CN116478934A - 一种杂交瘤细胞、鼠抗人铁蛋白轻链单克隆抗体及其制备方法及应用 - Google Patents
一种杂交瘤细胞、鼠抗人铁蛋白轻链单克隆抗体及其制备方法及应用 Download PDFInfo
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Abstract
本发明提供了一种能分泌特异性好、亲和力强鼠抗人铁蛋白轻链单克隆抗体杂交瘤细胞株,有效解决了现有铁蛋白单克隆抗体质量水平参差不齐,特异性及亲和力不高的问题。采用本发明的杂交瘤细胞获得的鼠抗人铁蛋白轻链单克隆抗体序列,可利用杂交瘤技术和重组技术大量生产抗体,所获得的抗体产物纯度高。所述鼠抗人铁蛋白轻链单克隆抗体可用于制备人铁蛋白轻链的检测试剂盒,特异性强、灵敏度高,可用于自身免疫病、感染性疾病、肿瘤性疾病等的辅助诊断。
Description
技术领域
本发明属于生物领域,具体涉及杂交瘤细胞、该杂交瘤细胞分泌的鼠抗人铁蛋白轻链单克隆抗体、制备方法及应用。
背景技术
铁蛋白是一种普遍存在且高度对称的蛋白质,是体内主要铁储存形式,对于调节体内铁稳态有重要作用,在炎症和氧化应激中起到使细胞免受氧化损伤作用。铁蛋白主要存在于细胞内,它由24个亚基构成蛋白外壳,能够以生物大分子的形式储存多达4500个三价铁离子。其亚基有两种类型,分别称为铁蛋白重链(H链,21kDa)和铁蛋白轻链(L链,19kDa),这两个亚基在氨基酸水平上具有55%的同一性,并采用相似的三维结构,允许它们共同组装成相同的铁蛋白分子。铁蛋白分子是含有这两个亚基的不同比例的杂聚物。在不同的组织中,H和L亚基的比例可能有所不同,在肝脏和脾脏等组织中占主导地位的是铁蛋白L链(FTL),而心脏和肾脏中以铁蛋白H链(FTH)为主。
FTL作为生物体内重要的内源性储铁蛋白的亚基,其广泛分布于细胞核、细胞质以及溶酶体,参与细胞的多项生理学功能,如自噬,氧化应激铁平衡,中性粒细胞脱颗粒作用,参与细胞骨架,对细胞损伤的反应。其通过直接影响细胞内外的铁离子浓度,改变细胞的氧化应激水平及通过转录调控水平影响相关神经退行性变相关典型蛋白质的转录修饰等分子细胞学功能影响到细胞状态而对疾病的发生发展产生相应影响。多项研究表明FTL异常与自身免疫病、感染性疾病、肿瘤性疾病等并发的免疫异常密切相关。
免疫学检测由于其灵敏度高,速度快的优势,已经在检测人血清铁蛋白上得到越来越广泛的应用。血清铁蛋白免疫学检测试剂盒质量的最主要制约因素就是生物活性原材料的质量。从方法学上来说,血清铁蛋白检测试剂盒大多采用双抗体夹心法,其生物活性原材料就是铁蛋白单克隆抗体,抗体性能是决定着整个检测盒灵敏度、特异性和准确性的最重要因素。市场现有的铁蛋白单克隆抗体质量水平参差不齐,特异性及亲和力不高。
发明内容
为了解决的现有技术中鼠抗人铁蛋白轻链单克隆抗体特异性以及亲和力不高的问题,本发明提供了一种杂交瘤细胞,用于制备高质量的鼠抗人铁蛋白轻链单克隆抗体。
本发明的目的是通过以下措施实现的:
本发明提供了一种杂交瘤细胞,保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:2022356,保藏日期为:2022年11月29号。该细胞是将人FTL蛋白免疫6-8周龄昆明白小鼠免疫小鼠,3次免疫后选择血清效价高的小鼠进行骨髓瘤(SP2/0)细胞和脾脾脏淋巴细胞进行电融合,间接ELISA方法检测阳性单克隆,最终获得特异性分泌鼠抗人铁蛋白轻链单克隆抗体的杂交瘤细胞株,该保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:2022356,保藏日期为:2022年11月29号。
本发明提供了一种鼠抗人铁蛋白轻链单克隆抗体,序列来源于上述杂交瘤细胞分泌的抗体,能特异性结合人铁蛋白轻链抗原。
本发明还提供了一种鼠抗人铁蛋白轻链单克隆抗体的制备方法,包含以下步骤:
步骤1,重组质粒制备:将权利要求1所述杂交瘤细胞用5’RACE测序列获得抗体的重链和轻链序列,将重链和轻链序列分别连接入真核表达载体pcDNA3.4中获得重组质粒。
步骤2,抗体表达:将重组重链和轻链质粒各10μg转染293F细胞,置于震动培养箱中培养,当活细胞量下降至60%左右时,400×g离心20min收取细胞上清液。
步骤3,抗体纯化:利用HITRAP PROTEIN A HP进行上清中蛋白的纯化,再将纯化后的蛋白用PBS透析过夜,超滤管浓缩并调整浓度至(0.1-1mg/mL)。
步骤4,抗体进行SDS-PAGE电泳鉴定。
本发明还提供了一种上述鼠抗人铁蛋白轻链单克隆抗体在制备检测人铁蛋白轻链试剂盒中的应用。
本发明还提供了一种检测人铁蛋白轻链的试剂盒,以上述鼠抗人铁蛋白轻链单克隆抗体作为检测抗体。
本发明还提供了一种检测人铁蛋白轻链的胶体金试剂条,以上述鼠抗人铁蛋白轻链单克隆抗体作为检测抗体或包被抗体。
有益效果
1、本发明基于现有的铁蛋白单克隆抗体质量水平参差不齐,特异性及亲和力不高的问题,筛选到了一种杂交瘤细胞株,不仅能分泌鼠抗人铁蛋白轻链单克隆抗体,而且得到的鼠抗人铁蛋白轻链单克隆抗体特异性好,亲和力强。
2、本发明将所述杂交瘤细胞株分泌的鼠抗人铁蛋白轻链单克隆抗体用于检测,所获取的检测试剂盒,检测人铁蛋白轻链特异性强、灵敏度高,可用于自身免疫病、感染性疾病、肿瘤性疾病等的辅助诊断。
3、采用本发明的杂交瘤细胞获得的抗体序列,可利用杂交瘤技术和重组技术大量生产抗体,所获得的抗体产物纯度高,达95%以上。
附图说明
图1.校准品检测杂交瘤细胞上清特异性的胶体金试剂条检测图(1:1-1A、2:1-6F、3:1-7D、4:1-12C、5:2-6C、6:2-6D、7:3-2F;从左向右依次为:稀释液、5ng/mL、15ng/mL、25ng/mL、50ng/mL、100ng/mL、250ng/mL、500ng/mL)
图2.抗原检测杂交瘤细胞上清特异性的胶体金试剂条检测图(1:1-1A、2:1-6F、3:1-7D、4:1-12C、5:2-6C、6:2-6D、7:3-2F;从左向右依次为:稀释液、稀释液、1000ng/mL、1000ng/mL、500ng/mL、500ng/mL、100ng/mL、100ng/mL、50ng/mL、50ng/mL、10ng/mL、10ng/mL)
图3.重组抗体SDS-PAGE电泳图(M:Marker;1:2-6C;2:2-6D;3:3-2F)
图4.重组抗体亲和力检测结果(KD)
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的实验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1
1.制备杂交瘤细胞
1)动物免疫
利用人FTL蛋白免疫6-8周龄昆明白小鼠,50μg/次/只,3次免疫后采集小鼠眼眶血,分离血清后用间接ELISA法进行免疫效价检测,选取血清效价高的小鼠进行加强免疫,50μg/只,4天后进行杂交瘤融合实验。
2)杂交瘤细胞融合及筛选
A.小鼠骨髓瘤(SP2/0)细胞培养:复苏SP2/0细胞,置于37℃,5%CO2的培养箱中培养,传代3次后调整细胞密度,当细胞密度达到70%-80%,留待使用。
B.脾脏淋巴细胞单细胞悬液制备:加强免疫第4天处死小鼠,分离小鼠脾脏,将小鼠脾脏轻轻研磨,使脾脏组织分散为单细胞悬液,研磨结束后,用移液枪取10mL RPMI 1640基础培养基冲洗细胞筛网(40μm),使上面的细胞通过筛网流至下面的培养皿中,并混匀所得细胞液,1200rpm离心5min,收集脾脏淋巴细胞。
C.杂交瘤细胞电融合:将步骤A培养的SP2/0细胞与步骤B得到的脾脏淋巴细胞按照1:5进行混合,1200rpm离心5min,弃上清,用RPMI 1640基础培养基重悬细胞沉淀,1200rpm离心5min,离心后弃上清,用电融合液10mL重悬细胞,1200rpm离心5min,最后再用9mL电融合液重悬细胞,将细胞悬液加入电融合槽,设置电压参数(800V,40μs,1次),点击融合按钮,进行电融合。
3)杂交瘤细胞筛选
A.获得单克隆杂交瘤细胞:将上一步得到的杂交瘤融合细胞转移至加有HAT的半固体培养基中,混匀后加入10cm培养皿,放于37℃,5%CO2培养箱中培养,7-10天后挑取单个克隆至含有HT培养基的96孔板中,继续培养2-3天用于后续检测。
B.筛选杂交瘤细胞:采用间接ELISA方法检测阳性单克隆杂交瘤细胞分泌抗体,包被抗原为FTL蛋白,检测样品为杂交瘤上清,酶标二抗为antimouse IgG(HRP),将检测阳性的单克隆从96孔板传至24孔板,待细胞生长占孔底面积的75%时,取细胞上清进行抗体配对检测。
共计挑取288个杂交瘤克隆进行ELISA检测,初步筛选出7株OD450nm>1.0的杂交瘤细胞(表1)。
表1杂交瘤上清与FTL蛋白结合ELISA检测结果
4)杂交瘤细胞上清特异性检测
将上述7株杂交瘤细胞上清进行抗体配对检测,评估其特异性和灵敏度。将筛选出的1-1A、1-6F、1-7D、1-12C、2-6C、2-6D、3-2F共7株杂交瘤细胞上清进行标记,喷金,用胶体金FER正常生产的抗体作为包被抗体进行划膜,将FER校准品(定值血样)稀释至500ng/mL、250ng/mL、50ng/mL、25ng/mL、15ng/mL、5ng/mL进行检测,胶体金免疫层析分析仪(单通道,PMDT8000)分析试剂卡,T线颜色越深,检测值越高。结果如表2所示,2-6C、2-6D、3-2F这三株杂交瘤细胞上清的检测值有梯度,而1-1A、1-6F、1-7D、1-12C这四株杂交瘤细胞上清的检测值无梯度,C线很浅或不显色(图1),可能是这四株杂交瘤细胞上清中含有的杂蛋白与胶体金进行了结合,或者FER抗体浓度太低没有结合上造成。
表2杂交瘤细胞上清特异性检测(校准品)
再对7株杂交瘤细胞上清进行抗原检测,即将7株杂交瘤细胞上清进行标记,喷金,用胶体金FER正常生产的抗体作为包被抗体进行划膜,将FER抗原(2mg/mL)稀释至1000ng/mL、500ng/mL、100ng/mL、50ng/mL、10ng/mL,同上进行检测。结果如表3所述,1-12C、2-6C、2-6D和3-2F这四株杂交瘤细胞上清的检测值梯度和CV较好,而1-1A、1-6F和1-7D的检测值梯度较差或无梯度,C线很浅或不显色(图2),可能是杂交瘤细胞里面的杂蛋白与胶体金进行了结合,而FER抗体浓度太低没有结合上造成,因此结合校准品和抗原的检测结果,最终筛选2-6C、2-6D和3-2F这3株杂交瘤细胞进行亲和力检测。
上述杂交瘤细胞分泌的单克隆抗体还可作为包被抗体,用于检测人铁蛋白轻链的胶体金检测试剂条。
表3杂交瘤细胞上清特异性检测(抗原)
实施例2制备鼠抗人铁蛋白轻链单克隆抗体
1.载体构建:将筛选的3株杂交瘤细胞用5’RACE方法进行序列测定,获得抗体的重链和轻链序列,将获得的重链和轻链序列分别连接入真核表达载体pcDNA3.4中获得重组质粒。
2.抗体小量表达:取对数生长期的293F细胞按照0.8×106cells/mL密度进行传代。第二天,待细胞长到1.5×106cells/mL,并且细胞活率大于95%后,将细胞稀释到1×106cells/mL后进行转染。取2个无菌的2mL EP管,一个加入200μL Opti-MEM培养液和重组重链和轻链质粒各10μg,另一个加入200μLOpti-MEM培养液和60μL转染试剂,各自混匀后室温孵育5min。再将混匀的PEI溶液快速加入混匀的重组质粒DNA中,用移液枪轻轻吹打混匀,室温孵育15min,再逐滴加入准备好的293F细胞中,边加边轻轻震荡细胞。将转染好的细胞至于震动培养箱中培养,每天用台盼蓝染色法计算细胞活率,当活细胞量下降至60%左右时,400×g离心20min收取细胞上清液。
3.抗体纯化:利用HITRAP PROTEIN A HP进行上清中蛋白的纯化。先用5倍柱体积的超纯水冲洗柱子,再用5倍柱体积的结合缓冲液(20mmol/L PB buffer,0.2mol/L NaCl,PH=7.2)平衡柱子。将收集的细胞上清用0.45μm滤膜过滤后与等体积的结合缓冲液混合,再上样,流速为1mL/min,收集流穿液。用10倍柱体积的结合缓冲液洗柱子,直到紫外和电导均为基线。用洗脱缓冲液(100mmol/L甘氨酸,PH=2.7)洗脱至中和缓冲液(1mol/L Tris-HCl,PH=9.0)中,流速1mL/min。洗脱后立即用10倍柱体积的结合缓冲液重新平衡柱子。再将纯化后的蛋白用PBS透析过夜后用超滤管浓缩并调整至(0.1-1mg/mL)。
将3个重组表达及纯化后的抗体进行SDS-PAGE电泳,结果显示,3个抗体经过还原Buffer处理后,重链均在50Kda左右,轻链在25Kda左右(图3),符合预期大小,且抗体纯度都在95%以上。
实施例3:抗体的特异性和亲和力检测
1.采用间接ELISA方法检测重组表达的单克隆抗体对抗原的结合能力:用包被液将FTL蛋白稀释至2μg/mL,100μL/孔,4度包被过夜。取出包被好的ELISA板,PBST(取250μL吐温-20至500mL PBS中)洗涤三次,在含1% BSA的PBS(取1g BSA溶解至100mL PBS)中37度封闭1h。取出封闭好的ELISA板,PBST洗涤三次,加入重组表达的抗体(稀释100倍),设置免疫小鼠和免疫前小鼠的血清为阳性和阴性对照(稀释100倍),加含1% BSA的PBS为空白对照,37℃孵育2h。取出孵育好的ELISA板,PBST洗涤三次,anti mouse IgG(HRP)(1:5000,用1%BSA PBS稀释),37度孵育2h。取出封闭好的ELISA板,PBST洗涤三次,加入TMB显色15min,用终止液(2.5M H2SO4)终止,酶标仪读取OD450数值。
根据表4中的ELISA检测结果可知,将重组表达抗体2-6C、2-6D、3-2F稀释至0.1mg/mL,3个重组抗体都能与FTL蛋白结合,但2-6D结合优于2-6C和3-2F。
表4重组抗体结合的ELISA检测
2.单克隆抗体亲和力检测
将纯化后的3个单克隆抗体进行KD检测,利用ForteBio Octet分子相互作用技术平台,将纯化后的3个单克隆抗体分别固相化到Anti Mouse IgG Fc传感器上,分别经过5个不同浓度(0、25、50、75或100nmol/L)FTL蛋白结合,计算KD值。
根据抗体亲和力的结果(图4)可知,2-6C、2-6D、3-2F 3个重组表达的抗体亲和力分别为:1.4×10-8M、2.8×10-9M、5.9×10-9M,其中2-6D亲和力最好,因此,综合ELISA和亲和力的结果,优选2-6D杂交瘤细胞株进行保藏。
Claims (6)
1.一种杂交瘤细胞,其特征在于:保藏号为CCTCC No:2022356。
2.一种鼠抗人铁蛋白轻链单克隆抗体,其特征在于:所述抗体来源于权利要求1所述杂交瘤细胞。
3.一种鼠抗人铁蛋白轻链单克隆抗体的制备方法,包含以下步骤:
步骤1,重组质粒制备:将权利要求1 所述杂交瘤细胞用5’ RACE 测序列获得抗体的重链和轻链序列,将重链和轻链序列分别连接入真核表达载体pcDNA3.4 中获得重组质粒;
步骤2,抗体表达:将重组重链和轻链质粒各10μg 转染293F 细胞,置于震动培养箱中培养,当活细胞量下降至60%左右时,400×g 离心20 min 收取细胞上清液;
步骤3, 抗体纯化:利用HITRAP PROTEIN A HP 进行上清中蛋白的纯化,再将纯化后的蛋白用PBS 透析过夜,超滤管浓缩并调整至0.1-1mg/mL;
步骤4, 抗体进行SDS-PAGE 电泳鉴定。
4.一种如权利要求1 所述杂交瘤细胞和/或权利要求2 所述抗体在制备检测人铁蛋白轻链的试剂盒中的应用。
5.一种检测人铁蛋白轻链的试剂盒,其特征在于:包括权利要求2 所述单克隆抗体。
6.一种检测人铁蛋白轻链的胶体金检测试剂条,其特征在于:检测抗体或包被抗体为权利要求2 所述单克隆抗体。
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