CN116693675B - 抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物及应用 - Google Patents
抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物及应用 Download PDFInfo
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Abstract
本发明公开了抗甲型流感病毒核衣壳蛋白的单克隆抗体及应用，包括1号克隆抗体和2号克隆抗体，1号克隆抗体的重链可变区氨基酸序列如SEQ ID NO.1所示，1号克隆抗体的轻链可变区氨基酸序列如SEQ ID NO.2所示；2号克隆抗体的重链可变区氨基酸序列如SEQ ID NO.3所示，2号克隆抗体的轻链可变区氨基酸序列如SEQ ID NO.4所示。此抗体组合具有特异性和高亲和力的特点，同时也提供了上述抗体的诊断检测试剂盒，从而提高诊断试剂的特异性和灵敏度。
Description
技术领域
本发明属于抗体的制备及序列测定领域，具体涉及抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物及应用。
背景技术
流感病毒（Influenza virus）属于正粘病毒科流感病毒属的单股负链RNA病毒，可根据流感病毒的核衣壳蛋白（Nucleoprotein，NP）和基质蛋白（Matrix，M）的不同特性，将流感病毒分为甲型流感病毒（Influenza A virus），乙型流感病毒（Influenza B virus），丙型流感病毒（Influenza C virus）和丁型流感病毒（Influenza D virus）。其中甲型流感病毒主要感染人类和各种动物，乙型和丙型主要感染的宿主是人类，丁型流感病毒主要感染猪和牛。甲型流感病毒最容易发生变异，流感大流行就是甲型流感病毒出现新亚型或旧亚型重现引起的。根据病毒表面的血凝素HA和神经氨酸酶NA抗原不同，将甲型流感分为许多亚型，H可分为18个亚型(H1-H18)，N有11个亚型(N1-N11 )。据统计20世纪期间有4次较大规模的甲型流感病毒大流行，分别是1918年西班牙H1N1流感，1957年H2N2亚洲流感，1968年H3N2流感和2009年H1N1墨西哥流感，近年来，高致病性禽流感（H5N1）也不断地威胁着人类的生命健康，流感的季节性流行和大流行造成全世界范围内的人类和动物的发病和死亡，对全球公共卫生安全、经济发展和社会稳定造成巨大的威胁。
流感病毒核蛋白（Nucleoprotein，NP）由第5条基因编码，498个氨基酸组成，是流感病毒的重要结构蛋白，约占病毒颗粒的25%，NP蛋白在不同亚型的甲流病毒中高度保守，氨基酸同源性很高，差异性不超过11%，具有种群和型的特异性，可用于流感病毒型的区别诊断，是诊断流感病毒最具代表性的蛋白。具有很高的免疫原性，可以用于制备诊断用单克隆抗体。
甲型流感诊断试剂的核心原料是NP蛋白的单克隆抗体，大部分依赖于进口，国内厂商的特异性和灵敏度不高，无法准确检测甲型流感病毒的含量，会有漏检的情况，所以开发开发性能优异的甲型流感诊断试剂非常有必要。
发明内容
为解决灵敏性、特异性的问题，本发明提供了抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物。该抗体性能优异、准确性高。
同时提供甲型流感病毒抗原检测试剂盒，能准确检测甲型流感病毒的含量，为甲型流感诊断提供依据。
本发明提供如下技术方案：
抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物，包括1号克隆抗体和2号克隆抗体，
1号克隆抗体的重链可变区氨基酸序列：
EVQLQQSGPELVKPGASVKMSCKASGYTFTIYFIHWVKQKPGQGLEWIAYINPYNDDIKYNEKFRGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAREDWLRFDNWGQGTTLTVSS
1号克隆抗体的轻链可变区氨基酸序列：
DIVLTQSPATLSVTPGDSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYTSQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPLTFGAGTKLELK
2号克隆抗体的重链可变区氨基酸序列：
EVQLQQSGPEVVKPGASVKISCKTSGYTFTEYTMHWLKQSHGKSLEWIGHINPNNGGTAYNQKFKGKATLTVGKSSSTAYMELRSLTSEDSAVYYCARRGYDYGRVHYFAMDYWGQGTSVTVSS
2号克隆抗体的轻链可变区氨基酸序列：
DIQLTQSPASLSASVGESVTITCRASENIYSFLAWYQQKQGKSPQLLVYTVKTLAEGVPSRFSGSGSGTQFSLKINSLHPEDFGSYYCQHHYGTPLTFGAGTKLELK
抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物的制备方法，将亲和纯化后的流感病毒核蛋白（NP蛋白）与弗氏佐剂进行等体积混合乳化，免疫计量为100ug/只小鼠，3周后进行加强免疫，免疫计量为50ug/只小鼠，后期间隔2周每次，采集小鼠血液分离血清，酶联免疫吸附测定抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过HAT筛选培养基筛选培养后，取上清进行酶联免疫吸附测定，筛选出阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株。
流感病毒核蛋白的亲和纯化：菌体用磷酸盐缓冲液重悬，高压均质机进行高压均质后，离心取上清，用重力柱进行亲和纯化，用梯度咪唑浓度进行洗杂和洗脱，进行SDS-PAGE电泳，检测蛋白纯度，将蛋白透析至磷酸盐缓冲液中，0.22um过滤器无菌过滤后，BCA测定蛋白浓度。
流感病毒核蛋白的表达生产：将pET30a-FluA NP表达质粒，转化大肠杆菌感受态细胞，挑取单菌落进行诱导表达，在含有卡那霉素的LB培养基中培养，培养至OD600nm至0.6-0.8时，加入异丙基硫代半乳糖苷进行诱导表达后收集菌体。
抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物在制备甲型流感诊断试剂中的应用。
单克隆抗体用于甲型流感病毒抗原检测试剂盒。试剂盒使用1号克隆抗体和2号克隆抗体分别作为检测抗体和包被抗体。
与现有技术相比，本发明的有益效果是：本发明提供了抗甲型流感病毒核衣壳蛋白，具有种群和型的特异性和很高的免疫原性，用于制备诊断用单克隆抗体，用于流感病毒型的区别诊断，本发明的抗甲型流感病毒核衣壳蛋白的单克隆抗体，此抗体组合具有特异性和高亲和力的特点，同时也提供了基于上述抗体的诊断检测试剂盒，从而提高诊断试剂的特异性和灵敏度。对于甲型流感病毒的诊断和流行病学调查具有很大的临床意义。
实施方式
下面将结合本发明实施例，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1 NP蛋白表达载体的构建
在NCBI官网上查找甲型流感病毒的NP蛋白氨基酸序列，在蛋白氨基酸加上组氨酸标签，利用密码子优化软件在大肠杆菌表达系统中优化密码子，基因合成后利用NdeI&XhoI限制性内切酶酶切位点，将基因克隆至pET30a表达载体中，构建pET30a-FluA NP表达质粒，并测序验证基因序列。
实施例2 NP蛋白的表达生产
将pET30a-Flu A NP表达质粒，转化大肠杆菌BL21(DE3)感受态细胞，挑取单菌落进行诱导表达，在含有50ug/ml的卡那霉素的LB培养基中培养，37℃，220rpm条件下培养至OD600nm至0.6-0.8时，加入0.2mM 异丙基硫代半乳糖苷（IPTG）进行诱导表达，37℃诱导6小时，8000g，离心10min收集菌体。
实施例3 NP蛋白的亲和纯化
菌体用20mM PB，300mM NaCl，10%甘油，pH8.0的缓冲液重悬，用800bar压力进行高压均质后，12000g，离心30min，取上清，用重力柱Ni Smart-6FF beads进行亲和纯化，20mMPB，300mM NaCl，10%甘油，50mM咪唑，pH8.0溶液进行洗杂，20mM PB，300mM NaCl，10%甘油，250mM咪唑，pH8.0溶液进行洗脱，进行SDS-PAGE电泳，检测蛋白纯度，将蛋白透析至20mMPB，300mM NaCl，10%甘油，pH8.0缓冲液中，0.22um过滤器无菌过滤后，BCA测定蛋白浓度。
实施例4 单克隆抗体的制备
将蛋白与弗氏佐剂进行等体积混合乳化，免疫计量为100ug/只小鼠，皮下多点注射，3周后进行加强免疫，免疫计量为50ug/只小鼠，皮下多点注射，后期间隔2周每次，采集小鼠血液分离血清，ELISA检测抗体效价，筛选免疫较好的小鼠进行杂交瘤融合实验，经过10天左右的HAT筛选培养基筛选培养后，取上清进行ELISA检测，筛选出性能较优的阳性细胞株，并进行两轮的细胞亚克隆，克隆出单克隆细胞株，用无血清培养和发酵细胞株，收集培养上清，进行抗体纯化，将纯化后的抗体进行诊断性能分析。
实施例5 单克隆抗体性能分析
结合垫的制备方法：
本操作规程以制备100ml成品乳胶颗粒为例，标记温度37℃。
（1）活化：将洁净烧杯放置到磁力搅拌器上，在容器中放入磁力搅拌子，加入40ml活化缓冲液(0.05M MES pH 6.0)，边搅拌边依次加入红色乳胶微球5ml、2ml 10mg/ml NHS、2ml 10mg/ml EDC，搅拌反应30min；
（2）标记：活化结束后转移乳胶至洁净离心管中，常温12000rpm离心30min，弃上清后加入40ml标记缓冲液20mM PB（pH7.5），涡旋加水浴超声使乳胶混合均匀。将乳胶转移至洁净容器中，在搅拌下加入1号克隆抗体20mg，持续反应1小时；
（3）封闭：在上述反应体系中加入2ml 10%BSA持续搅拌反应0.5小时；
（4）清洗：反应结束后将上述胶乳转移至离心管中，常温12000rpm离心30min，完全弃上清后加入40ml 20mmTris(pH 8.5)，涡旋加水浴超声使乳胶混合均匀；将上述胶乳再次常温12000rpm离心20min，完全弃上清后加入40ml 20mmTris+3%海藻糖，涡旋加水浴超声使乳胶混合均匀。使用超声波细胞粉碎机配置2号超声探头，设置30%功率、5秒超声、3秒间隔参数，超声处理上述标记物10min。将超声处理后的乳胶体积调整至100ml即完成标记工作；
（5）结合垫制备：将标记好的1号克隆抗体按照2ul/cm，使用喷金仪均匀的固定在结合垫上，37℃干燥20min，干燥密封保存。
包被方法：
（1）贴膜：将卷式NC膜使用剪刀剪切成31cm左右长度片段，在水平桌面上揭下PVC胶板中间2.5cm宽不干胶保护膜，两手轻拿硝酸纤维素膜（NC膜）两端，对齐刀口线后将NC膜整齐贴到PVC胶板上，将贴好NC膜的胶板两张面对面放置，成捆收集。
（2）划膜：将包被2号克隆抗体使用包被液（20mmPB 1%海藻糖）配制成抗体终浓度为1mg/ml的划膜液，使用连续划膜仪将包被液均匀的固定在NC膜上。
（3）干燥与封装：划线后，将NC膜放置至室温18-26℃、相对湿度20-28%房间内干燥30min后，将贴膜的一面两两相对，用橡皮筋包扎20-50张一捆；将每捆包被后的胶板放置到铝箔袋中，放入干燥剂后密封，封装完成后在铝箔袋上标明名称、生产日期、数量等，收入中间库，做好记录。
（4）组装：将吸水纸、NC膜、结合垫、样品垫组装成大板，将大板用切条机斩切成试剂条，并装入卡壳内，组装环境的相对湿度应在20%-28%范围。
配制参考品：
使用提取液将甲型流感N蛋白分别稀释至300、600、1200pg/ml，制备成最低检出限参考品。
使用提取液将甲型流感N蛋白稀分别释液至1200、11000 pg/ml，制备成重复性参考品。
使用提取液将甲型流感N蛋白稀分别释液至600、2660、10650、42600、170000 pg/ml，制备成阳性参考品。
使用提取液将10例未感染甲型流感的正常人鼻咽拭子，制备成阴性参考品。
上述提取液配方为100mM PB、150mM Nacl、5%BSA、10%Glycerin、0.5%procin300,pH=8.0。
检测试剂与同类产品对比：
目前国内外甲型流感检测试剂盒主要包括胶体金法和PCR法，实施例中的甲型流感病毒抗原检测试剂盒（胶乳法）在231例临床标本上进行了相关性对比，如表1所示，抗体临床性能测试结果如下：
分析结果：
灵敏度=93.97% (95%CI: 88.07%~97.05%)
特异性=97.39% (95%CI: 92.61%~99.11%)
准确性: 95.67% (95% CI: 92.22%~97.63%)
说明本发明的试剂能准确检测甲型流感病毒的含量，为甲型流感诊断提供依据，能够充分满足临床体外诊断检测需求。
灵敏度和精密度考察：
按照国家药监局器审中心《定性检测体外诊断试剂分析性能评估注册审查指导原则》对本发明中的灵敏度进行了研究，结果表明最低检出限分别为 600pg/mL。
按照国家药监局器审中心《定性检测体外诊断试剂分析性能评估注册审查指导原则》对本发明中的试剂盒的重复性研究，检测结果显色一致。
开发性能优异的甲型流感诊断试剂的核心原料NP蛋白的单克隆抗体，是需要排除乙型流感病毒等其他常见病毒的交叉反应，保证单克隆抗体的特异性和高亲和力，从而提高诊断试剂的特异性和灵敏度，对于甲型流感病毒的诊断和流行病学调查具有很大的临床意义。
检测试剂抗干扰能力分析：
与普通的呼吸道病毒和肺炎支原体无交叉反应。
测试的病毒和细菌如下：
-- 乙型流感病毒
-- 呼吸道合胞病毒
--副流感病毒
--腺病毒
--金黄色葡萄球菌
--脑膜炎奈瑟菌
--肺炎链球菌
与普通的呼吸道病毒和肺炎支原体无交叉反应。这类试剂盒做的是定性检测，在做以上交叉反应实验试剂盒只显示C线，即阴性。
Claims (5)
1.抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物，其特征在于：包括1号克隆抗体和2号克隆抗体，
所述1号克隆抗体的重链可变区氨基酸序列如SEQ ID NO.1所示，1号克隆抗体的轻链可变区氨基酸序列如SEQ ID NO.2所示；
所述2号克隆抗体的重链可变区氨基酸序列如SEQ ID NO.3所示，2号克隆抗体的轻链可变区氨基酸序列如SEQ ID NO.4所示。
2.权利要求1所述的抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物在制备甲型流感诊断试剂中的应用。
3.根据权利要求2所述的抗甲型流感病毒核衣壳蛋白的单克隆抗体组合物在制备甲型流感诊断试剂中的应用，其特征在于：单克隆抗体组合物用于甲型流感病毒抗原检测试剂盒。
4.甲型流感病毒抗原检测试剂盒，其特征在于：含有权利要求1中的1号克隆抗体和2号克隆抗体。
5.根据权利要求4所述的甲型流感病毒抗原检测试剂盒，其特征在于：试剂盒使用1号克隆抗体和2号克隆抗体分别作为检测抗体和包被抗体。
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