液和终止液。
进一步地限定，所述重组新型冠状病毒RBD蛋白是由的新型冠状病毒RBD蛋白、新型冠状病毒德尔塔变异株RBD蛋白和新型冠状病毒奥米克戎变异株RBD蛋白组成的。
进一步地限定，是所述新型冠状病毒RBD蛋白的氨基酸序列如SEQ ID NO.1所示；所述新型冠状病毒德尔塔变异株RBD蛋白的氨基酸序列如SEQ ID NO.2所示；新型冠状病毒奥米克戎变异株RBD蛋白的氨基酸序列如SEQ ID NO.3所示。
进一步地限定，新型冠状病毒RBD蛋白质量：新型冠状病毒德尔塔变异株RBD蛋白质量：新型冠状病毒奥米克戎变异株RBD蛋白质量＝1:1:1。
进一步地限定，所述编码SEQ ID No.1所示的氨基酸序列的核苷酸序列如SEQ IDNo.7 所示；所述编码SEQ ID No.2所示的氨基酸序列的核苷酸序列如SEQ ID No.8所示；所述编码SEQ ID No.3所示的氨基酸序列的核苷酸序列如SEQ ID No.9所示。
进一步地限定，所述重组新型冠状病毒RBD蛋白包被浓度为0.3～6μg/mL；所述阳性对照为新型冠状病毒重组S1蛋白免疫羊血清；所述阴性对照为含50％V/V胎牛血清；所述浓缩洗液为10×PBST；所述显色液为单组分TMB显色液；所述终止液为2M H2SO4溶液。
进一步地限定，所述酶标试剂由以下成分组成：酶标的重组新型冠状病毒RBD、酶标蛋白稳定剂和0.05％V/V防腐剂ProClin300。
进一步地限定，酶标利用的酶为辣根过氧化物酶、碱性磷酸酶或β-D-半乳糖甘酶；所述支持介质为96孔聚苯乙烯微孔板。
进一步地限定，所述重组新型冠状病毒RBD蛋白制备方法的步骤如下：
(1)将编码SEQ ID No.1所示的氨基酸序列的核苷酸序列进行优化获得核苷酸序列SEQ ID No.4，然后连接到pCAG-neo载体中，得到重组载体1，将重组载体1转染BHK-21细胞，提取纯化获得蛋白1；
(2)将编码SEQ ID No.2所示的氨基酸序列的核苷酸序列进行优化获得核苷酸序列SEQ ID No.5，然后连接到pCAG-neo载体中，得到重组载体2，将重组载体2转染BHK-21细胞，提取纯化获得蛋白2；
(3)将编码SEQ ID No.3所示的氨基酸序列的核苷酸序列进行优化获得核苷酸序列SEQ ID No.6，然后连接到pCAG-neo载体中，得到重组载体3，将重组载体3转染BHK-21细胞，提取纯化获得蛋白3；
(4)将步骤(1)-步骤(3)获得的蛋白用碳酸盐缓冲液进行混合。
本发明提供上述的试剂盒在制备新型冠状病毒疫苗免疫后的抗体检测试剂盒、野生动物新型冠状病毒疫情监测试剂盒、药物筛选试剂盒和疫苗研究评价试剂盒中的应用。
有益效果：本发明分析了多个突变株RBD蛋白序列，经分析后优化组合，由的新型冠状病毒RBD蛋白、新型冠状病毒德尔塔变异株RBD蛋白和新型冠状病毒奥米克戎变异株RBD进行组合作为包被抗原以及酶标试剂，对流行的毒株抗体具有更高的检测率。
中和抗体是当病原微生物侵入机体时会产生相应的抗体。病原微生物入侵细胞时需要依赖病原体自身表达的特定分子与细胞上的受体结合，才能感染细胞，并进一步扩增。中和抗体是B淋巴细胞产生的某些抗体，能够与病原微生物表面的抗原结合，从而阻止该病原微生物黏附靶细胞受体，防止侵入细胞。
本发明试剂盒适用于人以及不同动物血清、血浆中新冠病毒抗体的检测，不受物种种属限制，具有通用性，具有极大的使用便利性。本发明试剂盒对待检样品中的全部抗体均可检测，无论是IgG或是IgM或是IgA等都可以发生特异性反应。本发明试剂盒组组份简单，操作便利，结果直观，可实现定量分析。
附图说明
图1为间接免疫荧光检测S-RBD蛋白表达质粒转染BHK-21细胞的瞬时表达结果；
图2为Western blot检测克隆细胞培养上清液中RBD蛋白的表达结果；
图3为间接免疫荧光试验检测不同代次RBD蛋白表达细胞系的表达结果；
图4为纯化S-RBD蛋白的电泳分析图。
具体实施方式
质粒，菌株与细胞：真核表达载体质粒pCAG-neo、DH5α感受态细胞和BHK-21细胞商业购买，质粒提取试剂盒为QIAGEN公司产品，DNA凝胶回收试剂盒购自上海华舜生物技术有限公司，G418为Amresco公司产品，胰酶购自Gibco公司，限制性内切酶Ssp I购自NEB公司，抗His单克隆抗体购自金思瑞公司，FITC标记山羊抗小鼠IgG购自中杉金桥生物公司，
HD Transfection Reagent转染试剂盒购于Roche公司。
实施例1.稳定表达新型冠状病毒S-RBD蛋白重组细胞系的构建及鉴定
1.重组表达质粒的构建：参考新型冠状病毒S蛋白序列NCBI登录号:YP_009724390.1)， 将该选取该蛋白序列中RBD区域，即S-RBD蛋白，序列如SEQ ID NO.1所示。将S-RBD蛋白 基因编码序列按哺乳动物偏嗜性密码子进行基因序列优化，优化基因序列如SEQ ID NO.4所 示，密码子优化基因序列命名为opti-S-RBD。opti-S-RBD基因由南京金斯瑞生物科技有限公 司合成，合成的基因克隆到质粒pCAG-neo(Hua et al.,2014.Generation and characterization of a new mammalian cell linecontinuously expressing virus-like particles of Japanese encephalitis virusfor a subunit vaccine candidate.DOI:10.1186/1472-6750-14-62)中的Sac I与Xho I双酶切位 点之间，经测序验证后保留序列正确的克隆命名为pCAGneo-opti-S-RBD。
重组表达质粒的构建表达细胞系的构建：构建的重组表达质粒pCAGneo-opti-S-RBD经测序结果表明重组质粒中SacΙ﹑XhoΙ酶切位点间的序列和设计的编码S-RBD蛋白的基因序列完全一致。质粒经转染BHK-21细胞后48小时进行间接免疫荧光检测，结果表明转染质粒得到了表达，可以见到黄绿色荧光，而对照细胞则无特异性黄绿色荧光(图1)。表明所构建的质粒在BHK-21细胞中能得到表达，可以进行下一步转染与稳定表达细胞的筛选。
2.质粒提取：将序列正确的质粒转化DH5α感受态细胞后涂布含氨苄的LB平板，过夜培养后挑取单菌落扩增培养，再将过过夜培养的菌液按2％接种取新鲜的含氨苄的LB液体培养基中，振荡培养过夜后再用试剂盒提取质粒；提取的质粒用限制性内切酶SspI进行单酶切线性化，经胶回收后测定含量置-20℃保存备用。
3.细胞转染与筛选：选生长状态良好的BHK-21细胞消化传代至24孔板中，待BHK-21 细胞长至90％满时，按照
HD Transfection Reagent转染试剂盒操作说明用重组质粒pCAGneo-opti-S-RBD转染细胞。转染48h后加入含G418(1 000μg/m L)选择性培养基进行加压培养，4d后将细胞用胰酶消化，以有限稀释法传代于96孔板中继续培养，8d后在倒置显微镜下观察每孔细胞的克隆数目。挑选含有1个细胞集落(即1个细胞团块)的孔相继在24孔板、6孔板、细胞培养瓶中扩大培养，同时对各细胞进行IFA鉴定以及对表达蛋白进行western blot检测。筛选IFA信号较强及表达抗原量高的细胞克隆。
4.间接免疫荧光试验检测目的蛋白在转染细胞中的表达：将克隆细胞接种至24孔或12孔板中，24h后去除培养液，用PBS洗3次，4％多聚甲醛固定10min，PBS洗3次，用含0.1％Triton X-100的PBS作用细胞10min，PBS洗3次，用含4％BSA的PBS封闭2h，PBS洗1次，加入稀释致使用浓度的抗His蛋白单克隆抗体，4℃孵育过夜，PBS 洗3次，加入按1:200稀释的FITC标记山羊抗小鼠IgG，室温孵育2h，PBS洗3次，在荧光显微镜下观察结果。
5.克隆细胞表达RBD蛋白的western blot检测：克隆细胞在24孔板中培养72h后，收集上清液，用超滤管进行10倍浓缩，取浓缩后细胞培养液40μl，加入5×蛋白上样缓冲液，混匀后置沸水中煮10分钟，再进行蛋白电泳；电泳后将蛋白转印至硝酸纤维素膜中(NC膜)，用5％脱脂乳溶液封闭2小时后，用PBST洗3次，再用His标签单克隆抗体于37℃孵育1小时，PBST 洗3次后，用红外荧光标记羊抗鼠二抗孵育1小时，PBST洗5次后用LICOR Odyssey红外荧光扫描仪扫描成像。
表达细胞系的western blot表达量筛选：根据间接免疫荧光试验检测的结果，初步选出12 个克隆细胞进行进一步比较培养上清液中S-RBD蛋白表达量相对比较检测，结果表明经 western blot检测筛选出的12个克隆中10号克隆所测得的特异性条带灰度值最高(图2)，选取10 号细胞克隆进行传代培养与进一步特性分析，且命名为B-S-RBD细胞。10号克隆细胞经过不同代次传代后进行IFA检测，结果表明筛选出的克隆细胞系在多次传代后仍保持很好的阳性细胞率(图3)。结果表明构建的重组细胞系能稳定表达目的基因。
实施例2.细胞系重组表达蛋白的纯化
将B-S-RBD细胞用含10％胎牛血清与10μg/ml G418的DMEM培养基传代培养，扩增至足够数量后待细胞长至80％～90％满时换为维持表达培养基，维持表达培养基为含1％胎牛血清的DMEM培养液。维持表达培养4-5天后收获细胞培养上清液，细胞上清液用双层滤纸过滤后用Ni离子亲自层析柱进行蛋白纯化。结合缓冲液中含40mM咪唑，上清液过完柱后用20倍住体积的含80mM咪唑的洗涤缓冲液进行洗涤杂蛋白，洗涤完后用含500mM咪唑的吃洗脱缓冲液进行蛋白洗脱，收集蛋白洗脱液并取样进行电泳分析(图4)，用10kDa 超滤管进行离心浓缩。离心浓缩后的洗脱蛋白再用PD-10脱盐柱进行脱盐更换为pH值为7.4 的PBS缓冲液。用BCA法测定蛋白含量后置-70℃保存备用。
实施例3.重组蛋白的标记
采用改良过碘酸钠法对实施例1制备的新型冠状病毒重组蛋白S-RBD进行辣根过氧化物酶(HRP)的标记。称取2mg辣根过氧化物酶(HRP)溶于0.2ml超纯水中，加入0.2ml新鲜配制的NaIO4溶液(128.4mg NaIO4溶于10ml超纯水中)，混匀，2～8℃避光静置作用30分钟；在上述溶液中加入0.2ml乙二醇水溶液(0.16mol/L)，混匀，室温避光作用30分钟；加入含10mg/ml重组蛋白水溶液0.2ml，混匀后，加入到透析袋中，用碳酸盐缓冲液(0.05mol/L，pH9.6)于2～8℃搅拌透析6小时。整个操作需避光进行。将透析后的混合液转移至1.5ml EP管中，加入80μl新鲜配制的NaBH4溶液(5mg NaBH4溶于1ml超纯水)，室温作用2小时，每隔30分钟混匀一次。将得到的产物用用分子筛(Hiload 16/600，Superdex 200pg)层析去除游离的辣根过氧化物酶。先用去离子水清洗层析柱，再用磷酸盐缓冲液(20mmol/L NaH2PO4，150mmol/L NaCl，pH 7.4)平衡层析柱后，以1.5mL/min流速上样，上样结束后继续用磷酸盐缓冲液洗脱分离目的蛋白，收集第一蛋白峰，即为HRP标记的S-RBD蛋白，调整浓度至 1mg/ml后分装，置-70℃保存。
实施例4.新型冠状病毒德尔塔变异株RBD蛋白(DS-RBD)的表达纯化与酶标记
新冠病毒德尔塔变异株S蛋白RBD(DS-RBD)的表达、纯化与酶标记步骤与过程分别与实施例1，实施例2和实施例3相同，区别是DS-RBD蛋白序列如SEQ ID NO.2序列所示，密码子优化的DS-RBD蛋白基因编码序列opti-DS-RBD如SEQ ID NO.5所示。获得HRP 标记的DS-RBD蛋白。
实施例5.新型冠状病毒奥米克戎变异株RBD蛋白(0S-RBD)的表达纯化与酶标记
新冠病毒奥米克戎变异株S蛋白RBD(OS-RBD)的表达、纯化与酶标记步骤与过程分别与实施例1，实施例2和实施例3相同，区别是OS-RBD蛋白序列如SEQ ID NO.3序列所示，密码子优化的OS-RBD蛋白基因编码序列opti-OS-RBD如SEQ ID NO.6所示。获得 HRP标记的OS-RBD蛋白。
实施例6.双抗原夹心ELISA抗体检测试剂盒的制备及检测方法
一、试剂盒的制备
1.抗原包被板：用碳酸盐缓冲液(0.05mol/L,pH值9.6)将纯化的重组RBD蛋白(S-RBD,DS-RBD和OS-RBD按质量比1：1：1混合)稀释至优选的终浓度进行包被，100μl/ 孔，2～8℃包被过夜，用PBST洗液洗涤3次，加入封闭液(含1％BSA的PBS溶液)200μl/ 孔，37℃封闭2小时，弃去封闭液后再用含5％M/V蔗糖、20％V/V新生牛血清、0.05％V/V ProClin300的磷酸盐缓冲液于2～8℃作用过夜，次日弃去孔内液体，拍干后将包被板在37℃干燥3小时，密封后置2～8℃保存备用。
2.酶标试剂：将HRP标记RBD蛋白(HRP标记S-RBD、DS-RBD和OS-RBD按质量比 1:1:1混合)用酶标蛋白保护液
SELET Stabilizer按适当比例稀释后作为酶标试剂，分装于置2～8℃保存备用。
3.样品稀释液：取牛血清白蛋白10g、氯化钠8g、磷酸氢二钠2.9g、磷酸二氢钾0.24g、氯化钾0.2g、超纯水900ml、吐温20 0.5ml、ProClin300 1ml，完全溶解后用超纯水定容至 1000ml，0.22μm滤膜过滤，分装后置2～8℃存放备用。
4.浓缩洗涤液10×PBST：取氯化钠80g、磷酸氢二钠29g、磷酸二氢钾2.4g、氯化钾2g、超纯水900ml、吐温20 5ml，完全溶解后用超纯水定容至1000ml，用0.22μm滤膜过滤，分装后置2～8℃存放备用。使用时用蒸馏水稀释10倍。
5.阳性对照：将真核表达的新型冠状病毒S1蛋白(SEQ ID No.10)与弗氏佐剂混合乳化后皮内注射免疫山羊(500μl/只)，间隔21日后以相同剂量和免疫方式进行加强免疫，加强免疫2次后21日采血并分离血清，收集血清即为阳性血清，为新型冠状病毒重组S1蛋白免疫羊血清。取胎牛血清200ml、免疫羊血清10ml，0.5ml ProClin300混匀并用pH值为7.4的PBS定容至1000ml，0.45μm滤膜过滤后分装置-20℃存放备用。作为组装试剂盒组份时置2～8℃存放备用。
6.阴性对照：取胎牛血清200ml、0.5ml ProClin300混匀并用pH值为7.4的PBS定容至 1000ml，0.45μm滤膜过滤后分装置-20℃存放备用。作为组装试剂盒组份时置2～8℃存放备用。
7.显色液：为单组份TMB显色液，为SIGMA公司产品，分装后置2～8℃存放备用。
终止液：2M H2SO4溶液。
8.试剂盒组装后所有组份均置2～8℃存放。
9.检测操作步骤
(1)将试剂盒所有组份恢复至室温。
(2)加入样品稀释液，50μL/孔；加入阳性、阴性对照血清各2孔，50μL/孔；加入待检血清，50μL/孔；记录加样顺序；置密封盒中或封口袋中；室温作用2小时。
(3)用洗涤液洗3次，300μL/孔，每次洗液在孔中停留2min后拍干。
(4)加入酶标试剂，100μL/孔；室温作用30分钟；
(5)用洗涤液洗3次，300μL/孔，每次洗液在孔中停留2min后拍干。
(6)加入显色液显色，100μL/孔，室温避光显色10min；
(7)加入终止液，100μL/孔；10min内测定OD450值；
(8)结果判定：当阴性对照血清OD值小于0.1，阳性对照血清OD值大于0.5时试验成立，否则试验无效应重做。试验成立时，临界值(Cut off值)计算：临界值＝0.12+阴性对照孔平均OD值(阴性对照孔OD值低于0.05者以0.05计算)。
样品OD值≥临界值者为新型冠状病毒抗体阳性；样品OD值<临界值者为新型冠状病毒抗体阴性。
实施例7.抗原包被浓度的选择
用碳酸盐缓冲液(0.05mol/L,pH值9.6)将纯化的重组RBD蛋白(S-RBD、DS-RBD和OS-RBD按质量比1：1：1混合)分别稀释至终浓度为0.3、0.6、0.9、1.5、2、3、6μg/ml 进行包被制备抗原包被板，按照实施例6检测方法分别检测阳性对照、阴性对照血清样品，计算P/N值(阳性对照孔OD平均值/阴性对照孔OD平均值)，结果如表1所示，P/N值随抗原包被浓度增加而增加，至3μg/ml时最高，而当包被浓度达6μg/ml时P/N值略有下降，故选择抗原包被浓度为3μg/ml。
表1 RBD蛋白包被浓度选择试验结果
实施例8.酶标试剂工作液
按实施例7优化的抗原包被浓度，将纯化的重组RBD蛋白(S-RBD、DS-RBD和OS-RBD按质量比1：1：1混合)用包被液稀释至3ug/ml，包被ELISA板。检测阳性与阴性对照血清。再将实施例3、实施例4和实施例5标记的RBD蛋白按质量比1：1：1进行混合后，用酶标蛋白保护液
SELET Stabilizer分别以1：1000、1：4000、1：8000、1： 10000、1：20000进行稀释，按照实施例6检测方法分别检测阳性对照、阴性对照血清，计算P/N值(阳性对照孔OD平均值/阴性对照孔OD平均值)。结果显示从2000倍至10000 倍稀释时OD值保持相对稳定，只略有下降，且P/N值呈上升趋势，至10000倍时P/N值最高(表2)，所以选择酶标蛋白的工作浓度为1：10000倍稀释。
表2酶标蛋白工作浓度选择试验结果
实施例9.新型冠状病毒双抗原夹心ELISA抗体检测试剂盒对动物血清的检测
取SPF小鼠血清10份，SPF鸡血清10份，SPF猪血清10份，健康水貂血清5份，用组装的试剂盒进行检测。结果均为阴性(表3)，结果表明该试剂盒与多种动物血清均无非特异性反应。
表3试验盒对新冠病毒抗体阴性动物血清检测结果
实施例10.试剂盒对新型冠状病毒抗体阳性人血清、新型冠状病毒抗体阳性动物血清的检测
取用重组冠状病毒S1蛋白免疫小鼠血清5份，重组新冠病毒S1蛋白免疫水貂血清5份，重组新冠病毒S1蛋白免疫猫血清，疫苗加强免疫后人血清5份，这些血清已经实验室测定为新冠病毒中和抗体阳性样品，再用组装的试剂盒进行检测。结果均为阳性(表4)，结果表明该试剂盒对新型冠状病毒抗体阳性血清均能检出。
表4动物血清检测结果
SEQ ID No.1(新冠病毒S-RBD蛋白的氨基酸序列)
VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNL DSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNG VGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN
SEQ ID No.2(新冠病毒德尔塔变异株DS-RBD蛋白的氨基酸序列)
VQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNL DSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNG VGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN
SEQ ID No.3(新冠病毒奥米克戎变异株OS-RBD蛋白的氨基酸序列)
VQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYG VSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKL DSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYG VGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN
SEQ ID No.4(密码子优化的编码opti-S-RBD蛋白的基因序列)
gtgcagcccaccgagtccatcgtgcgcttccccaacatcaccaacctgtgccccttcggcgaggtgttcaacgccacccgcttcgcctccgtgt acgcctggaaccgcaagcgcatctccaactgcgtggccgactactccgtgctgtacaactccgcctccttctccaccttcaagtgctacggcgt gtcccccaccaagctgaacgacctgtgcttcaccaacgtgtacgccgactccttcgtgatccgcggcgacgaggtgcgccagatcgcccccg gccagaccggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgcgtgatcgcctggaactccaacaacctggactc caaggtgggcggcaactacaactacctgtaccgcctgttccgcaagtccaacctgaagcccttcgagcgcgacatctccaccgagatctacca ggccggctccaccccctgcaacggcgtggagggcttcaactgctacttccccctgcagtcctacggcttccagcccaccaacggcgtgggct accagccctaccgcgtggtggtgctgtccttcgagctgctgcacgcccccgccaccgtgtgcggccccaagaagtccaccaacctggtgaa gaacaagtgcgtgaac
SEQ ID No.5(密码子优化的编码opti-DS-RBD蛋白的基因序列)
gtgcagcccaccgagtccatcgtgcgcttccccaacatcaccaacctgtgccccttcggcgaggtgttcaacgccacccgcttcgcctccgtgt acgcctggaaccgcaagcgcatctccaactgcgtggccgactactccgtgctgtacaactccgcctccttctccaccttcaagtgctacggcgt gtcccccaccaagctgaacgacctgtgcttcaccaacgtgtacgccgactccttcgtgatccgcggcgacgaggtgcgccagatcgcccccg gccagaccggcaagatcgccgactacaactacaagctgcccgacgacttcaccggctgcgtgatcgcctggaactccaacaacctggactc caaggtgggcggcaactacaactaccgctaccgcctgttccgcaagtccaacctgaagcccttcgagcgcgacatctccaccgagatctacc aggccggctccaagccctgcaacggcgtggagggcttcaactgctacttccccctgcagtcctacggcttccagcccaccaacggcgtggg ctaccagccctaccgcgtggtggtgctgtccttcgagctgctgcacgcccccgccaccgtgtgcggccccaagaagtccaccaacctggtga agaacaagtgcgtgaac
SEQ ID No.6(密码子优化的编码opti-OS-RBD蛋白的基因序列)
gtgcagcccaccgagtccatcgtgcgcttccccaacatcaccaacctgtgccccttcgacgaggtgttcaacgccacccgcttcgcctccgtgt acgcctggaaccgcaagcgcatctccaactgcgtggccgactactccgtgctgtacaacctggcccccttcttcaccttcaagtgctacggcgt gtcccccaccaagctgaacgacctgtgcttcaccaacgtgtacgccgactccttcgtgatccgcggcgacgaggtgcgccagatcgcccccg gccagaccggcaacatcgccgactacaactacaagctgcccgacgacttcaccggctgcgtgatcgcctggaactccaacaagctggactc caaggtgtccggcaactacaactacctgtaccgcctgttccgcaagtccaacctgaagcccttcgagcgcgacatctccaccgagatctacca ggccggcaacaagccctgcaacggcgtggccggcttcaactgctacttccccctgcgctcctactccttccgccccacctacggcgtgggcc accagccctaccgcgtggtggtgctgtccttcgagctgctgcacgcccccgccaccgtgtgcggccccaagaagtccaccaacctggtgaa gaacaagtgcgtgaac
SEQ ID No.7(编码S-RBD蛋白的基因序列)
gtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccag atttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcat cattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattca tttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattacc agatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattacctgt atagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacacct tgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttacca accatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatt tggttaaaaacaaatgtgtcaat
SEQ ID No.8(编码DS-RBD蛋白的基因序列)
gtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccag atttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcat cattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattca tttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattacc agatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattaccggt atagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcaaacct tgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttacca accatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatt tggttaaaaacaaatgtgtcaat
SEQ ID No.9(编码OS-RBD蛋白的基因序列)
gtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttgatgaagtttttaacgccaccaaatttgcatctgtttatgcttg gaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataatctcgcaccatttttcacttttaagtgttatggagtgtctcctactaaa ttaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaatattgct gattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaagcttgattctaaggttagtggtaattataattacctgta tagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtaacaaaccttgtaatggtgttgcaggttt taattgttactttcctttacgatcatatagtttccgacccacttatggtgttggtcaccaaccatacagagtagtagtactttcttttgaacttctacatgc accagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaat
SEQ ID No.10(新型冠状病毒S1蛋白的氨基酸序列)
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNV TWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNAT NVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQ GNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHR SYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFT VEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLY NSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFT GCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFP LQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGV LTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQD VNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQ TQTNSP。
SEQUENCE LISTING
<110> 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心）
<120> 一种广谱性通用型新型冠状病毒双抗原夹心ELISA抗体检测试剂盒及其应用
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 221
<212> PRT
<213> 人工合成
<400> 1
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
1 5 10 15
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
20 25 30
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
35 40 45
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
50 55 60
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
65 70 75 80
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
85 90 95
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
100 105 110
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
115 120 125
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
130 135 140
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
145 150 155 160
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
165 170 175
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
210 215 220
<210> 2
<211> 221
<212> PRT
<213> 人工合成
<400> 2
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
1 5 10 15
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
20 25 30
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
35 40 45
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
50 55 60
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
65 70 75 80
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
85 90 95
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
100 105 110
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
115 120 125
Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
130 135 140
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro
145 150 155 160
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
165 170 175
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
210 215 220
<210> 3
<211> 221
<212> PRT
<213> 人工合成
<400> 3
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
1 5 10 15
Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
20 25 30
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
35 40 45
Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser
50 55 60
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
65 70 75 80
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
85 90 95
Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
100 105 110
Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly
115 120 125
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
130 135 140
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro
145 150 155 160
Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr
165 170 175
Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val
180 185 190
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
195 200 205
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
210 215 220
<210> 4
<211> 663
<212> DNA
<213> 人工合成
<400> 4
gtgcagccca ccgagtccat cgtgcgcttc cccaacatca ccaacctgtg ccccttcggc 60
gaggtgttca acgccacccg cttcgcctcc gtgtacgcct ggaaccgcaa gcgcatctcc 120
aactgcgtgg ccgactactc cgtgctgtac aactccgcct ccttctccac cttcaagtgc 180
tacggcgtgt cccccaccaa gctgaacgac ctgtgcttca ccaacgtgta cgccgactcc 240
ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg caagatcgcc 300
gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg gaactccaac 360
aacctggact ccaaggtggg cggcaactac aactacctgt accgcctgtt ccgcaagtcc 420
aacctgaagc ccttcgagcg cgacatctcc accgagatct accaggccgg ctccaccccc 480
tgcaacggcg tggagggctt caactgctac ttccccctgc agtcctacgg cttccagccc 540
accaacggcg tgggctacca gccctaccgc gtggtggtgc tgtccttcga gctgctgcac 600
gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa caagtgcgtg 660
aac 663
<210> 5
<211> 663
<212> DNA
<213> 人工合成
<400> 5
gtgcagccca ccgagtccat cgtgcgcttc cccaacatca ccaacctgtg ccccttcggc 60
gaggtgttca acgccacccg cttcgcctcc gtgtacgcct ggaaccgcaa gcgcatctcc 120
aactgcgtgg ccgactactc cgtgctgtac aactccgcct ccttctccac cttcaagtgc 180
tacggcgtgt cccccaccaa gctgaacgac ctgtgcttca ccaacgtgta cgccgactcc 240
ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg caagatcgcc 300
gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg gaactccaac 360
aacctggact ccaaggtggg cggcaactac aactaccgct accgcctgtt ccgcaagtcc 420
aacctgaagc ccttcgagcg cgacatctcc accgagatct accaggccgg ctccaagccc 480
tgcaacggcg tggagggctt caactgctac ttccccctgc agtcctacgg cttccagccc 540
accaacggcg tgggctacca gccctaccgc gtggtggtgc tgtccttcga gctgctgcac 600
gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa caagtgcgtg 660
aac 663
<210> 6
<211> 663
<212> DNA
<213> 人工合成
<400> 6
gtgcagccca ccgagtccat cgtgcgcttc cccaacatca ccaacctgtg ccccttcgac 60
gaggtgttca acgccacccg cttcgcctcc gtgtacgcct ggaaccgcaa gcgcatctcc 120
aactgcgtgg ccgactactc cgtgctgtac aacctggccc ccttcttcac cttcaagtgc 180
tacggcgtgt cccccaccaa gctgaacgac ctgtgcttca ccaacgtgta cgccgactcc 240
ttcgtgatcc gcggcgacga ggtgcgccag atcgcccccg gccagaccgg caacatcgcc 300
gactacaact acaagctgcc cgacgacttc accggctgcg tgatcgcctg gaactccaac 360
aagctggact ccaaggtgtc cggcaactac aactacctgt accgcctgtt ccgcaagtcc 420
aacctgaagc ccttcgagcg cgacatctcc accgagatct accaggccgg caacaagccc 480
tgcaacggcg tggccggctt caactgctac ttccccctgc gctcctactc cttccgcccc 540
acctacggcg tgggccacca gccctaccgc gtggtggtgc tgtccttcga gctgctgcac 600
gcccccgcca ccgtgtgcgg ccccaagaag tccaccaacc tggtgaagaa caagtgcgtg 660
aac 663
<210> 7
<211> 663
<212> DNA
<213> 人工合成
<400> 7
gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg cccttttggt 60
gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa gagaatcagc 120
aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac ttttaagtgt 180
tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta tgcagattca 240
tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg aaagattgct 300
gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg gaattctaac 360
aatcttgatt ctaaggttgg tggtaattat aattacctgt atagattgtt taggaagtct 420
aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg tagcacacct 480
tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg tttccaaccc 540
actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga acttctacat 600
gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa caaatgtgtc 660
aat 663
<210> 8
<211> 663
<212> DNA
<213> 人工合成
<400> 8
gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg cccttttggt 60
gaagttttta acgccaccag atttgcatct gtttatgctt ggaacaggaa gagaatcagc 120
aactgtgttg ctgattattc tgtcctatat aattccgcat cattttccac ttttaagtgt 180
tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta tgcagattca 240
tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg aaagattgct 300
gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg gaattctaac 360
aatcttgatt ctaaggttgg tggtaattat aattaccggt atagattgtt taggaagtct 420
aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg tagcaaacct 480
tgtaatggtg ttgaaggttt taattgttac tttcctttac aatcatatgg tttccaaccc 540
actaatggtg ttggttacca accatacaga gtagtagtac tttcttttga acttctacat 600
gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa caaatgtgtc 660
aat 663
<210> 9
<211> 663
<212> DNA
<213> 人工合成
<400> 9
gtccaaccaa cagaatctat tgttagattt cctaatatta caaacttgtg cccttttgat 60
gaagttttta acgccaccaa atttgcatct gtttatgctt ggaacaggaa gagaatcagc 120
aactgtgttg ctgattattc tgtcctatat aatctcgcac catttttcac ttttaagtgt 180
tatggagtgt ctcctactaa attaaatgat ctctgcttta ctaatgtcta tgcagattca 240
tttgtaatta gaggtgatga agtcagacaa atcgctccag ggcaaactgg aaatattgct 300
gattataatt ataaattacc agatgatttt acaggctgcg ttatagcttg gaattctaac 360
aagcttgatt ctaaggttag tggtaattat aattacctgt atagattgtt taggaagtct 420
aatctcaaac cttttgagag agatatttca actgaaatct atcaggccgg taacaaacct 480
tgtaatggtg ttgcaggttt taattgttac tttcctttac gatcatatag tttccgaccc 540
acttatggtg ttggtcacca accatacaga gtagtagtac tttcttttga acttctacat 600
gcaccagcaa ctgtttgtgg acctaaaaag tctactaatt tggttaaaaa caaatgtgtc 660
aat 663
<210> 10
<211> 681
<212> PRT
<213> 人工合成
<400> 10
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro
675 680
Claims (10)
1.一种新型冠状病毒双抗原夹心ELISA抗体检测试剂盒，其特征在于，所述试剂盒由以下部分组成：包被重组新型冠状病毒RBD蛋白的支持介质、酶标试剂、阴性对照、阳性对照、浓缩洗液、显色液和终止液。
2.根据权利要求1所述的试剂盒，其特征在于，所述重组新型冠状病毒RBD蛋白是由的新型冠状病毒RBD蛋白、新型冠状病毒德尔塔变异株RBD蛋白和新型冠状病毒奥米克戎变异株RBD蛋白组成的。
3.根据权利要求2所述的三聚体蛋白，其特征在于，是所述新型冠状病毒RBD蛋白的氨基酸序列如SEQ ID NO.1所示；所述新型冠状病毒德尔塔变异株RBD蛋白的氨基酸序列如SEQ ID NO.2所示；新型冠状病毒奥米克戎变异株RBD蛋白的氨基酸序列如SEQ ID NO.3所示。
4.根据权利要求2所述的试剂盒，其特征在于，新型冠状病毒RBD蛋白质量：德尔塔变异株RBD蛋白质量：奥米克戎变异株RBD蛋白质量＝1:1:1。
5.根据权利要求2所述的三聚体蛋白，其特征在于，所述编码SEQ ID No.1所示的氨基酸序列的核苷酸序列如SEQ ID No.4所示；所述编码SEQ ID No.2所示的氨基酸序列的核苷酸序列如SEQ ID No.5所示；所述编码SEQ ID No.3所示的氨基酸序列的核苷酸序列如SEQID No.6所示。
6.根据权利要求1所述的试剂盒，其特征在于，所述重组新型冠状病毒RBD蛋白包被浓度为0.3～6μg/mL；所述阳性对照为新型冠状病毒重组S1蛋白免疫羊血清；所述阴性对照为含50％V/V胎牛血清；所述支持介质为96孔聚苯乙烯微孔板；所述浓缩洗液为10×PBST；所述显色液为单组分TMB显色液；所述终止液为2M H2SO4溶液。
7.根据权利要求1所述的试剂盒，其特征在于，所述酶标试剂由以下成分组成：酶标的重组新型冠状病毒RBD、酶标蛋白稳定剂和0.05％V/V防腐剂ProClin300。
8.根据权利要求7所述的试剂盒，其特征在于，酶标利用的酶为辣根过氧化物酶、碱性磷酸酶或β-D-半乳糖甘酶。
9.根据权利要求1所述的试剂盒，其特征在于，所述重组新型冠状病毒RBD蛋白制备方法的步骤如下：
(1)将编码SEQ ID No.1所示的氨基酸序列的核苷酸序列进行优化获得核苷酸序列SEQID No.4，然后连接到pCAG-neo载体中，得到重组载体1，将重组载体1转染BHK-21细胞，提取纯化获得蛋白1；
(2)将编码SEQ ID No.2所示的氨基酸序列的核苷酸序列进行优化获得核苷酸序列SEQID No.5，然后连接到pCAG-neo载体中，得到重组载体2，将重组载体2转染BHK-21细胞，提取纯化获得蛋白2；
(3)将编码SEQ ID No.3所示的氨基酸序列的核苷酸序列进行优化获得核苷酸序列SEQID No.6，然后连接到pCAG-neo载体中，得到重组载体3，将重组载体3转染BHK-21细胞，提取纯化获得蛋白3；
(4)将步骤(1)-步骤(3)获得的蛋白用碳酸盐缓冲液进行混合。
10.权利要求1-9任一项所述的试剂盒在制备新型冠状病毒疫苗免疫后的抗体检测试剂盒、野生动物新型冠状病毒疫情监测试剂盒、药物筛选试剂盒和疫苗研究评价试剂盒中的应用。
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