CN114395039A - 一种针对人细胞周期蛋白1的单克隆抗体及其制备方法、免疫检测试剂及应用 - Google Patents
一种针对人细胞周期蛋白1的单克隆抗体及其制备方法、免疫检测试剂及应用 Download PDFInfo
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Abstract
本发明属于单克隆抗体领域，具体涉及一种针对人细胞周期蛋白1(Cyclin D1)的单克隆抗体及其制备方法、免疫检测试剂及应用。该针对Cyclin D1的单克隆抗体包含氨基酸序列如SEQ NO ID:1‑3所示的VHCDR1、VHCDR2和VHCDR3，和氨基酸序列如SEQ ID NO:4‑6所示的VLCDR1、VLCDR2和VLCDR3。本发明的单克隆抗体，具有较好的特异性与亲和力，能够准确识别石蜡组织与冰冻组织中表达的Cyclin D1蛋白。并且在较低的浓度下，可达到与进口试剂SP4兔单克隆单体一致或更好的染色效果，预示该单克隆抗体具有更好的亲和力，可替代进口试剂使用。
Description
技术领域
本发明属于单克隆抗体领域，具体涉及一种针对人细胞周期蛋白1(Cyclin D1)的单克隆抗体及其制备方法、免疫检测试剂及应用。
背景技术
Cyclin D1的编码基因位于11q13上，有5个外显子，全长约15kb。与其他Cyclin相比，Cyclin D1的N末端缺少一个"见解盒"片段，因此是最小的Cyclin。Cyclin D1是一种与细胞增殖密切相关的周期蛋白，是细胞周期中与恶性肿瘤关系最为密切的癌基因之一，可作用于G1期细胞，在调节细胞周期G1/S调控点中有重要作用。Cyclin D1高表达能加速细胞增生分化，通过激活CdK4或CdK6等作用，促进DNA合成，加速细胞增殖，还能经由调节基因转录程序对染色体稳定性产生影响，致使染色体不稳定及肿瘤发生。
研究表明CyclinD1与淋巴瘤、乳腺癌、前列腺癌、肝癌和肺癌等肿瘤发生发展密切相关，在肿瘤组织中高表达。针对CyclinD1的病理诊断，无论是在临床还是在药物开发、病理研究等领域都具有重要意义。
目前市场上用于病例诊断的CyclinD1单抗均为进口试剂，国际质控委员会NordiQC对Cyclin D1单抗的质控评比活动中(如图1所示)，克隆号为SP4与EP12的兔单克隆抗体具有较好的表现，以兔单克隆抗体SP4为例，其在免疫诊断时的应用效果优秀，是世界范围内广泛应用的单抗品种之一。目前，EP12、SP4的售价较高，这大大增加了国内临床诊断及研究成本，不利于国内CyclinD1病理诊断及相关研究的长期高效发展。
发明内容
本发明的目的是提供一种针对人细胞周期蛋白1的单克隆抗体，以准确识别石蜡组织与冰冻组织中表达的Cyclin D1蛋白，具有较好的特异性与亲和力。
本发明的第二个目的是提供含有上述单克隆抗体的免疫检测试剂。
本发明的第三个目的是提供上述单克隆抗体在免疫组化方面的应用。
本发明的第四个目的是提供上述单克隆抗体的制备方法。
为了实现以上目的，本发明所采用的技术方案是：
一种针对人细胞周期蛋白1的单克隆抗体，其包含氨基酸序列如SEQ NO ID:1-3所示的VHCDR1、VHCDR2和VHCDR3，和氨基酸序列如SEQ ID NO:4-6所示的VLCDR1、VLCDR2和VLCDR3。
VHCDR1的氨基酸序列为GFSLSSYD，VHCDR2的氨基酸序列为IWNSGGA，VHCDR3的氨基酸序列为AG。
VLCDR1的氨基酸序列为EPWYNS，VLCDR2的氨基酸序列为PAS。VHCDR3的氨基酸序列为LQMDSKDITD。
本发明的针对人细胞周期蛋白1的单克隆抗体，具有较好的特异性与亲和力，能够准确识别石蜡组织与冰冻组织中表达的Cyclin D1蛋白。并且在较低的浓度下，可达到与进口试剂SP4兔单克隆单体一致或更好的染色效果，预示该单克隆抗体具有更好的亲和力，可替代进口试剂使用，有利于大幅降低CyclinD1的临床诊断成本。
优选地，所述单克隆抗体包含氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区。采用上述重链可变区、轻链可变区的单克隆抗体，可进一步优化单克隆抗体在免疫组化方面的应用。
一种含有上述单克隆抗体的免疫检测试剂。
在以上单克隆抗体的基础上，可按常规方式构建免疫检测试剂，如抗体工作液、免疫分析试剂盒等，从而使上述单克隆抗体可方便的用于CyclinD1的免疫分析。
上述针对人细胞周期蛋白1的单克隆抗体在免疫组化方面的应用。
免疫组化实验表明，在检测结果的一致性方面，上述人细胞周期蛋白1的单克隆抗体与进口试剂SP4相同。亲和力方面，上述单克隆抗体在较低浓度下即可达到一致或更好的染色效果，具有良好的亲和力。
一种针对人细胞周期蛋白1的单克隆抗体的制备方法，包括以下步骤：
(1)使用人细胞周期蛋白1免疫原免疫兔子，采集兔外周血，收集兔外周血单个核细胞，分离出抗原特异性B淋巴细胞；
(2)针对步骤(1)得到的抗原特异性B淋巴细胞，经过培养鉴定筛选出阳性细胞孔，进而获取抗体重链、轻链基因，扩增抗体重链、轻链目的基因，经重组、表达获得兔源单克隆抗体。
本发明的针对人细胞周期蛋白1的单克隆抗体的制备方法，以兔子为免疫动物，兔子个体免疫器官发达，可耐受的免疫剂量较高，便于免疫效果评价和验证，最终筛选获得实际应用效果较佳的优势抗体。
可采用人细胞周期蛋白1为单独免疫原免疫兔子。为进一步提高单克隆抗体的筛选效率，优选地，步骤(1)中，所述人细胞周期蛋白1免疫原包括MLH1、CyclinD1、p57、CK19和WT1。
优选地，步骤(2)中，在抗体重链、轻链基因5’端均增加一段如SEQ ID NO：13所示的核苷酸序列，然后进行所述扩增。在上游5’端均同时增加的有一段可增加表达量的信号区碱基序列，能够提高后续工程化抗体的表达量。
进一步优选地，步骤(2)中，扩增抗体重链、轻链目的基因时，重链上游引物的核苷酸序列如SEQ ID NO：9所示，重链下游引物的核苷酸序列如SEQ ID NO：10所示；轻链上游引物的核苷酸序列如SEQ ID NO：11所示；轻链下游引物的核苷酸序列如SEQ ID NO：12所示。
附图说明
图1为NordiQC对CyclinD1单克隆抗体的质控评比结果；
图2为本发明的单克隆抗体B17在食管癌组织中的免疫组化结果；
图3为进口SP4单抗在食管癌组织中的免疫组化结果；
图4为本发明的单克隆抗体B17在乳腺癌组织中的免疫组化结果；
图5为进口SP4单抗在乳腺癌组织中的免疫组化结果；
图6为本发明的单克隆抗体B17在胃癌组织中的免疫组化结果；
图7为进口SP4单抗在胃癌组织中的免疫组化结果。
具体实施方式
下面结合具体实施例对本发明的实施过程进行说明。
实施例1单克隆抗体
本实施例的针对人细胞周期蛋白1的单克隆抗体，其重链可变区的氨基酸序列如SEQ ID NO：7所示，轻链可变区的氨基酸序列如SEQ ID NO：8所示。重链可变区包含氨基酸序列如SEQ NO ID:1-3所示的VHCDR1、VHCDR2和VHCDR3，轻链可变区包含氨基酸序列如SEQID NO:4-6所示的VLCDR1、VLCDR2和VLCDR3。
实施例2单克隆抗体的制备方法
本实施例的单克隆抗体的制备方法，包括以下步骤：
(1)动物免疫
将5种免疫原(免疫原I、II、III、IV、V分别为MLH1、CyclinD1、p57、CK19和WT1)和免疫伴侣(CSF2蛋白)加入水溶性佐剂中，得到免疫试剂，控制免疫伴侣在免疫试剂中的浓度为50μg/mL，5种免疫原的免疫剂量均为100μg/只进行免疫。然后采用免疫试剂免疫新西兰大白兔，在评估免疫效果达到预期后，对兔子进行免疫原冲击免疫，以达到调动兔子机体针对目标免疫原的B淋巴细胞快速激活。
动物免疫时以腿部肌肉方式进行免疫，周期为两个月。免疫完成后，利用B(CyclinD1)蛋白对该兔的耳缘静脉采集血液进行抗体滴度检测，以检测结果达到6×104以上为合格。效价达到要求之后，对兔子进行B(Cyclin D1)免疫原冲击免疫，待第3天通过耳缘静脉采集10mL血液。
添加CSF2细胞因子作为免疫伴侣可进一步优化多免疫原免疫的效果，为优选的实施方案。其中CSF2蛋白可采用包括以下步骤的方法制得：
1)首先通过查阅Uniprot数据库，确定天然CSF2蛋白的全长基因，含启动子与终止密码子。
2)利用传统基因扩增与载体构建手段，将CSF2基因构建到pcDNA3.1等哺乳动物表达系统载体上，同时需要保证CSF2构建成功后不含其它非自身基因(例如标签蛋白基因)。
3)利用瞬转表达方法将构建测序正确的目的载体(pcDNA3.1-CSF2)转染至经WB验证无相关蛋白表达的兔肾细胞RK-13等兔源哺乳动物表达系统。利用常规WB手段检测表达水平。
4)待表达水平达到要求后，利用免疫亲和层析手段，将抗CFS2抗体偶联至纯化基质上，利用抗原抗体结合特点，将破碎及上清离心过滤的目标溶液经该方法纯化，将洗脱下来的CFS2依据等电点调保存pH，储存在PBS缓冲体系条件下分装，-80℃条件保存。
(2)兔外周血采集与PBMCs分离、B淋巴细胞分离与培养
从冲击免疫后兔子的外周血中先利用淋巴细胞分离液分离获得PBMCs细胞(即外周血单个核细胞；可利用商品化兔外周血淋巴细胞分离试剂盒实现)，随后利用B淋巴细胞表面标志物CD4、CD8、CD14、CD28、CD80进行负筛，去除T、单核等细胞，提高B淋巴细胞的丰度。
然后利用荧光染料FITC标记的山羊抗兔IgG挑选表达识别免疫原的B淋巴细胞。
再利用商品化的生物素标记试剂盒，按照说明书分别对免疫原I、II、III、IV、V进行标记，各免疫原按摩尔量1：1：1：1：1进行混合，混匀后与商品化的亲和素标记偶联荧光素(FITC)试剂，在2～8℃条件下孵育1h(期间需要晃动搅拌)，得到生物素-亲和素筛选试剂。将孵育完成的生物素-亲和素筛选试剂分装放在2～8℃条件下保存，使用时按照细胞量(1×106个细胞/5μL)进行添加。利用生物素-亲和素筛选试剂再次对B淋巴细胞进行正筛，得到识别各种免疫原的B淋巴细胞群。
(3)B淋巴细胞培养上清ELISA检测
对于分选出的B淋巴细胞群进行铺板培养，按照1～2个细胞/孔铺入预先含有滋养层细胞的96板中，同时添加IL-2、IL-10细胞因子进行共培养，待培养时间达到7～14天后，分别利用5种免疫原(免疫原I、II、III、IV、V)分别进行ELISA检测，选择免疫前兔子采集的血清为阴性对照孔，以大于阴性对照孔2.1倍定义为阳性，选择阳性孔。通过该步骤可以筛选出分别针对各免疫原的抗体，一次分选培养可以得到识别多种免疫原的细胞阳性孔。
利用间接ELISA检测方法对上清进行检测，以B(Cyclin D1)蛋白包被ELISA检测板筛选出分泌有目标抗体的细胞培养孔。
(4)cDNA获取、重轻链基因扩增并测序
对筛选获得的抗体细胞株孔进行细胞裂解，提取RNA并通过RT-PCR方法获得cDNA，利用PCR扩增方法对重轻链基因分别进行扩增，经测序确定序列。具体序列信息如实施例1所示。
在抗体重链、轻链目的基因上游5’端均增加一段可增加表达量的信号区碱基序列：AAACCACAAGACAGACTTGCAAAAGAAGGC，如SEQ ID NO：13所示。然后设计抗体重链与轻链上下游引物进行目的基因扩增，从而在目的基因上游增加一段信号区，方便提高后期目的基因表达量。
重链上游引物的核苷酸序列如SEQ ID NO：9所示；重链下游引物的核苷酸序列SEQID NO：10所示；轻链上游引物的核苷酸序列如SEQ ID NO：11所示；轻链下游引物的核苷酸序列如SEQ ID NO：12所示。
PCR扩增循环设定为：I:95℃5min；II：95℃10s；III:56℃10s；IV：72℃60s；II～IV：循环35次；72℃：5min。1％核酸电泳目的条带并切胶回收目的片段，该操作参考Axygen切胶回收试剂盒说明书要求进行操作。
(5)构建重链抗体重组质粒、轻链抗体重组质粒
扩大培养pTT5阳性DH5α菌株，进行质粒(pTT5质粒)提取及双酶切(EcoRI与BamHI)操作。质粒提取按照Axygen质粒提取试剂盒说明书操作要求进行。双酶切反应体系为：pTT5质粒2μg，EcoRI 1.5μL，BamHI 1.5μL，Buffer 5μL，ddH2O 40μL；酶切条件为37℃水浴，酶切时间为2h。对酶切后的体系进行核酸电泳并切胶回收双酶切质粒片段，该操作参考Axygen切胶回收试剂盒说明书要求进行操作。
将目的基因片段和双酶切质粒片段利用无缝克隆试剂盒进行同源重组，该步骤参考近岸无缝克隆试剂盒(货号：NR005-01A)说明书操作要求进行。
无缝重组后转DH5α感受态细胞，涂氨苄抗性的LB平板。37℃条件下温箱培养过夜。第二日挑单克隆5个进行基因测序，对测序结果正确的扩大培养提取重轻链质粒。
(6)质粒转染
分别将经过扩大培养获得的高浓度质粒，按照PEI转染试剂操作说明书进行操作，抗体重链与轻链按照摩尔质量1：1进行预先混合，利用CHO(或293F)细胞基础培养基稀释质粒，同时等体积的该培养基稀释PEI，质粒：PEI＝1：2，W/W。转染到培养至对数生长期的CHO细胞(或293F细胞)中。待培养至48h后，经过间接ELISA方法对上清分泌的抗体滴度进行检测，确定表达量相对较高的细胞系。
(7)抗体工程化表达、纯化
对确定的细胞系进行扩大培养至500mL培养基，调整细胞生长状态至对数生长期，转染质粒并培养48～72h后，收集细胞。上清通过离心去细胞后，经过Protein G柱进行亲和层析纯化，对结合吸附获得的抗体通过pH3.0的柠檬酸缓冲液进行洗脱，收集洗脱液，利用pH8.8的Tris-HCl溶液迅速中和到pH7.2～7.4之间。纯化得到的抗体再经过透析处理浓缩至1mg/mL浓度以上。
实施例3免疫组化检测试剂
本实施例为Cyclin D1免疫组化检测试剂，为Cyclin D1单克隆抗体溶液，其浓度0.10μg/mL，溶剂为TBS缓冲液，含保护性蛋白BSA为10mg/mL，进一步添加防腐剂Proclin-300与Proclin-950等辅助成分(防腐剂均按1:1000(V/V)的比例添加)。
在本实施例的基础上，配套酶标检测板、辣根过氧化物酶标记二抗、标准品、标准品稀释液、TMB显色底物、终止液，可构建ELISA检测试剂盒。
实施例4免疫组化应用
应用本实施例的单克隆抗体对癌组织或正常组织进行免疫组化染色，染色过程如下所示：
1、不同乳腺癌石蜡组织样本3μm切片，65℃条件下烤片2h。
2、脱蜡与水化：石蜡切片以下经以下处理：二甲苯15min-二甲苯15min-无水乙醇5min-无水乙醇5min-90％乙醇5min-80％乙醇5min-70％乙醇5min，纯化水浸泡5min。
3、抗原修复：利用pH9.0的EDTA溶液加热煮沸20min，自然冷却5min后，用自来水使之冷却，液体冷却后取出切片，纯水浸泡5min，TBS冲洗浸泡5min共2次。
4、加过氧化物酶封闭剂100μL/张，室温下孵育5min，TBS冲洗浸泡5min共2次。
5、一抗孵育：加入上述实施例的抗体工作液，37℃条件下孵育30min，TBS冲洗浸泡2次，5min/次。
6、二抗孵育：滴加100μL二抗，37℃孵育30min，TBS冲洗浸泡2次，5min/次。
7、显色：除去TBS溶液，滴加100μL DAB显色液，室温孵育5min，纯化水浸泡2次，5min/次。
8、复染：滴加100μL苏木素复染液室温孵育3min，纯化水浸泡2次，5min/次。
9、脱水透明：常规梯度乙醇脱水、二甲苯透明。
10、中性树胶封片观察。
以上染色过程中，同时以进口产品兔单克隆抗体SP4作为平行对照，实施例的单克隆抗体(B17)与进口产品SP4在食管癌组织中的染色结果分别如图2和图3所示。在乳腺癌组织中的染色结果分别如图4和图5所示。在胃癌组织中的染色结果分别如图6和图7所示。
经计算，以上免疫组化染色过程中，实施例的单克隆抗体(B17)的浓度为0.1μg/mL。对照SP4的浓度为0.35μg/mL。单克隆抗体(B17)的浓度不到对照SP4的1/3，但单克隆抗体(B17)的染色效果与对照SP4相同甚至稍好，背景较佳，显示出良好的特异性和亲和力。
进一步扩大两种单克隆抗体在不同组织中的染色结果，如下表1所示。评价组织包括食管癌组织、胃癌组织、扁桃体、阑尾(急性坏疽性阑尾炎)、胎盘、正常脾脏、正常结肠、肝脏-中分化肝细胞癌、胰腺、肺(支气管，肺泡)、正常肾脏、甲状腺(滤泡)、混合性生殖细胞肿瘤、卵巢浆液性癌、右侧卵巢囊肿、嗜铬细胞瘤、左前臂恶性黑色素瘤、尿路上皮癌、胶质母细胞瘤、滑膜肉瘤、乳腺纤维腺瘤、孤立性纤维瘤、浆液性癌、输尿管-小细胞肿瘤、胚胎性肿瘤、梭型细胞肿瘤、弥漫型星形细胞瘤、急性化脓性阑尾炎、宫颈、胸腺、弥散性大B细胞淋巴瘤、精原细胞癌、胸腺瘤、神经鞘瘤、宫颈-鳞状细胞癌等，评价数量共64个。
表1两种单克隆抗体的组织评价统计结果
结果显示，二种抗体在阴阳一致性评价方面无显著差异(P＜0.05)。
<110> 河南赛诺特生物技术有限公司
<120> 一种针对人细胞周期蛋白1的单克隆抗体及其制备方法、免疫检测试剂及应用
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<213> 人工序列
<221> 重链上游引物
<400> 9
gtttaaacgg atctctagcg aattcaaacc acaagacaga 40
<210> 10
<211> 40
<212> DNA
<213> 人工序列
<221> 重链下游引物
<400> 10
agaggtcgag gtcgggggat ccctatttac ccggagagcg 40
<210> 11
<211> 40
<212> DNA
<213> 人工序列
<221> 轻链上游引物
<400> 11
gtttaaacgg atctctagcg aattcaaacc acaagacaga 40
<210> 12
<211> 40
<212> DNA
<213> 人工序列
<221> 轻链下游引物
<400> 12
agaggtcgag gtcgggggat ccctagcagt cacccctatt 40
<210> 13
<211> 30
<212> DNA
<213> 人工序列
<221> 信号区
<400> 13
aaaccacaag acagacttgc aaaagaaggc 30
Claims (8)
1.一种针对人细胞周期蛋白1的单克隆抗体，其特征在于，其包含氨基酸序列如SEQ NOID:1-3所示的VHCDR1、VHCDR2和VHCDR3，和氨基酸序列如SEQ ID NO:4-6所示的VLCDR1、VLCDR2和VLCDR3。
2.如权利要求1所述的针对人细胞周期蛋白1的单克隆抗体，其特征在于，所述单克隆抗体包含氨基酸序列如SEQ ID NO：7所示的重链可变区，和氨基酸序列如SEQ ID NO：8所示的轻链可变区。
3.一种含有如权利要求1或2所述的单克隆抗体的免疫检测试剂。
4.一种如权利要求1或2所述的针对人细胞周期蛋白1的单克隆抗体在免疫组化方面的应用。
5.一种如权利要求1或2所述的针对人细胞周期蛋白1的单克隆抗体的制备方法，其特征在于，包括以下步骤：
(1)使用人细胞周期蛋白1免疫原免疫兔子，采集兔外周血，收集兔外周血单个核细胞，分离出抗原特异性B淋巴细胞；
(2)针对步骤(1)得到的抗原特异性B淋巴细胞，经过培养鉴定筛选出阳性细胞孔，进而获取抗体重链、轻链基因，扩增抗体重链、轻链目的基因，经重组、表达获得兔源单克隆抗体。
6.如权利要求5所述的针对人细胞周期蛋白1的单克隆抗体的制备方法，其特征在于，步骤(1)中，所述人细胞周期蛋白1免疫原包括MLH1、CyclinD1、p57、CK19和WT1。
7.如权利要求5所述的针对人细胞周期蛋白1的单克隆抗体的制备方法，其特征在于，步骤(2)中，在抗体重链、轻链基因5’端均增加一段如SEQ ID NO：13所示的核苷酸序列，然后进行所述扩增。
8.如权利要求7所述的针对人细胞周期蛋白1的单克隆抗体的制备方法，其特征在于，步骤(2)中，扩增抗体重链、轻链目的基因时，重链上游引物的核苷酸序列如SEQ ID NO：9所示，重链下游引物的核苷酸序列如SEQ ID NO：10所示；轻链上游引物的核苷酸序列如SEQID NO：11所示；轻链下游引物的核苷酸序列如SEQ ID NO：12所示。
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| CN101280014A (zh) * | 2008-02-29 | 2008-10-08 | 吉林大学 | 抗CyclinD1人源单链抗体 |
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| CN112964877A (zh) * | 2021-03-09 | 2021-06-15 | 河南赛诺特生物技术有限公司 | 一种用于鉴别套细胞淋巴瘤的免疫组化多重染色试剂盒及染色程序 |
| CN113621069A (zh) * | 2021-09-06 | 2021-11-09 | 福州迈新生物技术开发有限公司 | 抗her-2蛋白单克隆抗体及其制备方法和应用 |
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| CN101280014A (zh) * | 2008-02-29 | 2008-10-08 | 吉林大学 | 抗CyclinD1人源单链抗体 |
| CN111499746A (zh) * | 2020-04-28 | 2020-08-07 | 优睿赛思(武汉)生物科技有限公司 | 一种针对人白介素-2的高亲和力兔单克隆抗体及其应用 |
| CN112964877A (zh) * | 2021-03-09 | 2021-06-15 | 河南赛诺特生物技术有限公司 | 一种用于鉴别套细胞淋巴瘤的免疫组化多重染色试剂盒及染色程序 |
| CN113621069A (zh) * | 2021-09-06 | 2021-11-09 | 福州迈新生物技术开发有限公司 | 抗her-2蛋白单克隆抗体及其制备方法和应用 |
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| 郑航;梁继珍;苏宁;陈逢生;李荣;: "兔抗人Cyclin D1b多克隆抗体的制备与鉴定", 中国肿瘤生物治疗杂志, vol. 17, no. 05, pages 559 - 561 * |
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