CN115287266A - 一种靶向于猪RORγt的单克隆抗体及其杂交瘤细胞株和应用 - Google Patents
一种靶向于猪RORγt的单克隆抗体及其杂交瘤细胞株和应用 Download PDFInfo
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Abstract
本发明提供了一种靶向于猪RORγt的单克隆抗体及其杂交瘤细胞株和应用，属于医药技术领域。本发明提供了一种靶向于猪RORγt的单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株保藏于中国典型培养物保藏中心，地址：中国.武汉.武汉大学，保藏日期为：2022年7月27日，保藏编号为：CCTCCNO：C2022219。本发明提供的杂交瘤细胞株用于生产靶向于猪RORγt的单克隆抗体，所得抗体经验证对猪RORγt蛋白具有较高的特异性。
Description
技术领域
本发明涉及医药技术领域，尤其涉及一种靶向于猪RORγt的单克隆抗体及其杂交瘤细胞株和应用。
背景技术
视黄酸相关的孤儿受体(RAR-relatedorphanreceptor，RORs)是核受体的一个家族，是细胞内转录因子的一员。有三个ROR家族成员：ROR-α、ROR-β和ROR-γ分别由基因RORA、RORB、RORC编码。ROR调节了几种重要的生理过程，并涉及许多病症。ROR-α对小脑发育和骨形成至关重要，而ROR-β调节脑和视网膜的功能，ROR-γ在淋巴发育和胸腺生成中起关键作用。其中RORγ又可以分为RORγ1和RORγ2(RORγt)，它们表现出不同的组织特异性，RORγ1表达于多种组织，包括肝脏、脂肪、骨骼肌和肾脏。RORγt主要表达于免疫系统的一些不同类型细胞，如促进Th17细胞分化并产生关键因子IL-17，在自身免疫疾病的发生和发展中发挥关键作用。RORs的不寻常之处在于它们是以单体形式结合到激素反应元件上的，而其它大部分核受体都是以二聚体形式发挥作用。
在小鼠中对类似基因的研究表明，RORγt基因可能对淋巴器官发生至关重要，并且可能在胸腺生成中发挥重要的调节作用。此外，小鼠研究表明，该基因编码的蛋白质可能抑制Fas配体和IL2的表达。已为该基因发现了两种编码不同同种型的转录变体。目前，RORγt被认为是抗自身免疫疾病，特别是银屑病药物研发的新靶点，RORγt小分子抑制剂最近几年已成为学术界乃至国际制药公司研究的热点领域，极具开发潜力。其相关通路包括免疫应答IL-23信号通路和Th17细胞分化。与该基因相关的基因本体(GO)注释包括DNA结合转录因子活性和类固醇激素受体活性。
因此，本领域仍需要开发能够与RORγt高亲和力结合，并且能不与ROR-α、ROR-β发生反应，可以特异性结合RORγt受体的抗体。
发明内容
本发明的目的在于提供一种对RORγt具有高亲和力、高生物活性的单克隆抗体及杂交瘤细胞株和应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种生产靶向于猪RORγt的单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株保藏于中国典型培养物保藏中心，地址：中国.武汉.武汉大学，保藏日期为：2022年7月27日，保藏编号为：CCTCC NO：C2022219。
本发明还提供了一种所述的杂交瘤细胞株生产的靶向于猪RORγt的单克隆抗体。
本发明还提供了一种所述的单克隆抗体在制备检测猪RORγt基因表达蛋白的试剂中的应用。
本发明提供的杂交瘤细胞株用于生产靶向于猪RORγt的单克隆抗体，所得抗体经验证不仅对猪RORγt蛋白具有较高的特异性，而且还具有高亲和力、高生物活性的特点，猪RORγt单克隆抗体能够与猪RORγt蛋白高亲和力结合，可用于ELISA和FLOW中对于转录因子RORγt基因表达蛋白的检测。
附图说明
图1为实施例2中猪RORγt单克隆抗体的聚丙烯酰胺电泳图；
图2为实施例2中猪RORγt单克隆抗体的Western实验结果；
图3为实施例3中猪RORγt单克隆抗体的流式细胞检验结果；
图4为实施例4中猪RORγt单克隆抗体的免疫荧光检验结果；
图5为实施例4中图4的细节位置展示图。
具体实施方式
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
保藏说明
杂交瘤细胞株11F4，保藏于中国典型培养物保藏中心，地址：中国.武汉.武汉大学，保藏日期为：2022年7月27日，保藏编号为：CCTCC NO：C2022219。
实施例1
免疫血清的制备
选择5只6周龄雌性BalB/C小鼠，对小鼠进行标记并编号(1～5号)。然后进行首次免疫，将抗原蛋白HisSumo-RORC(上海捷禺生物科技有限公司提供，编号P300271)与弗氏完全佐剂(厂家Sigma，货号F5881)等体积混合，进行抗原乳化，用于皮下注射，每只小鼠100μl。两周后进行第二次免疫，将抗原蛋白HisSumo-RORC与弗氏不完全佐剂(厂家Sigma，货号F5506)等体积混合，进行抗原乳化，用于皮下注射，每只小鼠50μl(二免后第5天3号小鼠死亡，属于实验过程中可预期的死亡)。第二次免疫一周后对剩余的四只小鼠进行第三次免疫，将抗原蛋白HisSumo-RORC与弗氏不完全佐剂等体积混合，进行抗原乳化，用于皮下注射，每只小鼠50μl(三免后第4天5号小鼠死亡，属于实验过程中可预期的死亡)。第三次免疫一周后，对剩余的三只小鼠尾静脉采血40μl，凝血后离心收集免疫血清。用ELISA检测免疫血清的效价，以未经免疫的小鼠血清为空白血清。效价检测结果如表1所示：
表1三次免疫后剩余3只小鼠的血清效价
| 小鼠编号 | 1号 | 2号 | 4号 | 空白血清 |
| 1/1000 | 2.0044 | 1.9703 | 1.9021 | 0.1046 |
| 1/3000 | 1.9011 | 1.8601 | 1.8024 | 0.0833 |
| 1/9000 | 1.8633 | 1.6803 | 1.5554 | 0.0705 |
| 1/27000 | 1.3096 | 1.2699 | 1.1975 | 0.0669 |
| 1/81000 | 0.9852 | 0.8212 | 0.8023 | 0.0660 |
| 1/243000 | 0.4484 | 0.3864 | 0.3550 | 0.0614 |
| 1/792000 | 0.2237 | 0.2114 | 0.1876 | 0.0430 |
结果显示，经三次免疫后的3只小鼠血清效价相近，均可达到1:792000，满足后续细胞融合的要求。
细胞融合
在细胞融合的前一周准备小鼠骨髓瘤细胞SP2/0，调整细胞状态至对数生长期，并选择ELISA效价最高的1号小鼠，进行加强免疫(HisSumo-RORC与弗氏不完全佐剂等体积混合，50μl/只，腹腔注射)。
一周后，取小鼠脾脏，机械破碎后收集脾细胞，200目筛网过滤，PBS洗涤3次；收集小鼠骨髓瘤细胞SP2/0，PBS洗涤3次；分别对小鼠脾细胞和SP2/0细胞进行计数，然后按SP2/0：脾细胞＝1：2的比例进行混合，混合后离心弃去PBS，使用电融合仪进行细胞融合，融合结束，细胞离心后加入含HAT(厂商Gibco，货号20160017)的完全培养基，重悬混匀细胞后铺板于96孔板中，融合后5天进行一次全换液，继续培养3天用于杂交瘤细胞的筛选。
杂交瘤细胞的筛选采用ELISA检测法，筛选单克隆阳性孔，经过初筛、复筛、反筛，得到本发明的杂交瘤细胞株，并将其命名为11F4，保藏于中国典型培养物保藏中心，地址：中国.武汉.武汉大学，保藏日期为：2022年7月27日，保藏编号为：CCTCC NO：C2022219。
实施例2抗体的制备
将实施例1筛选得到的杂交瘤细胞注射至小鼠腹腔，1×106个细胞/只小鼠，10天后小鼠腹腔明显膨胀后处死小鼠，收集腹水，并进行SDS-PAGE检测，检测结果如图1所示。从图1中可以看出，电泳结果中出现了两个条带，它们是所制备抗体的重链和轻链，分别为50kDa和21kDa，与猪RORγt抗体预计的蛋白大小一致，表明本发明制备得到的RORγt单克隆抗体为猪RORγt单克隆抗体，且该RORγt单克隆抗体可以与猪RORγt蛋白在体外特异性结合。
将制备得到的猪RORγt单克隆抗体纯化后检测抗体效价，结果如表2所示。从表2可知，本发明提供的猪RORγt单克隆抗体效价良好。
表2纯化的猪RORγt单克隆抗体效价检测结果(间接ElisaA450)
并对纯化的猪RORγt单克隆抗体进行Western实验，结果如图2所示，用猪RORγt单克隆抗体孵育猪RORγt抗原，可见70kDa处有明显条带，与猪RORγt蛋白大小完全符合，表明本发明制备的猪RORγt单克隆抗体能够与猪RORγt蛋白结合，Western效价良好。
实施例3
使用Abcam公司的抗体偶联试剂盒(ab102918)，在猪RORγt单克隆抗体上偶联藻红蛋白，得到藻红蛋白偶联RORγt抗体。
在超净台中，用眼科剪，眼科镊(已高压灭菌)剥离猪脾脏，将多余的脂肪摘除。将叠好的200目无菌滤网后放置于无菌小平皿中，加入1mL RPMI-1640培养液。取20g猪脾脏组织放入滤网中，用无菌1mL注射器末端轻轻研磨，直到研磨充分后，将液体吸到1.5mL EP管中，放入预冷的离心机中，4℃，2000rpm，离心5min。然后弃上清获得淋巴细胞。再用FACSbuffer洗涤两次，最后用1mLPBS重悬细胞，稀释后细胞计数板计数。
取1×106个细胞加入1μL的CD16/32(BD，553142)在4℃状态下孵5min；2mLFACS(invitrogen，2270788)洗液重悬细胞之后用eBioscienceTM Foxp3/TranscriptionFactorStaining Buffer Set(invitrogen，2254161)进行固定破膜处理，避光4℃45min，加入2mLFACS洗液清洗；加入0.5μL的藻红蛋白偶联RORγt抗体在4℃状态下孵育25min，然后加入2mLFACS洗液重悬细胞之后上机检测。
结果如图3所示。图3中的对照为同型对照，同型对照是使用与猪RORγt单克隆抗体相同种属来源、相同亚型、相同剂量和相同的免疫球蛋白及亚型的免疫球蛋白，用于消除由于抗体非特异性与细胞结合而产生的背景染色。从图3的实验结果中可以看出，本发明提供的猪RORγt单克隆抗体能够检测出猪RORγt蛋白，表明本发明提供的猪RORγt单克隆抗体是猪RORγt蛋白的特异性抗体，能够用于猪RORγt蛋白的流式检测。
实施例4
采用本发明提供的猪RORγt单克隆抗体作为一抗进行免疫荧光实验，结果如图4、图5所示(图5为图4的细节位置展示图)。从图4的猪肠道的切片染色结果中可以看出，本发明提供的猪RORγt单克隆抗体在猪肠道固有层中表达，这与预期结果一致，表达位置正确。且与白细胞共表达抗原CD45共定位，可以证明该抗体可以用于免疫荧光实验中对猪RORγt蛋白的验证。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
Claims (3)
1.一种生产靶向于猪RORγt的单克隆抗体的杂交瘤细胞株，其特征在于，所述杂交瘤细胞株保藏于中国典型培养物保藏中心，地址：中国.武汉.武汉大学，保藏日期为：2022年7月27日，保藏编号为：CCTCC NO：C2022219。
2.一种权利要求1所述的杂交瘤细胞株生产的靶向于猪RORγt的单克隆抗体。
3.一种权利要求2所述的单克隆抗体在制备检测猪RORγt基因表达蛋白的试剂中的应用。
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