CN112574306A - 脂联素单克隆抗体、抗体对及其制备方法和用途 - Google Patents
脂联素单克隆抗体、抗体对及其制备方法和用途 Download PDFInfo
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- CN112574306A CN112574306A CN202011504233.7A CN202011504233A CN112574306A CN 112574306 A CN112574306 A CN 112574306A CN 202011504233 A CN202011504233 A CN 202011504233A CN 112574306 A CN112574306 A CN 112574306A
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- adiponectin
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Abstract
本发明涉及脂联素单克隆抗体及抗体对，所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB‑DA120AmN①、CSB‑DA120AmN③和CSB‑DA120AmN④；所述抗体对为CSB‑DA120AmN③‑CSB‑DA120AmN④。本发明还提供了上述脂联素单克隆抗体及抗体对的制备方法，并保护其在在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。本发明通过基因重组，获得性能更为优异的脂联素单克隆抗体和抗体对，稳定、高效、批间差小，适于作为诊断试剂的抗体原料。
Description
技术领域
本发明属生物技术领域，具体涉及脂联素单克隆抗体、抗体对及其制备方法和用途。
背景技术
脂联素(Adiponectin/ADPN)是脂肪细胞分泌的一种内源性生物活性多肽或蛋白质。脂联素是一种胰岛素增敏激素(An Insulin-sensitizing Hormone)，能改善小鼠的胰岛素抗性(Insulin resistance)和动脉硬化症；对人体的研究发现，脂联素水平能预示II型糖尿病和冠心病的发展。
脂联素由APM1基因编码，该基因位于3号染色体长臂第27区域内，此区域亦存在2型糖尿病及代谢综合征的易感基因，全长17kb，包含2个内含子和3个外显子，其转录由5端胞嘧啶开始，多肽编码的区域起始于外显子2，终止于外显子3。
心血管系统疾病是威胁人类生命健康的首敌，彻底阐明其发病机理并寻求相应的治疗措施，是当今世界医学界亟待攻克的一项重大课题。近年来的研究证明，以糖尿病为突出临床特征的代谢综合征是心血管疾病的“原凶”。控制糖尿病及代谢综合征的药物正成为心血管系统疾病防治的第一战场。脂联素主要是由脂肪细胞合成和分泌的细胞因子，能够调节糖脂代谢、抗炎症、抗凋亡、抑制氧化/硝化反应，从而直接或者间接地保护心血管系统。肥胖、2型糖尿病患者血浆脂联素下降，其对心血管保护作用也减弱。
目前，脂联素已经开始用于II型糖尿病和冠心病的检测，然而现有的脂联素检测试剂灵敏度和精度都还不能满足临床诊断的需求。
发明内容
本发明针对现有技术中存在的不足，提供一种脂联素单克隆抗体、抗体对及其制备方法和用途，通过基因重组，获得性能更为优异的脂联素单克隆抗体和抗体对，稳定、高效、批间差小，适于作为诊断试剂的抗体原料。
为解决上述问题，本发明的技术方案如下：
本发明保护脂联素单克隆抗体，所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④；
所述CSB-DA120AmN①轻链可变区部分核苷酸序列为SEQ ID NO.4和SEQ ID NO.5，所述CSB-DA120AmN①重链可变区部分核苷酸序列为SEQ ID NO.6和SEQ ID NO.7；
所述CSB-DA120AmN③轻链可变区部分核苷酸序列为SEQ ID NO.8和SEQ ID NO.9，所述CSB-DA120AmN③重链可变区部分核苷酸序列为SEQ ID NO.10和SEQ ID NO.11；
所述CSB-DA120AmN④轻链可变区部分核苷酸序列为SEQ ID NO.12和SEQ IDNO.13，所述CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15。
本发明还保护上述脂联素单克隆抗体的制备方法，包括以下步骤：
步骤1)，采用酵母表达系统表达脂联素重组蛋白，蛋白的序列为SEQ ID NO.1；
步骤2)，分离纯化步骤1)得到的脂联素重组蛋白；
步骤3)，将步骤2)得到的脂联素重组蛋白免疫宿主动物；
步骤4)，获取步骤3)得到的免疫宿主动物的脾脏细胞，并通过脾脏细胞制备融合细胞；
步骤5)，采用步骤4)制得的融合细胞制备脂联素单克隆抗体。
在上述技术方案的基础上，本发明还能够有如下改进。
进一步，所述步骤1)中，采用酵母表达系统表达，包括以下步骤：
步骤a，合成序列SEQ ID NO.1的基因序列以及对应的亚克隆引物，并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含卡拉霉素和氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h以上，挑选单菌落PCR和双酶切验证正确后，进行测序验证，得到测序正确的重组子；
步骤b，将步骤a得到的三个重组子接种到含LB液体培养基，37℃培养8～16h，无菌条件下吸取菌液保种，剩余菌液接种于含卡拉霉素和氨苄青霉素的LB液体培养基，37℃培养12h以上，然后大提质粒；
步骤c，用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化线性化质粒于GS115感受态中，涂布、转化后的菌液于RBD固体培养基，30℃培养4～5天；
步骤d，从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5ml YPG液体培养基的试管中，30℃，培养48h以上，吸取适量菌液与甘油均匀混合后冷冻保存备用，将得到7种阳性株接种于1.5ml YPG液体培养基，30℃，培养24h以上，然后加入1％甲醇诱导，培养24h以上，再加入1％甲醇诱导，继续培养24h以上，最后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量；
步骤e，将产量最高的细胞株经前培养24h以上，按1％接种量接种至1L的YPG液体培养基中，30℃，培养24h以上，然后装入500ml Backman离心瓶，离心处理，用含1％甲醇的YP重悬菌体，倒入原三角瓶中，并加入含1％甲醇的YP和200ul消泡剂，30℃，培养72h以上，每隔24h加入10ml甲醇诱导，最后离心取上清。
进一步，所述步骤2)中，所述分离纯化，包括以下步骤：将最终得到的上清过Ni-NTA层析柱，然后用0～500mM NaCl洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
优选的，所述引物序列为SEQ ID NO.2和SEQ ID NO.3。
进一步，所述步骤3)中，所述宿主动物为Balb/C雌性小鼠，所述融合用细胞系SP2/0。
优选的，所述步骤3)中，所述脂联素重组蛋白免疫宿主动物包括以下步骤：以每只老鼠50ug脂联素重组蛋白加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次所述小鼠，每次免疫取50ug脂联素重组蛋白与弗氏不完全佐剂混匀；免疫四次后测效价，待效价达到1:10000以上时，按50ug/mouse腹腔冲击免疫。
进一步，所述步骤4)中，所述获取免疫宿主动物的脾脏细胞采用以下步骤，腹腔冲击免疫三天后，在无菌条件下取出小鼠脾脏，并制成单个脾细胞悬液。
进一步，所述步骤4)中，所述制备融合细胞工序，包括细胞融合和细胞建株，最后得到杂交瘤细胞。
优选的，所述步骤4)中，所述制备融合细胞工序包括以下步骤：
步骤A，细胞融合：制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，脾细胞和SP2/0以10:1的比例混合并离心，离心后滴干混合细胞，轻敲弹松细胞团块，在37℃水浴中加入1ml PEG1450，加完后在37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI-1640终止液，融合细胞终止后于800rpm离心5min，并吸干残留液体，HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基；
步骤B，细胞建株：待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价，从检测为阳性对应的微孔取阳性细胞进行亚克隆，利用HT培养基通过有限稀释法将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待单克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次选取其中阳性细胞，重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样的单克隆，直至有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得的阳性细胞株即为杂交瘤细胞。
进一步，所述步骤5)中，融合细胞制备抗体，包括无血清制备和纯化步骤，纯化后得到不同的单克隆抗体。
优选的，所述步骤5)中，无血清制备包括以下步骤：在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例，逐渐增加代用的添加物，使杂交瘤细胞适应并继续生长；用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱，流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE并鉴定其纯度。
本发明还保护一种脂联素单克隆抗体对，所述脂联素单克隆抗体对为CSB-DA120AmN③-CSB-DA120AmN④。
本发明还保护上述脂联素单克隆抗体对的制备方法，包括如下步骤：
将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
此外，本发明还保护上述脂联素单克隆抗体和/或脂联素单克隆抗体对，在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。
本发明的有益效果是：本发明采用酵母表达系统表达脂联素重组蛋白，并免疫小鼠，得到脾脏细胞，然后用脾脏细胞制备融合细胞即为杂交瘤细胞，杂交瘤细胞用无血清驯化、培养，上清过滤后纯化得到不同的单克隆抗体，进行效价验证之后取优选的抗体，后对单克隆抗体进行DNA测序，确定脂联素抗体可变区序列，本发明所述制备方法稳定，得到产物纯度程度高，批间差小；本发明的脂联素单克隆抗体、抗体对，具有稳定、高效等诸多优良特点，适用于作为诊断试剂盒开发的抗体原料，应用范围也较广，尤其适用于胶乳增强比浊、化学发光、免疫层析等高端诊断试剂盒开发的抗体原料；本发明的脂联素单克隆抗体、抗体对灵敏度较高，对各种浓度的脂联素重组蛋白进行定量检测，均能够满足临床检验的要求。
附图说明
图1为本发明实施例1抗体对11例健康志愿者血样和21例糖尿病患者血样在胶乳比浊平台进行检测的检测结果图；
图2为本发明实施例2抗体对14例健康志愿者血样和25例糖尿病患者血样在化学发光平台进行检测的检测结果图；
图3为本发明实施例2抗体对14例健康志愿者血样和25例糖尿病患者血样在免疫层析平台进行检测的检测结果图。
具体实施方式
以下对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
本发明设计了脂联素单克隆抗体，所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④；
所述CSB-DA120AmN①轻链可变区部分核苷酸序列为SEQ ID NO.4和SEQ ID NO.5，所述CSB-DA120AmN①重链可变区部分核苷酸序列为SEQ ID NO.6和SEQ ID NO.7；
所述CSB-DA120AmN③轻链可变区部分核苷酸序列为SEQ ID NO.8和SEQ ID NO.9，所述CSB-DA120AmN③重链可变区部分核苷酸序列为SEQ ID NO.10和SEQ ID NO.11；
所述CSB-DA120AmN④轻链可变区部分核苷酸序列为SEQ ID NO.12和SEQ IDNO.13，所述CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15。
目前，定量检测脂联素的检测试剂普遍采用脂联素单克隆抗体。如公开号为CN111239421A，发明名称为一种定量检测脂联素ADPN的胶乳增强免疫比浊试剂盒及其制备与应用方法的中国专利，比如公开号为CN104694478B，发明名称为抗人脂联素的单克隆抗体及其应用的中国专利，再比如公开号为CN110372783A，发明名称为一种脂联素抗原、抗体及其胶乳试剂的制备方法以及脂联素检测试剂盒的中国专利均采用了脂联素单克隆抗体，上述抗体均是依赖脂联素制备的，而且在制备过程中并未对脂联素的基因序列进行改造。
发明人通过对上述现有脂联素检测产品进行实验研究，发现上述产品脂联素的单克隆抗体的应用范围有限，而且检测灵敏度也存在一定问题。
针对上述问题，发明人对脂联素的基因进行了相关研究，并设计了脂联素单克隆抗体的基因重组方案，以克服上述问题。在研究过程中，发明人发现采用双抗夹心法免疫检测，效果较佳，并基于此发现设计了脂联素单克隆抗体对，以确保能够实现双抗夹心法免疫检测。
本发明所设计的脂联素单克隆抗体对为CSB-DA120AmN③-CSB-DA120AmN④。
采用抗体对后，抗体对的两个抗体分别用作捕获抗体和标记抗体，能够提供极高的检测灵敏度，可以对脂联素进行精确的定量检测。
上述脂联素单克隆抗体的制备方法，包括以下步骤：
步骤1)，采用酵母表达系统表达脂联素重组蛋白，蛋白的序列为SEQ ID NO.1；
步骤2)，分离纯化步骤1)得到的脂联素重组蛋白；
步骤3)，将步骤2)得到的脂联素重组蛋白免疫宿主动物；
步骤4)，获取步骤3)得到的免疫宿主动物的脾脏细胞，并通过脾脏细胞制备融合细胞；
步骤5)，采用步骤4)制得的融合细胞制备脂联素单克隆抗体。
本发明采用了酵母表达系统表达脂联素重组蛋白，可以成功实现胞内表达或是分泌表达，且其放大培养基相对廉价，培养条件要求不高，适宜工业放大，所得重组蛋白与天然蛋白十分接近。
同时，本发明通过脾脏细胞制备融合细胞，能够实现无血清驯化、培养，制备方法稳定，得到产物纯度程度高，批间差小。
具体的，所述步骤1)中，采用酵母表达系统表达，可以采用以下优化的工艺步骤：
步骤a，合成序列SEQ ID NO.1的基因序列以及对应的亚克隆引物，并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含卡拉霉素和氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h以上，挑选单菌落PCR和双酶切验证正确后，进行测序验证，得到测序正确的重组子；
步骤b，将步骤a得到的三个重组子接种到含LB液体培养基，37℃培养8～16h，无菌条件下吸取菌液保种，剩余菌液接种于含卡拉霉素和氨苄青霉素的LB液体培养基，37℃培养12h以上，然后大提质粒；
步骤c，用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化线性化质粒于GS115感受态中，涂布、转化后的菌液于RBD固体培养基，30℃培养4～5天；
步骤d，从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5ml YPG液体培养基的试管中，30℃，培养48h以上，吸取适量菌液与甘油均匀混合后冷冻保存备用，将得到7种阳性株接种于1.5ml YPG液体培养基，30℃，培养24h以上，然后加入1％甲醇诱导，培养24h以上，再加入1％甲醇诱导，继续培养24h以上，最后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量；
步骤e，将产量最高的细胞株经前培养24h以上，按1％接种量接种至1L的YPG液体培养基中，30℃，培养24h以上，然后装入500ml Backman离心瓶，离心处理，用含1％甲醇的YP重悬菌体，倒入原三角瓶中，并加入含1％甲醇的YP和200ul消泡剂，30℃，培养72h以上，每隔24h加入10ml甲醇诱导，最后离心取上清。
在本发明较为优选的实施方式中，所述步骤2)中，所述分离纯化，包括以下步骤：将最终得到的上清过Ni-NTA层析柱，然后用0～500mM NaCl洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
在本发明较为优选的实施方式中，所述引物序列为SEQ ID NO.2和SEQ ID NO.3。
在本发明较为优选的实施方式中，所述步骤3)中，所述宿主动物为Balb/C雌性小鼠，所述融合用细胞系SP2/0。
在本发明更较为优选的实施方式中，所述步骤3)中，所述脂联素重组蛋白免疫宿主动物包括以下步骤：以每只老鼠50ug脂联素重组蛋白加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次所述小鼠，每次免疫取50ug脂联素重组蛋白与弗氏不完全佐剂混匀；免疫四次后测效价，待效价达到1:10000以上时，按50ug/mouse腹腔冲击免疫。
选择采用Balb/C雌性小鼠作为宿主动物，融合用细胞系SP2/0，宿主动物个体差异小，遗传基因更纯，整体素质更好，融合产生杂交瘤细胞分裂能力更强，使单克隆抗体迅速大量增殖，有效提高制备效率和制备的稳定性。
在本发明较为优选的实施方式中，所述步骤4)中，所述获取免疫宿主动物的脾脏细胞采用以下步骤，腹腔冲击免疫三天后，在无菌条件下取出小鼠脾脏，并制成单个脾细胞悬液。
在本发明较为优选的实施方式中，所述步骤4)中，所述制备融合细胞工序，包括细胞融合和细胞建株，最后得到杂交瘤细胞。
具体的，所述制备融合细胞工序可以采用以下优化的工艺步骤：
步骤A，细胞融合：制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，脾细胞和SP2/0以10:1的比例混合并离心，离心后滴干混合细胞，轻敲弹松细胞团块，在37℃水浴中加入1ml PEG1450，加完后在37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI-1640终止液，融合细胞终止后于800rpm离心5min，并吸干残留液体，HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基；
步骤B，细胞建株：待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价，从检测为阳性对应的微孔取阳性细胞进行亚克隆，利用HT培养基通过有限稀释法将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待单克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次选取其中阳性细胞，重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样的单克隆，直至有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得的阳性细胞株即为杂交瘤细胞。
在本发明较为优选的实施方式中，所述步骤5)中，融合细胞制备抗体，包括无血清制备和纯化步骤，纯化后得到不同的单克隆抗体。
具体的，所述无血清制备可以采用以下优化的工艺步骤：
在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例，逐渐增加代用的添加物，使杂交瘤细胞适应并继续生长；用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱，流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE并鉴定其纯度。
上述脂联素单克隆抗体对的制备方法，包括如下步骤：
将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
此外，本发明还保护上述脂联素单克隆抗体和/或脂联素单克隆抗体对，在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。
实施例1
脂联素重组蛋白制备以及分离纯化；
A、合成序列表1的基因序列以及相应亚克隆引物(引物序列如SEQ ID NO.2和SEQID NO.3所述)并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含50ug/ml卡拉霉素和1mg/ml氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h，挑选单菌落PCR和双酶切验证正确后，进行测序，得到测序正确的重组子；
B、将上述正确的三个重组子接种到含3ml LB液体培养基，200rpm，37℃培养8-16h，无菌条件下吸取200ul菌液保种，剩下的2.8ml菌液接种于300ml LB液体培养基(含50ug/ml卡拉霉素和1mg/ml氨苄青霉素)，200rpm，37℃培养12h，然后大提质粒，质粒纯度99％以上，浓度在10ug/ul左右；
C、用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化(电压1500V，时间5ms)线性化质粒于GS115感受态中；涂布150ul转化后的菌液于(1mg/ml G418)RBD固体培养基，30℃培养4-5天；
D、小量表达筛选：从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5ml YPG液体培养基的试管中，30℃，200rpm培养48h，吸取一定量菌液与甘油均匀混合后保存于-20℃备用，将得到7种阳性株接种于1.5ml YPG液体培养基，30℃，200rpm培养24h，然后加入1％甲醇诱导，培养24h再加入1％甲醇诱导，继续培养24h后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量，最高产量约3mg/ml。
E、摇瓶发酵：将产量最高的细胞株经前培养24h，按1％接种量接种至1L的YPG液体培养基中，30℃，200rpm培养24h，然后装入500ml Backman离心瓶，8000rpm，3min离心，用YP(含1％甲醇)重悬菌体，倒入原来的三角瓶中，并加入YP(含1％甲醇)和200ul消泡剂，30℃，200rpm培养72h，每隔24h加入10ml甲醇诱导；最后离心取上清，浓缩后纯化。
F、将上清过Ni-NTA层析柱，然后用0-500mM NaCl(PH8.0)洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
实施例2
脂联素单克隆抗体制备
2.1脂联素重组蛋白免疫小鼠
按每只老鼠50ug脂联素重组蛋白，加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次，每次免疫取50ug酵母表达重组蛋白与弗氏不完全佐剂混匀。免疫四次后测效价，若效价达到1:10000以上，按50ug/mouse腹腔冲击免疫，三天后在无菌条件下取出小鼠脾脏，并制成单个细胞悬液。
2.2细胞融合
制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，按脾细胞：SP2/0＝10:1混合并离心，1000rpm离心5min后滴干混合细胞，轻敲弹松细胞团快。在37℃水浴中加入1mlPEG1450，加完后再37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI—1640终止液。
融合细胞终止后于800rpm离心5min，并吸干残留液体。HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基。
2.3细胞建株
待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价(即阴性对照<1.0，阳性对照>1.0)，从检测为阳性对应的微孔中取阳性细胞进行亚克隆。利用HT培养基通过有限稀释法，将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次取其中阳性细胞重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样单克隆，如果有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得到的的阳性细胞株即为杂交瘤细胞。
2.4无血清培养杂交瘤细胞及纯化抗体
在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例(如20％血清降至15％血清降至10％血清依次到无血清)，逐渐增加代用的添加物，使杂交瘤细胞能够很快适应并继续生长。在制备单克隆抗体时，用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱(HiTrapProtein G,Pharmacia Biotech)流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE鉴定其纯度。
2.5抗体验证
CSB-DA120AmN①；
CSB-DA120AmN③—CSB-DA120AmN④
脂联素单克隆抗体对的制备方法，包括如下步骤：将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对应用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④：
CSB-DA120AmN①轻链可变区部分核苷酸序列如SEQ ID NO.4和SEQ ID NO.5所述：
CSB-DA120AmN①重链可变区部分核苷酸序列如SEQ ID NO.6和SEQ ID NO.7所述：
CSB-DA120AmN③轻链可变区部分核苷酸序列如SEQ ID NO.8和SEQ ID NO.9所述：
CSB-DA120AmN③重链可变区部分核苷酸序列如SEQ ID NO.10和SEQ ID NO.11所述：
CSB-DA120AmN④轻链可变区部分核苷酸序列如SEQ ID NO.12和SEQ ID NO.13所述；
CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15所述。
本发明实施例制备的抗体临床应用效果评价如下：
为验证本发明实施例制备的抗体对适用于临床检测，利用抗体对组合对14例健康志愿者血样和25例糖尿病患者血样进行检测，其检测结果如附图2所示，此结果表明本发明实施例所产生的抗体能区分正常组和病例组样本，能够满足临床检测需求。
检测的实验流程如下：
A、用0.05M PH9.0碳酸盐包被缓冲液将抗体稀释至浓度为1ug/ml，后在每个聚苯乙烯板的反应孔中加0.1ml抗体溶液，4℃过夜。次日，弃去孔内溶液，用洗涤缓冲液洗3次，每次3分钟；
B、加稀释后的待检样本50ul于上述已包被的反应孔中，置37℃孵育1小时，然后洗涤；
C、于个反应孔中，加入稀释的酶标抗体100ul，37℃孵育1小时，然后洗涤；
D、加入化学发光底物液50ul，避光放置于微孔板化学发光仪上，静置5min，依序测量各孔的相对发光单位(RLU)。
由图1～图3的数据结果可知，采用本发明实施例的方法制备的脂联素单克隆抗体，具有稳定、高效等诸多优良特点，适用于作为诊断试剂盒开发的抗体原料，尤其适用于胶乳增强比浊、化学发光、免疫层析等高端诊断试剂盒开发的抗体原料。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 武汉华美生物工程有限公司
<120> 脂联素单克隆抗体、抗体对及其制备方法和用途
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 226
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Glu Thr Thr Thr Gln Gly Pro Gly Val Leu Leu Pro Leu Pro Lys Gly
1 5 10 15
Ala Cys Thr Gly Trp Met Ala Gly Ile Pro Gly His Pro Gly His Asn
20 25 30
Gly Ala Pro Gly Arg Asp Gly Arg Asp Gly Thr Pro Gly Glu Lys Gly
35 40 45
Glu Lys Gly Asp Pro Gly Leu Ile Gly Pro Lys Gly Asp Ile Gly Glu
50 55 60
Thr Gly Val Pro Gly Ala Glu Gly Pro Arg Gly Phe Pro Gly Ile Gln
65 70 75 80
Gly Arg Lys Gly Glu Pro Gly Glu Gly Ala Tyr Val Tyr Arg Ser Ala
85 90 95
Phe Ser Val Gly Leu Glu Thr Tyr Val Thr Ile Pro Asn Met Pro Ile
100 105 110
Arg Phe Thr Lys Ile Phe Tyr Asn Gln Gln Asn His Tyr Asp Gly Ser
115 120 125
Thr Gly Lys Phe His Cys Asn Ile Pro Gly Leu Tyr Tyr Phe Ala Tyr
130 135 140
His Ile Thr Val Tyr Met Lys Asp Val Lys Val Ser Leu Phe Lys Lys
145 150 155 160
Asp Lys Ala Met Leu Phe Thr Tyr Asp Gln Tyr Gln Glu Asn Asn Val
165 170 175
Asp Gln Ala Ser Gly Ser Val Leu Leu His Leu Glu Val Gly Asp Gln
180 185 190
Val Trp Leu Gln Val Tyr Gly Glu Gly Glu Arg Asn Gly Leu Tyr Ala
195 200 205
Asp Asn Asp Asn Asp Ser Thr Phe Thr Gly Phe Leu Leu Tyr His Asp
210 215 220
Thr Asn
225
<210> 2
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Gly Ala Arg Ala Cys Asn Ala Cys Asn Ala Cys Asn Cys Ala Arg Gly
1 5 10 15
Gly Asn Cys Cys Asn Gly Gly Asn Gly
20 25
<210> 3
<211> 25
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Cys Asn Cys Cys Asn Gly Gly Asn Cys Cys Tyr Thr Gly Asn Gly Thr
1 5 10 15
Asn Gly Thr Asn Gly Thr Tyr Thr Cys
20 25
<210> 4
<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Ala Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Cys Thr Gly Gly Gly Ala
35 40 45
Gly Gly Cys Ala Ala Thr Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Ala Ala Gly Cys Cys Ala
65 70 75 80
Ala Gly Ala Cys Ala Thr Thr Ala Ala Cys Cys Ala Gly Thr Ala Thr
85 90 95
Ala Thr Ala Gly Gly Thr Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys
100 105 110
Ala Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly
115 120 125
Thr Cys Cys Thr Ala Gly Gly Cys Cys Gly Cys Thr Cys Ala Thr Ala
130 135 140
Cys Ala Thr Thr Ala Cys Ala Cys Ala Thr Cys Thr Ala Ala Ala Thr
145 150 155 160
Thr Ala Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Gly Ala Gly
195 200 205
Ala Thr Thr Ala Thr Thr Cys Cys Thr Thr Cys Ala Gly Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Cys Thr Thr
245 250 255
Ala Thr Thr Ala Thr Thr Gly Thr Cys Thr Ala Cys Ala Ala Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys Gly Gly Ala Cys Gly
275 280 285
Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Ala Ala
290 295 300
Ala Gly Thr Thr Gly Gly Ala Ala Ala Thr Ala Ala Ala Ala Cys Gly
305 310 315 320
Gly
<210> 5
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Asn Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gln Tyr
20 25 30
Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile
35 40 45
His Tyr Thr Ser Lys Leu Leu Ala Gly Ile Pro Ser Arg Tyr Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 6
<211> 348
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Ala Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Gly Gly Ala Cys Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly
145 150 155 160
Gly Cys Ala Gly Thr Gly Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Thr Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
210 215 220
Cys Ala Thr Gly Ala Ala Ala Ala Thr Cys Cys Thr Gly Thr Thr Cys
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys
245 250 255
Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Ala Gly Cys Cys Ala Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Ala Cys Ala Ala Gly Gly Gly Ala Thr Cys Thr Gly Thr Thr Cys Thr
290 295 300
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys
325 330 335
Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345
<210> 7
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Arg Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Lys Ile Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Asp Leu Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 8
<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Ala Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Cys Thr Gly Gly Gly Ala
35 40 45
Gly Gly Cys Ala Ala Thr Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Ala Ala Gly Cys Cys Ala
65 70 75 80
Ala Gly Ala Cys Ala Thr Thr Ala Ala Cys Cys Ala Gly Thr Ala Thr
85 90 95
Ala Thr Ala Gly Gly Thr Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys
100 105 110
Ala Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly
115 120 125
Thr Cys Cys Thr Ala Gly Gly Cys Cys Gly Cys Thr Cys Ala Thr Ala
130 135 140
Cys Ala Thr Thr Ala Cys Ala Cys Ala Thr Cys Thr Ala Ala Ala Thr
145 150 155 160
Thr Ala Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Gly Ala Gly
195 200 205
Ala Thr Thr Ala Thr Thr Cys Cys Thr Thr Cys Ala Gly Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Cys Thr Thr
245 250 255
Ala Thr Thr Ala Thr Thr Gly Thr Cys Thr Ala Cys Ala Ala Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys Gly Gly Ala Cys Gly
275 280 285
Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Ala Ala
290 295 300
Ala Gly Thr Thr Gly Gly Ala Ala Ala Thr Ala Ala Ala Ala Cys Gly
305 310 315 320
Gly
<210> 9
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Asn Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gln Tyr
20 25 30
Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile
35 40 45
His Tyr Thr Ser Lys Leu Leu Ala Gly Ile Pro Ser Arg Tyr Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 10
<211> 348
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Ala Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Gly Gly Ala Cys Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly
145 150 155 160
Gly Cys Ala Gly Thr Gly Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Thr Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
210 215 220
Cys Ala Thr Gly Ala Ala Ala Ala Thr Cys Cys Thr Gly Thr Thr Cys
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys
245 250 255
Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Ala Gly Cys Cys Ala Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Ala Cys Ala Ala Gly Gly Gly Ala Thr Cys Thr Gly Thr Thr Cys Thr
290 295 300
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys
325 330 335
Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345
<210> 11
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Arg Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Lys Ile Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Asp Leu Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
<210> 12
<211> 321
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Gly Ala Thr Ala Thr Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Cys
1 5 10 15
Ala Gly Thr Cys Thr Cys Cys Ala Thr Cys Cys Thr Cys Ala Cys Thr
20 25 30
Gly Thr Cys Thr Gly Cys Ala Thr Cys Thr Cys Thr Gly Gly Gly Ala
35 40 45
Gly Gly Cys Ala Ala Thr Gly Thr Cys Ala Cys Cys Ala Thr Cys Ala
50 55 60
Cys Thr Thr Gly Cys Ala Ala Gly Gly Cys Ala Ala Gly Cys Cys Ala
65 70 75 80
Ala Gly Ala Cys Ala Thr Thr Ala Ala Cys Cys Ala Gly Thr Ala Thr
85 90 95
Ala Thr Ala Gly Gly Thr Thr Gly Gly Thr Ala Cys Cys Ala Ala Cys
100 105 110
Ala Cys Ala Ala Gly Cys Cys Thr Gly Gly Ala Ala Ala Ala Ala Gly
115 120 125
Thr Cys Cys Thr Ala Gly Gly Cys Cys Gly Cys Thr Cys Ala Thr Ala
130 135 140
Cys Ala Thr Thr Ala Cys Ala Cys Ala Thr Cys Thr Ala Ala Ala Thr
145 150 155 160
Thr Ala Cys Thr Gly Gly Cys Ala Gly Gly Cys Ala Thr Cys Cys Cys
165 170 175
Ala Thr Cys Ala Ala Gly Gly Thr Ala Cys Ala Gly Thr Gly Gly Ala
180 185 190
Ala Gly Thr Gly Gly Gly Thr Cys Thr Gly Gly Gly Ala Gly Ala Gly
195 200 205
Ala Thr Thr Ala Thr Thr Cys Cys Thr Thr Cys Ala Gly Cys Ala Thr
210 215 220
Cys Ala Gly Cys Ala Ala Cys Cys Thr Gly Gly Ala Gly Cys Cys Thr
225 230 235 240
Gly Ala Ala Gly Ala Thr Ala Thr Thr Gly Cys Gly Ala Cys Thr Thr
245 250 255
Ala Thr Thr Ala Thr Thr Gly Thr Cys Thr Ala Cys Ala Ala Thr Ala
260 265 270
Thr Gly Ala Thr Ala Ala Thr Cys Thr Thr Cys Gly Gly Ala Cys Gly
275 280 285
Thr Thr Cys Gly Gly Thr Gly Gly Ala Gly Gly Cys Ala Cys Ala Ala
290 295 300
Ala Gly Thr Thr Gly Gly Ala Ala Ala Thr Ala Ala Ala Ala Cys Gly
305 310 315 320
Gly
<210> 13
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Gly Asn Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Gln Tyr
20 25 30
Ile Gly Trp Tyr Gln His Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile
35 40 45
His Tyr Thr Ser Lys Leu Leu Ala Gly Ile Pro Ser Arg Tyr Ser Gly
50 55 60
Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln Tyr Asp Asn Leu Arg Thr
85 90 95
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 14
<211> 348
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Gly Ala Gly Gly Thr Gly Ala Ala Gly Cys Thr Gly Gly Thr Gly Gly
1 5 10 15
Ala Ala Thr Cys Thr Gly Gly Gly Gly Gly Ala Gly Gly Cys Thr Thr
20 25 30
Ala Gly Thr Gly Ala Ala Gly Cys Cys Thr Gly Gly Ala Gly Gly Gly
35 40 45
Thr Cys Cys Cys Gly Gly Ala Cys Ala Cys Thr Cys Thr Cys Cys Thr
50 55 60
Gly Thr Gly Cys Ala Gly Cys Cys Thr Cys Thr Gly Gly Ala Thr Thr
65 70 75 80
Cys Ala Cys Thr Thr Thr Cys Ala Gly Thr Gly Ala Cys Thr Ala Thr
85 90 95
Gly Gly Ala Ala Thr Gly Cys Ala Cys Thr Gly Gly Gly Thr Cys Cys
100 105 110
Gly Thr Cys Ala Gly Gly Cys Thr Cys Cys Ala Gly Ala Gly Ala Ala
115 120 125
Gly Gly Gly Gly Cys Thr Gly Gly Ala Gly Thr Gly Gly Ala Thr Thr
130 135 140
Gly Cys Ala Thr Ala Cys Ala Thr Thr Ala Gly Thr Ala Gly Thr Gly
145 150 155 160
Gly Cys Ala Gly Thr Gly Gly Thr Ala Cys Cys Ala Thr Cys Thr Ala
165 170 175
Cys Thr Ala Thr Gly Cys Ala Gly Ala Cys Ala Cys Ala Thr Thr Gly
180 185 190
Ala Ala Gly Gly Gly Cys Cys Gly Ala Thr Thr Cys Ala Cys Cys Ala
195 200 205
Thr Cys Thr Cys Cys Ala Gly Ala Gly Ala Cys Ala Ala Thr Gly Cys
210 215 220
Cys Ala Thr Gly Ala Ala Ala Ala Thr Cys Cys Thr Gly Thr Thr Cys
225 230 235 240
Cys Thr Gly Cys Ala Ala Ala Thr Gly Ala Cys Cys Ala Gly Thr Cys
245 250 255
Thr Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly Gly Ala Cys Ala Cys
260 265 270
Ala Gly Cys Cys Ala Thr Thr Thr Ala Thr Thr Ala Cys Thr Gly Thr
275 280 285
Ala Cys Ala Ala Gly Gly Gly Ala Thr Cys Thr Gly Thr Thr Cys Thr
290 295 300
Ala Cys Thr Thr Thr Gly Ala Cys Thr Ala Cys Thr Gly Gly Gly Gly
305 310 315 320
Cys Cys Ala Ala Gly Gly Cys Ala Cys Cys Ala Cys Thr Cys Thr Cys
325 330 335
Ala Cys Ala Gly Thr Cys Thr Cys Cys Thr Cys Ala
340 345
<210> 15
<211> 116
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Arg Thr Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Glu Lys Gly Leu Glu Trp Ile
35 40 45
Ala Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Lys Ile Leu Phe
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ser Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Thr Arg Asp Leu Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu
100 105 110
Thr Val Ser Ser
115
Claims (15)
1.脂联素单克隆抗体，其特征在于：所述脂联素单克隆抗体有三种，所述三种脂联素单克隆抗体分别为CSB-DA120AmN①、CSB-DA120AmN③和CSB-DA120AmN④；
所述CSB-DA120AmN①轻链可变区部分核苷酸序列为SEQ ID NO.4和SEQ ID NO.5，所述CSB-DA120AmN①重链可变区部分核苷酸序列为SEQ ID NO.6和SEQ ID NO.7；
所述CSB-DA120AmN③轻链可变区部分核苷酸序列为SEQ ID NO.8和SEQ ID NO.9，所述CSB-DA120AmN③重链可变区部分核苷酸序列为SEQ ID NO.10和SEQ ID NO.11；
所述CSB-DA120AmN④轻链可变区部分核苷酸序列为SEQ ID NO.12和SEQ ID NO.13，所述CSB-DA120AmN④重链可变区部分核苷酸序列如SEQ ID NO.14和SEQ ID NO.15。
2.如权利要求1所述脂联素单克隆抗体的制备方法，其特征在于包括以下步骤：
步骤1)，采用酵母表达系统表达脂联素重组蛋白，蛋白的序列为SEQ ID NO.1；
步骤2)，分离纯化步骤1)得到的脂联素重组蛋白；
步骤3)，将步骤2)得到的脂联素重组蛋白免疫宿主动物；
步骤4)，获取步骤3)得到的免疫宿主动物的脾脏细胞，并通过脾脏细胞制备融合细胞；
步骤5)，采用步骤4)制得的融合细胞制备脂联素单克隆抗体。
3.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤1)中，采用酵母表达系统表达，包括以下步骤：
步骤a，合成序列SEQ ID NO.1的基因序列以及对应的亚克隆引物，并连接至pPIC9K质粒，得到的连接产物转化到感受态E.coli TOP10，转化后将菌液涂布于含卡拉霉素和氨苄青霉素的LB固体培养基，将培养皿置于37℃培养箱培养12h以上，挑选单菌落PCR和双酶切验证正确后，进行测序验证，得到测序正确的重组子；
步骤b，将步骤a得到的三个重组子接种到含LB液体培养基，37℃培养8～16h，无菌条件下吸取菌液保种，剩余菌液接种于含卡拉霉素和氨苄青霉素的LB液体培养基，37℃培养12h以上，然后大提质粒；
步骤c，用Sac1酶对上述大提质粒进行线性化，37℃水浴过夜后回收线性化质粒，然后用Eppendor电转化仪分别电转化线性化质粒于GS115感受态中，涂布、转化后的菌液于RBD固体培养基，30℃培养4～5天；
步骤d，从RDB平板上挑选20株单菌落，PCR扩增目的基因APM1，琼脂糖凝胶电泳大小正确的条带鉴定为阳性，得到17个阳性菌落，挑取这些菌落接种于1.5mlYPG液体培养基的试管中，30℃，培养48h以上，吸取适量菌液与甘油均匀混合后冷冻保存备用，将得到7种阳性株接种于1.5mlYPG液体培养基，30℃，培养24h以上，然后加入1％甲醇诱导，培养24h以上，再加入1％甲醇诱导，继续培养24h以上，最后离心收集上清，TCA沉淀后，SDS-PAGE分析重组脂联素表达量；
步骤e，将产量最高的细胞株经前培养24h以上，按1％接种量接种至1L的YPG液体培养基中，30℃，培养24h以上，然后装入500ml Backman离心瓶，离心处理，用含1％甲醇的YP重悬菌体，倒入原三角瓶中，并加入含1％甲醇的YP和200ul消泡剂，30℃，培养72h以上，每隔24h加入10ml甲醇诱导，最后离心取上清。
4.根据权利要求3所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤2)中，所述分离纯化，包括以下步骤：将最终得到的上清过Ni-NTA层析柱，然后用0～500mM NaCl洗脱，收集A280吸收峰的蛋白，最后SDS-PAGE分析蛋白纯度，纯化蛋白浓缩后备用。
5.根据权利要求3所述的脂联素单克隆抗体的制备方法，其特征在于，所述引物序列为SEQ ID NO.2和SEQ ID NO.3。
6.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤3)中，所述宿主动物为Balb/C雌性小鼠，所述融合用细胞系SP2/0。
7.根据权利要求6所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤3)中，所述脂联素重组蛋白免疫宿主动物包括以下步骤：以每只老鼠50ug脂联素重组蛋白加等量的弗氏完全佐剂，制成油包水乳剂，免疫6-8周龄Balb/C雌性小鼠；每两周免疫一次所述小鼠，每次免疫取50ug脂联素重组蛋白与弗氏不完全佐剂混匀；免疫四次后测效价，待效价达到1:10000以上时，按50ug/mouse腹腔冲击免疫。
8.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤4)中，所述获取免疫宿主动物的脾脏细胞采用以下步骤，腹腔冲击免疫三天后，在无菌条件下取出小鼠脾脏，并制成单个脾细胞悬液。
9.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤4)中，所述制备融合细胞工序，包括细胞融合和细胞建株，最后得到杂交瘤细胞。
10.根据权利要求9所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤4)中，所述制备融合细胞工序包括以下步骤：
步骤A，细胞融合：制备脾细胞悬液并用PBS洗涤干净后混入SP2/0细胞，脾细胞和SP2/0以10:1的比例混合并离心，离心后滴干混合细胞，轻敲弹松细胞团块，在37℃水浴中加入1ml PEG1450，加完后在37℃水浴中反应2min，沿管壁缓慢加入20ml RPMI-1640终止液，融合细胞终止后于800rpm离心5min，并吸干残留液体，HAT培养基重悬融合细胞，转入96孔细胞培养板培养，待融合细胞在HAT中培养7天左右后换成HT培养基；
步骤B，细胞建株：待孔中融合版细胞长至中等大小约104个细胞以上时开始检测，分别取50ul细胞培养液加入预先包被脂联素蛋白的微孔中，检测细胞上清抗体效价，从检测为阳性对应的微孔取阳性细胞进行亚克隆，利用HT培养基通过有限稀释法将筛选得到细胞稀释至1个/孔，将细胞铺于96孔细胞培养板，待单克隆细胞长至中等大小，密度约104个细胞以上即可检测效价，然后再次选取其中阳性细胞，重复一次亚克隆筛选，待检测到所有微孔中细胞上清都是阳性后，再进行一次同样的单克隆，直至有限稀释法后再次得到全阳性结果，能确定此时筛选到阳性细胞株，所得的阳性细胞株即为杂交瘤细胞。
11.根据权利要求2所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤5)中，融合细胞制备抗体，包括无血清制备和纯化步骤，纯化后得到不同的单克隆抗体。
12.根据权利要求11所述的脂联素单克隆抗体的制备方法，其特征在于，所述步骤5)中，无血清制备包括以下步骤：在有血清条件下复苏杂交瘤细胞，在连续几次细胞传代过程中，梯度减少血清的比例，逐渐增加代用的添加物，使杂交瘤细胞适应并继续生长；用无血清培养基复苏杂交瘤细胞，在细胞状态良好时，加大培养基的用量及培养细胞的容器，待细胞大量死亡时，收夜，并离心取上清，过滤收集，取待纯化的样品上样Protein A-琼脂糖亲和层析柱，流速为0.5ml/min，使抗体与Protein A结合，最后用洗脱液进行洗脱，得到抗体SDS-PAGE并鉴定其纯度。
13.脂联素单克隆抗体对，其特征在于：所述脂联素单克隆抗体对为CSB-DA120AmN③-CSB-DA120AmN④。
14.如权利要求13所述脂联素单克隆抗体对的制备方法，其特征在于，包括如下步骤：将所述的单克隆抗体CSB-DA120AmN③、CSB-DA120AmN④，分别用作捕获抗体和标记抗体，组合成抗体对用于双抗夹心法的免疫检测，将抗体对对各种浓度的脂联素重组蛋白进行检测，然后将单克隆抗体对应的细胞株提取RNA，经RT-PCR反转录得到cDNA，利用小鼠IgG抗体轻链和重链可变区通用引物进行DNA测序，确定抗体的可变区序列。
15.一种权利要求1和/或权利要求13所述的脂联素单克隆抗体和/或脂联素单克隆抗体对，在胶乳增强比浊、双抗夹心法的免疫检测产品中，或在制备检测脂联素的试剂、药剂或试剂盒中的应用。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113671196A (zh) * | 2021-07-29 | 2021-11-19 | 中国人民解放军空军军医大学 | Lair-1分子与脂联素的相互作用对于t细胞活化作用影响的研究方法 |
| CN114149507A (zh) * | 2021-12-31 | 2022-03-08 | 武汉华美生物工程有限公司 | 抗fdp单克隆抗体、抗体对及其制备方法和用途 |
| CN116396384A (zh) * | 2023-01-17 | 2023-07-07 | 武汉爱博泰克生物科技有限公司 | 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒 |
| CN116626309A (zh) * | 2023-06-16 | 2023-08-22 | 浙江伊利康生物技术有限公司 | 一种脂联素检测试剂盒及其制备方法 |
| CN117327177A (zh) * | 2022-06-24 | 2024-01-02 | 东莞市朋志生物科技有限公司 | 抗脂联素抗体、检测脂联素的试剂和试剂盒 |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003055916A2 (en) * | 2001-12-21 | 2003-07-10 | Maxygen Aps | Adiponectin fragments and conjugates |
| WO2004022596A1 (en) * | 2002-09-05 | 2004-03-18 | Komed Co., Ltd. | Monoclonal antibody against adiponectin, preparation method and use thereof |
| US20050074762A1 (en) * | 2001-02-01 | 2005-04-07 | Yusuke Nakamura | Adiponectin-associated protein |
| US20100111977A1 (en) * | 2008-10-15 | 2010-05-06 | 4S3 Bioscience Inc. | Methods and compositions for treatment of myotonic dystrophy |
| US20120070875A1 (en) * | 2009-05-28 | 2012-03-22 | Weisbart Richard H | Amino Acid Sequences which Enhance Peptide Conjugate Solubility |
| CN102517256A (zh) * | 2011-10-12 | 2012-06-27 | 陶凌 | 人脂联素elisa试剂盒 |
| CN104694478A (zh) * | 2014-11-06 | 2015-06-10 | 中国医学科学院北京协和医院 | 抗人脂联素的单克隆抗体及其应用 |
| CN106604740A (zh) * | 2014-02-14 | 2017-04-26 | 宏观基因有限公司 | 用于治疗血管化癌症的改进的方法 |
| CN110372783A (zh) * | 2019-04-15 | 2019-10-25 | 杭州博谱医药科技有限公司 | 一种脂联素抗原、抗体及其胶乳试剂的制备方法以及脂联素检测试剂盒 |
-
2020
- 2020-12-17 CN CN202011504233.7A patent/CN112574306B/zh active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050074762A1 (en) * | 2001-02-01 | 2005-04-07 | Yusuke Nakamura | Adiponectin-associated protein |
| WO2003055916A2 (en) * | 2001-12-21 | 2003-07-10 | Maxygen Aps | Adiponectin fragments and conjugates |
| WO2004022596A1 (en) * | 2002-09-05 | 2004-03-18 | Komed Co., Ltd. | Monoclonal antibody against adiponectin, preparation method and use thereof |
| US20100111977A1 (en) * | 2008-10-15 | 2010-05-06 | 4S3 Bioscience Inc. | Methods and compositions for treatment of myotonic dystrophy |
| US20120070875A1 (en) * | 2009-05-28 | 2012-03-22 | Weisbart Richard H | Amino Acid Sequences which Enhance Peptide Conjugate Solubility |
| CN102517256A (zh) * | 2011-10-12 | 2012-06-27 | 陶凌 | 人脂联素elisa试剂盒 |
| CN106604740A (zh) * | 2014-02-14 | 2017-04-26 | 宏观基因有限公司 | 用于治疗血管化癌症的改进的方法 |
| CN104694478A (zh) * | 2014-11-06 | 2015-06-10 | 中国医学科学院北京协和医院 | 抗人脂联素的单克隆抗体及其应用 |
| CN110372783A (zh) * | 2019-04-15 | 2019-10-25 | 杭州博谱医药科技有限公司 | 一种脂联素抗原、抗体及其胶乳试剂的制备方法以及脂联素检测试剂盒 |
Non-Patent Citations (1)
| Title |
|---|
| 赵满仓等: "脂联素单链抗体基因的克隆及表达", 《河北医药》 * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113671196A (zh) * | 2021-07-29 | 2021-11-19 | 中国人民解放军空军军医大学 | Lair-1分子与脂联素的相互作用对于t细胞活化作用影响的研究方法 |
| CN113671196B (zh) * | 2021-07-29 | 2023-09-12 | 中国人民解放军空军军医大学 | Lair-1分子与脂联素的相互作用对于t细胞活化作用影响的研究方法 |
| CN114149507A (zh) * | 2021-12-31 | 2022-03-08 | 武汉华美生物工程有限公司 | 抗fdp单克隆抗体、抗体对及其制备方法和用途 |
| CN117327177A (zh) * | 2022-06-24 | 2024-01-02 | 东莞市朋志生物科技有限公司 | 抗脂联素抗体、检测脂联素的试剂和试剂盒 |
| CN116396384A (zh) * | 2023-01-17 | 2023-07-07 | 武汉爱博泰克生物科技有限公司 | 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒 |
| CN116396384B (zh) * | 2023-01-17 | 2023-11-17 | 武汉爱博泰克生物科技有限公司 | 针对小鼠脂联素的兔单克隆抗体及其应用和双抗夹心法酶联免疫检测试剂盒 |
| CN116626309A (zh) * | 2023-06-16 | 2023-08-22 | 浙江伊利康生物技术有限公司 | 一种脂联素检测试剂盒及其制备方法 |
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