CN113583124A - 一种抗前胃泌素释放肽单克隆抗体及其制备方法 - Google Patents
一种抗前胃泌素释放肽单克隆抗体及其制备方法 Download PDFInfo
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- CN113583124A CN113583124A CN202110665746.4A CN202110665746A CN113583124A CN 113583124 A CN113583124 A CN 113583124A CN 202110665746 A CN202110665746 A CN 202110665746A CN 113583124 A CN113583124 A CN 113583124A
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Abstract
本发明属于抗体药物技术领域,涉及一种抗前胃泌素释放肽单克隆抗体及其制备方法。所述的单克隆抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO.5‑10所示。利用本发明的抗前胃泌素释放肽单克隆抗体及其制备方法,能够更好的制备抗前胃泌素释放肽单克隆抗体,制得抗前胃泌素释放肽单克隆抗体可更好的用于SCLC靶向诊治。
Description
技术领域
本发明属于抗体药物技术领域,涉及一种抗前胃泌素释放肽单克隆抗体及其制备方法。
背景技术
肺癌(Lung Cancer)是发病率和死亡率都极高的恶性肿瘤,其中小细胞肺癌(Small Cell Lung Cancer,SCLC)是恶性度较高,治愈率较低的一种肺癌,约占肺癌的15%-20%。SCLC的特点是瘤体倍增时间短、预后差、分化程度最低、极易转移,5年生存率仅为1%-7%。但SCLC对放化疗敏感,如早期发现采用全身化疗伴局部放疗等综合治疗手段,3年生存率可达30%以上,因此SCLC的早期诊断和治疗至关重要。
前胃泌素释放肽(Pro-Gastrin Releasing Peptide,PGRP)是SCLC的可靠、灵敏、特异性的肿瘤标志物,在血液中相对稳定。在物理诊断尚不明确前即可检测到PGRP,阳性率高达76%,已被用于SCLC早期临床诊断。PGRP由信号肽、胃泌素释放肽(Gastrin ReleasingPeptide,GRP)、酶切位点、恒定区以及可变的羧基末端组成。
随着基因工程技术的发展,抗体药物不断用于恶性肿瘤的早期诊断和治疗,并且取得良好的经济效益和社会效益。抗前胃泌素释放肽抗体的研制是SCLC靶向诊治方向之一,可为SCLC病人的诊断和治疗奠定一定的技术和物质基础。
发明内容
本发明的首要目的是提供一种抗前胃泌素释放肽单克隆抗体,以能够更好的用于SCLC靶向诊治。
为实现此目的,在基础的实施方案中,本发明提供一种抗前胃泌素释放肽单克隆抗体,所述的单克隆抗体的HCDR1(重链互补决定区1)、HCDR2(重链互补决定区2)、HCDR3(重链互补决定区3)、LCDR1(轻链互补决定区1)、LCDR2(轻链互补决定区2)、LCDR3(轻链互补决定区3)的氨基酸序列分别如SEQ ID NO.5-10所示。
在一种优选的实施方案中,本发明提供一种抗前胃泌素释放肽单克隆抗体,其中所述的单克隆抗体的重链可变区VH的氨基酸序列如SEQ ID NO.3所示,轻链可变区VL的氨基酸序列如SEQ ID NO.4所示。
在一种优选的实施方案中,本发明提供一种抗前胃泌素释放肽单克隆抗体,其中所述的单克隆抗体为小鼠单克隆抗体。
本发明的第二个目的是提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,以能够更好的制备上述抗前胃泌素释放肽单克隆抗体,制得抗前胃泌素释放肽单克隆抗体可更好的用于SCLC靶向诊治。
为实现此目的,在基础的实施方案中,本发明提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,所述的制备方法包括如下步骤:
(1)以鼠前胃泌素释放肽或其片段免疫小鼠后取其脾细胞;
(2)将步骤(1)得到的脾细胞与骨髓瘤细胞融合,并通过选择培养和ELISA检测筛选出杂交瘤细胞;
(3)对步骤(2)筛选出的杂交瘤细胞进行克隆化培养和稳定性培养,获得表达抗前胃泌素释放肽抗体的单克隆细胞株;
(4)将步骤(3)获得的单克隆细胞株注射至小鼠腹腔,抽取腹水进行单克隆抗体纯化及鉴定。
在一种优选的实施方案中,本发明提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,其中步骤(1)中,
所述的鼠前胃泌素释放肽或其片段为鼠前胃泌素释放肽的恒定区氨基酸肽段,
所述的小鼠为Balb/c小鼠。
在一种优选的实施方案中,本发明提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,其中步骤(2)中,所述的骨髓瘤细胞为SP2/0细胞,所述的选择培养所用培养基为HAT选择培养基。
在一种优选的实施方案中,本发明提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,其中步骤(4)中,利用蛋白A-琼脂糖亲和层析柱进行单克隆抗体纯化。
在一种优选的实施方案中,本发明提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,其中所述的制备方法还进一步包括如下步骤:
(5)对步骤(3)获得的单克隆细胞株进行总RNA的提取,并反转录成cDNA,扩增抗体重链和轻链的可变区,分别连接至载体,转化至感受态细胞后提质粒进行测序。
在一种更加优选的实施方案中,本发明提供一种上述抗前胃泌素释放肽单克隆抗体的制备方法,其中步骤(5)中,连接所用载体为pMD 19-T,所用感受态细胞为DH5α。
本发明的第三个目的是提供上述抗前胃泌素释放肽单克隆抗体用于制备诊断或治疗肺癌的试剂或药物的用途,以能够更好的用于肺癌,尤其是SCLC的靶向诊治。
为实现此目的,在基础的实施方案中,本发明提供上述抗前胃泌素释放肽单克隆抗体用于制备诊断或治疗肺癌的试剂或药物的用途。
本发明的有益效果在于,利用本发明的抗前胃泌素释放肽单克隆抗体及其制备方法,能够更好的制备抗前胃泌素释放肽单克隆抗体,制得抗前胃泌素释放肽单克隆抗体可更好的用于SCLC靶向诊治。
本发明通过免疫小鼠、细胞融合、克隆化培养及一系列细胞筛选实验,获得了抗前胃泌素释放肽单克隆抗体及其轻链可变区VL和重链可变区VH序列。实验证明本发明的单克隆抗体具有特异性,为肿瘤的靶向性治疗提供了一种新的方法。
本发明获得了抗体的序列,经后期的重组表达可直接获得大量抗体,无需进行动物实验,效率高、成本低。
附图说明
图1为实施例中蛋白A-琼脂糖亲和层析洗涤和洗脱产物的紫外吸收检测图谱,其中I为杂蛋白洗涤峰,II为单克隆抗体洗脱峰。
图2为实施例中蛋白A-琼脂糖抗前胃泌素释放肽单克隆抗体腹水纯化前后SDS-PAGE鉴定图,其中1为腹腔积液,2-4为洗涤蛋白(2为峰I顶支,3和4为峰I降支),5-8为单克隆抗体(5为峰II升支,6和7为峰II顶支,8为峰II降支,两条蛋白带分别为抗体重链和轻链),M为蛋白分子量标准。
图3为实施例中纯化的单克隆抗体倍比稀释SDS-PAGE纯度鉴定图,其中1:1×;2:10×;3:20×;4:40×;5:80×;6:160×;7:320×。
具体实施方式
以下结合实施例和附图对本发明的具体实施方式作出进一步的说明。
实施例1:抗前胃泌素释放肽单克隆抗体的制备
1、免疫原(抗原)
鼠前胃泌素释放肽(rPGRP)的恒定区氨基酸肽段,按照中国专利申请CN200510065889.2具体实施方式部分公开的方法制备。
2、小鼠免疫
用rPGRP按100ug/只/次的剂量免疫Balb/c小鼠。将抗原与等量福氏完全佐剂混匀乳化,腹腔注射免疫小鼠;两周后取相同剂量抗原与等量福氏不完全佐剂混匀乳化,背部皮下多点及腹腔注射免疫小鼠;两周后取相同剂量抗原溶于生理盐水中尾静脉注射加强免疫;融合前三天取相同剂量静脉注射免疫。
3、杂交瘤细胞制备
(1)饲养细胞的制备
将Balb/c小鼠脱颈椎处死,浸泡于75%酒精溶液中5分钟,置超净工作台内,腹部朝上固定于解剖台板上。剪开腹部皮肤充分暴露腹膜;用注射器吸取基础培养液-8ml,注入小鼠腹腔,固定注射器,使针头留在腹腔内,手持酒精棉球轻轻按摩腹部1-2分钟;用原注射器抽回腹腔内液体,注入离心管内,1000rpm离心10分钟,弃上清;用5ml培养液将沉淀细胞悬浮并混匀,作细胞计数,根据计数结果,调整细胞浓度为2×105cells/ml。将细胞悬液加入96孔培养板中,每孔0.1ml,于含5%CO2的37℃培养箱内培养。使用前一天制备。
(2)细胞融合
以基础培养液制备脾细胞和骨髓瘤细胞悬液,按白细胞计数法计数。分别取含1×108脾细胞和2-3×107个骨髓瘤细胞的悬液混入同一离心管离心,轻弹离心管底使细胞成糊状,加入0.7ml浓度50%PEG溶液使细胞融合,90s后加入25ml不完全培养液稀释,离心除去PEG。将细胞加入HAT培养液10ml,接种到96孔培养板中,每孔0.1ml,置于含5%CO2的37℃培养箱中培养。融合成功后对杂交瘤细胞克隆化培养,以筛选分泌特异性单克隆抗体细胞株。杂交瘤细胞于融合后第4天开始,每隔3天用HAT选择性培养液以1/2-2/3量换液,持续两周后改用HT培养液,每隔2天换液,培养两周。再转入普通培养液,于含5%CO2的37℃的培养箱中连续培养。通过ELISA筛选出表达抗前胃泌素释放肽抗体的杂交瘤细胞,进行克隆化培养。
4、单克隆细胞株克隆化培养和稳定性培养
取阳性孔细胞进行准确计数,稀释后使细胞浓度调至10cells/ml,于96孔培养板每孔加0.1ml,使每孔平均1个细胞。适时进行换液和检测,取单个克隆的阳性孔再次进行克隆化培养。所有产生细胞克隆孔均为阳性时则可认为筛选出一株分泌单克隆抗体的细胞株。将已筛选出的可分泌单克隆抗体细胞株继续培养3个月,根据细胞生长状况2-3天换液一次,并检测其上清中单克隆抗体的OD值,每周检测一次,OD值下降的放弃,最终获得可稳定表达抗前胃泌素释放肽单克隆抗体的细胞株F-B6、E-B5、F-B3、D-D3、E-C6。
5、腹水制备及纯化
(1)腹水制备
在接种杂交瘤细胞前一周,给小鼠腹腔注射原倍液体石蜡0.5ml。1000rpm离心10分钟,收集培养的杂交瘤细胞,弃上清液,用无血清培养液将细胞悬浮混匀,细胞数调至1-2×105cells/ml,每只小鼠经腹腔注射0.5ml。收集腹水,接种后1-2周,可见小鼠腹部明显膨大,消毒下腹部皮肤,用消毒注射器抽取腹水,每只小鼠一次可抽取3-5ml,间隔1-2天,待腹水再生积聚后,同法再抽,将抽取的腹水经3000rpm离心15分钟,收集上清液,测效价、分装冻存备用。
(2)腹水纯化
将已制备好的1.5ml左右的小鼠腹水以10000g离心10min,去除其中胶状物沉淀,用0.02mol/L的PBS将样品稀释至2ml备用;将蛋白A-琼脂糖亲和层析柱固定好,用25ml的双蒸水洗出酒精防腐剂,再用0.1mol/L的柠檬酸钠缓冲液(pH=4.6)10ml过柱,接着用0.02mol/L的磷酸盐缓冲液(pH=7.0)20ml冲洗柱子,用3mol/L NaCl、0.02mol/L PBS 25ml平衡柱子;将已准备好的腹水样品2ml缓慢注入到柱子中;先后用25ml0.5mol/L NaCl、0.02mol/L的PBS和25ml 0.02mol/L的磷酸盐缓冲液(pH=7.0)洗涤,除去未结合的蛋白,同时收集各自的流出液,以备检测;用30ml pH=2.4的甘氨酸-盐酸缓冲液洗脱已结合的单克隆抗体,收集流出液,每管2ml,同时尽快用pH=9.0的Tris-HCl缓冲液将每管洗脱收集液调至中性(pH=7.0),以备检测(以上纯化过程中的流速均为2ml/min);采用紫外吸收法,在280nm处监测亲和层析样本洗涤和洗脱过程并记录。
结果如图1所示,经纯化后腹水样本分离为2个紫外光吸收峰,即2个可分离蛋白组分。
分步收集各组分,通过间接ELISA法测定免疫活性,并进行SDS-PAGE检测。结果如表1所示,组分II比活度明显高于组分I和腹水,表明其含有大量的抗体,为分离纯化的单克隆抗体;组分Ⅰ抗体含量极低,为其它杂蛋白质,同时也表明纯化后抗体比活度显著高于纯化前。SDS-PAGE检测结果如图2所示,条带2-4(峰I)含有大量的杂蛋白;条带5-8(峰II)有两条明显的蛋白带,结合ELISA实验结果,可说明峰II为纯化的单克隆抗体,两条蛋白带分别为抗体重链和轻链。
表1纯化前后不同组分ELISA检测结果(OD值)
阴性对照:0.125,阳性对照:1.243,cutoff:0.125×2.1=0.2625
阴性对照为0.01mol/L PBS;阳性对照为已知抗体阳性培养上清液。
实施例2:实施例1制备的单克隆抗体的鉴定
(1)用间接酶联免疫吸附(ELISA)法测定单克隆抗体效价,以纯化抗体浓度/效价计算比活度,结果如表2所示,表明纯化后抗体比活度均高于纯化前。
(2)采用紫外吸收法测定蛋白浓度,计算蛋白回收率,结果如表3所示,表明腹水单克隆抗体质量浓度平均为5.21mg/ml,蛋白回收率平均为98.56%。
(3)纯化的单克隆抗体倍比稀释后进行SDS-PAGE检测,以鉴定纯化抗体的纯度,结果如图3所示,表明成功纯化出较纯的抗体蛋白,可清晰观察到解链后的抗体轻、重链。
(4)以骨髓瘤细胞培养上清液为阴性对照,抗HBs的IgG1 MAb亚类为阳性对照,按照美国Sigma公司小鼠单克隆抗体检测试剂盒鉴定单克隆抗体的亚型,结果如表4所示,表明MAb细胞株F-B6、E-A6、F-B3和E-C6为IgG2b,D-D3为IgG1。
(5)通过间接ELISA法测定单抗与乙型肝炎表面抗原(HBSAg)的交叉反应,HBSAg阴性和阳性对照分别为乙型肝炎表面抗体阴性和阳性样本,rPGRP阴性为细胞原初培养液,阳性为已知抗体阳性培养上清液,PGRP(42~53)、PGRP(68~82)阳性对照为其免疫小鼠血清,结果如表5所示,表明5株单克隆抗体与乙型肝炎表面抗原无交叉反应,E-C6抗体可与PGRP(68~82)合成肽发生反应,D-D3与PGRP(42~53)抗原反应,其余3株与PGRP(42~53)和PGRP(68~82)均不发生反应。
表2单克隆抗体纯化前后比活度比较
表3腹水纯化后回收率及单抗含量
表4单克隆抗体亚类鉴定结果(OD值)(n=5)
表5单克隆抗体特异性检测结果(OD值)(n=5)
实施例3:抗前胃泌素释放肽单克隆抗体序列分析
1、抗前胃泌素释放肽单克隆抗体基因序列获取
(1)RNA提取及cDNA转化
培养新鲜的E-B5杂交瘤细胞,离心收集107以上细胞;用Trizol试剂提取细胞总RNA;取9μL总RNA,2.5μL oligo(dT)12~18primer(10mM),及5μL dNTPs混合,70℃保温5分钟后冰置5分钟,或依照使用的逆转录酶(BPI)进行变性操作;随后,加入5μL RT buffer(5×),2.5μL DTT(0.1M)及1μL逆转录酶,42℃反应1小时;70℃孵育15分钟以终止反应,获得的cDNA保存在-20℃。
(2)基因获取
将获得的第一链cDNA进行PCR扩增:在50μL反应体系中加入引物各25pmol,dNTP及Buffer均按照常规加入,cDNA模板加入1μL;程序:95℃3min,95℃30s、退火温度55℃60s、72℃40s,25个循环,72℃3min。
2、抗前胃泌素释放肽单克隆抗体基因测序
(1)连接
1μl载体(pMD 19-T Vector,TaKaRa),3μl回收产物,4μl连接酶混合物(TaKaRa),混匀,16℃反应2h。
(2)转化
取出-80℃保存的感受态细胞(DH5α),放在冰上缓慢解冻;将连接产物加入感受态细胞中混匀,冰上放置30min;42℃热激90s;冰浴2min后,加入800μl无抗性的LB培养基;37℃培养45min;8000rpm离心1min,弃大部分上清,留约50~100μl,重悬菌体,选择有相应抗性的LB平板,涂板;晾干,于37℃培养箱中倒置培养过夜。
(3)鉴定
从平板上挑单克隆摇菌,取1μl菌液做模板,M13F/R为引物,PCR验证(程序:95℃3min,95℃30s、退火温度55℃60s、72℃40s,25个循环,72℃3min)。
(4)测序
重组质粒测序由北京华大蛋白质研发中心有限公司完成,测序结果如下:
(1)表达单克隆抗体重链可变区VH和轻链可变区VL的核苷酸序列分别如SEQ IDNO.1和SEQ ID NO.2所示。
(2)单克隆抗体的重链可变区VH的氨基酸序列如SEQ ID NO.3所示,轻链可变区VL的氨基酸序列如SEQ ID NO.4所示。
(3)单克隆抗体的HCDR1(重链互补决定区1)、HCDR2(重链互补决定区2)、HCDR3(重链互补决定区3)、LCDR1(轻链互补决定区1)、LCDR2(轻链互补决定区2)、LCDR3(轻链互补决定区3)的氨基酸序列分别如SEQ ID NO.5-10所示。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若对本发明的这些修改和变型属于本发明权利要求及其同等技术的范围之内,则本发明也意图包含这些改动和变型在内。上述实施例或实施方式只是对本发明的举例说明,本发明也可以以其它的特定方式或其它的特定形式实施,而不偏离本发明的要旨或本质特征。因此,描述的实施方式从任何方面来看均应视为说明性而非限定性的。本发明的范围应由附加的权利要求说明,任何与权利要求的意图和范围等效的变化也应包含在本发明的范围内。
序列表
<110> 中国辐射防护研究院
<120> 一种抗前胃泌素释放肽单克隆抗体及其制备方法
<130> -
<141> 2021-06-16
<160> 10
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<400> 6
Ile Tyr Trp Asp Asp Asp Lys
1 5
<210> 7
<211> 13
<212> PRT
<213> Mus musculus
<400> 7
Ala Arg Arg Ser Tyr Asp Gly Gly Tyr Ala Met Gly Tyr
1 5 10
<210> 8
<211> 11
<212> PRT
<213> Mus musculus
<400> 8
Gln Ser Leu Val His Ser Asn Gly Asn Thr Tyr
1 5 10
<210> 9
<211> 3
<212> PRT
<213> Mus musculus
<400> 9
Lys Val Ser
1
<210> 10
<211> 9
<212> PRT
<213> Mus musculus
<400> 10
Ser Gln Thr Thr His Val Pro Trp Thr
1 5
Claims (10)
1.一种抗前胃泌素释放肽单克隆抗体,其特征在于:所述的单克隆抗体的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2、LCDR3的氨基酸序列分别如SEQ ID NO.5-10所示。
2.根据权利要求1所述的单克隆抗体,其特征在于:所述的单克隆抗体的重链可变区VH的氨基酸序列如SEQ ID NO.3所示,轻链可变区VL的氨基酸序列如SEQ ID NO.4所示。
3.根据权利要求1或2所述的单克隆抗体,其特征在于:所述的单克隆抗体为小鼠单克隆抗体。
4.一种根据权利要求3所述的单克隆抗体的制备方法,其特征在于,所述的制备方法包括如下步骤:
(1)以鼠前胃泌素释放肽或其片段免疫小鼠后取其脾细胞;
(2)将步骤(1)得到的脾细胞与骨髓瘤细胞融合,并通过选择培养和ELISA检测筛选出杂交瘤细胞;
(3)对步骤(2)筛选出的杂交瘤细胞进行克隆化培养和稳定性培养,获得表达抗前胃泌素释放肽抗体的单克隆细胞株;
(4)将步骤(3)获得的单克隆细胞株注射至小鼠腹腔,抽取腹水进行单克隆抗体纯化及鉴定。
5.根据权利要求4所述的制备方法,其特征在于:步骤(1)中,
所述的鼠前胃泌素释放肽或其片段为鼠前胃泌素释放肽的恒定区氨基酸肽段,
所述的小鼠为Balb/c小鼠。
6.根据权利要求4所述的制备方法,其特征在于:步骤(2)中,所述的骨髓瘤细胞为SP2/0细胞,所述的选择培养所用培养基为HAT选择培养基。
7.根据权利要求4所述的制备方法,其特征在于:步骤(4)中,利用蛋白A-琼脂糖亲和层析柱进行单克隆抗体纯化。
8.根据权利要求4所述的制备方法,其特征在于,所述的制备方法还进一步包括如下步骤:
(5)对步骤(3)获得的单克隆细胞株进行总RNA的提取,并反转录成cDNA,扩增抗体重链和轻链的可变区,分别连接至载体,转化至感受态细胞后提质粒进行测序。
9.根据权利要求8所述的制备方法,其特征在于:步骤(5)中,连接所用载体为pMD 19-T,所用感受态细胞为DH5α。
10.根据权利要求1-3之一所述的单克隆抗体用于制备诊断或治疗肺癌的试剂或药物的用途。
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