CN114395040B - 再生蛋白reg1a单克隆抗体及其应用 - Google Patents
再生蛋白reg1a单克隆抗体及其应用 Download PDFInfo
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- CN114395040B CN114395040B CN202210122652.7A CN202210122652A CN114395040B CN 114395040 B CN114395040 B CN 114395040B CN 202210122652 A CN202210122652 A CN 202210122652A CN 114395040 B CN114395040 B CN 114395040B
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Abstract
本发明涉及再生蛋白REG1A单克隆抗体及其应用。该抗体包括重链和轻链，至少具有以下技术特征之一：i、重链包括重链CDR1，序列为VKQSH；ii、重链包括重链CDR2，序列为GGNVYNQKFKIKATLT；iii、重链包括重链CDR3，序列为PGTGYFDV；iv、轻链包括轻链CDR1，序列为RASQDISNYLN；v、轻链包括轻链CDR2，序列为YTSRLHS；vi、轻链包括轻链CDR3，序列为QQGHTLPRT。本发明的再生蛋白REG1A单克隆抗体能够有效检测再生蛋白REG1A水平，从而可以用以辅助诊断和治疗，以及预测疾病的发生和发展。
Description
技术领域
本发明涉及一种再生蛋白REG1A单克隆抗体及其应用，属于生物医药技术领域。
背景技术
再生基因(regenerating gene,Reg)是从再生胰岛中分离出来的一种基因，近年来，越来越多的研究表明再生基因家族蛋白的异常表达与炎症反应及肿瘤等多种疾病密切相关。其中再生蛋白(REG1)最早在大鼠再生胰岛中发现，后被证实与一种能够抑制胰腺结石形成的胰石蛋白(PSP)为同一种蛋白质，统称为PSP/reg。REG1A位于2p12染色体上，编码166个氨基酸，属于钙依赖性凝集素超家族。REG1A蛋白主要由胰腺腺泡细胞合成、分泌，是胰腺外分泌产物，生理状态下REG1A以可溶性形式主要分布于胰腺、消化道组织、胰液及血液中。REG1A具有促进增殖，诱导再生等作用，主要参与炎症、糖尿病、胰腺炎、肿瘤等疾病的发生。已有多项研究表明，REG1A的水平变化与这些疾病的进展密切相关，其在疾病的早期诊断及治疗中的作用越来越受到人们的重视。
经检索发现，申请号CN202010856716.7、申请公布号CN111909266A的发明专利申请公开了及一种再生基因蛋白REG1A单克隆抗体的制备方法，包括以下步骤：⑴制备免疫接种的小鼠；⑵骨髓瘤细胞培养3-4天，得到培养后的小鼠骨髓瘤细胞；⑶将REG1A蛋白溶解向免疫接种的小鼠腹腔接种免疫，并且获得脾细胞，经分散脾细胞得到脾细胞沉淀物；⑷培养后的小鼠骨髓瘤细胞离心得到骨髓瘤细胞沉淀物；⑸用DMEM高糖培养液分别悬浮脾细胞沉淀物、骨髓瘤细胞沉淀物并混合，经离心得到沉淀物；⑹沉淀物离心、悬浮，即得融合细胞；⑺将融合细胞进行培养、测定，确定杂交瘤细胞；⑻单抗的筛选分离；⑼大量制备单克隆抗体：将杂交瘤细胞系按常规方法进行单克隆抗体生产即得。然而，该技术方案并未能提供明确的单克隆抗体序列。
发明内容
本发明的主要目的是：克服现有技术存在的问题，提供一种再生蛋白REG1A单克隆抗体，能有效检测血清和组织中的REG1A；同时还提供该抗体的应用。
本发明解决其技术问题的技术方案如下：
一种再生蛋白REG1A单克隆抗体，所述抗体包括重链和轻链，其特征是，所述抗体至少具有以下技术特征之一：
i、所述重链包括重链CDR1，其氨基酸序列为VKQSH；
ii、所述重链包括重链CDR2，其氨基酸序列为GGNVYNQKFKIKATLT；
iii、所述重链包括重链CDR3，其氨基酸序列为PGTGYFDV；
iv、所述轻链包括轻链CDR1，其氨基酸序列为RASQDISNYLN；
v、所述轻链包括轻链CDR2，其氨基酸序列为YTSRLHS；
vi、所述轻链包括轻链CDR3，其氨基酸序列为QQGHTLPRT。
优选地，所述抗体至少具有以下技术特征之一：
i、所述重链包括重链CDR1，其氨基酸序列为VKQSH；所述重链包括重链CDR2，其氨基酸序列为GGNVYNQKFKIKATLT；所述重链包括重链CDR3，其氨基酸序列为PGTGYFDV；
ii、所述轻链包括轻链CDR1，其氨基酸序列为RASQDISNYLN；所述轻链包括轻链CDR2，其氨基酸序列为YTSRLHS；所述轻链包括轻链CDR3，其氨基酸序列为QQGHTLPRT。
优选地，所述抗体具有以下技术特征：
所述重链包括重链CDR1，其氨基酸序列为VKQSH；所述重链包括重链CDR2，其氨基酸序列为GGNVYNQKFKIKATLT；所述重链包括重链CDR3，其氨基酸序列为PGTGYFDV；
所述轻链包括轻链CDR1，其氨基酸序列为RASQDISNYLN；所述轻链包括轻链CDR2，其氨基酸序列为YTSRLHS；所述轻链包括轻链CDR3，其氨基酸序列为QQGHTLPRT。
优选地，所述重链的氨基酸序列如SEQ ID NO:1所示；所述轻链的氨基酸序列如SEQ ID NO:2所示。
优选地，所述抗体的重链或轻链上具有标记，所述标记包括荧光标记、酶标记、以及放射性标记。
本发明还提供：
编码前文所述再生蛋白REG1A单克隆抗体的核酸。
优选地，所述核酸包括编码重链的核酸且序列如SEQ ID NO:3所示，以及编码轻链的核酸且序列如SEQ ID NO:4所示。
本发明还提供：
前文所述再生蛋白REG1A单克隆抗体用于制备试剂盒的用途。
其中，所述试剂盒为针对再生蛋白REG1A的检测试剂盒或诊断试剂盒。
前文所述核酸用于制备再生蛋白REG1A单克隆抗体的用途。
本发明的再生蛋白REG1A单克隆抗体能够有效检测再生蛋白REG1A水平，从而可以用以辅助诊断和治疗，以及预测疾病的发生和发展。
附图说明
图1为本发明实施例1的SDS-PAGE分析结果图，其中1为纯化前样品，2为纯化后样品。
图2为本发明实施例1的Western blot分析结果图，其中1为REG1A重组蛋白，2为空载体质粒对照。
图3为本发明实施例2的单克隆抗体亚型鉴定分析结果图。
图4为本发明实施例2的Western blot分析结果图。
图5为本发明实施例2的细胞株单抗的效价检测结果图。
图6为本发明实施例2的再生蛋白REG1A单克隆抗体重链的具体可变区信息图。
图7为本发明实施例2的再生蛋白REG1A单克隆抗体轻链的具体可变区信息图。
图8为本发明实施例3的Western blot分析结果图。
图9为本发明实施例4的十二指肠免疫组化分析图。
具体实施方式
下面结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。
实施例1、原核表达带有组氨酸标签的重组再生蛋白REG1A。
1、获得目的基因人REG1A
通过本领域内常用方法，从人胰腺组织或人胰腺来源细胞系提取mRNA，逆转录得到cDNA，以编码REG1A蛋白的序列为基础设计特异性引物，在REG1A的N端添加6个组氨酸标签，并进行PCR扩增及扩增产物的琼脂糖凝胶电泳鉴定。
用于PCR扩增的引物为：
上游引物：5’-cagtggtggtggtggtggtgatggctcagaccagctcatacttc-3’
下游引物：5’-taccgacgacgacgacaaggctagtttttgaacttgcagacaaagg-3’
按以下程序进行PCR扩增：
(1)95℃预变性3min。(2)95℃变性15s。(3)58℃退火15s。(4)72℃延伸40s。(5)重复步骤(2)-(4)40次。(6)72℃延伸5min。
2、构建表达载体
(1)连接：以一种带有组氨酸标签的质粒为载体，如pET32a，经过PCR扩增得到载体cDNA。通过同源重组的方法连接线性化载体与目的基因片段，得到重组表达质粒pET32a-REG1A。
(2)转化：在冰上解冻克隆用的感受态细胞大肠杆菌DH5α。取适量重组质粒加入到感受态细胞中，冰上静置30min，后42℃水浴热激45s，立即置于冰上冷却2-3min；LB培养基(不含抗生素)，37℃摇菌1小时(转速200-250rpm)。将含有氨苄抗性的LB平板在37℃培养箱中预热；菌液5000rpm离心5min，弃掉部分上清，重悬菌体，用无菌涂布棒在LB平板上轻涂均匀；在37℃培养箱中倒置培养12-16小时，长出的菌斑既为阳性克隆。
(3)筛选：挑选若干单克隆菌落，37℃摇菌3-4小时，后进行菌落PCR及基因测序鉴定。
3、表达菌株
将鉴定成功的菌液扩大培养，提取重组质粒pET32a-REG1A，并将其转化至表达蛋白的宿主菌体大肠杆菌BL21中，得到重组菌株BL21-REG1A。
4、重组蛋白的表达
将重组菌株接种于LB培养基中(含100mg/ml氨苄霉素)，37℃，200rpm摇床过夜。次日按合适比例接种于新的LB培养基中，37℃，200rpm摇床培养至OD600为0.6-0.8，加入诱导剂IPTG继续培养。收集菌液，4℃，5000rpm离心20min，弃上清。经超声裂解菌体后分别取上清与沉淀进行12％SDS-PAGE电泳，考马斯亮蓝染色鉴定，确定重组蛋白HIS-REG1主要以可溶性蛋白形式存在于裂解液上清中。分别用不同温度、不同诱导剂浓度诱导菌体，确定融合蛋白表达的最佳条件，并进行蛋白的大量表达。
5、镍柱亲和层析法纯化蛋白
收集大量表达的菌体裂解液上清，过Ni琼脂糖凝胶亲和层析柱，收集洗脱峰流出液；使用超滤管(截留量10KDa)于4℃、4000×g离心20-30min进行浓缩、脱盐，得到纯化融合蛋白浓缩液。
将纯化前后的样品进行12％SDS-PAGE电泳，考马斯亮蓝染色鉴定，结果如图1所示，可见得到纯化的大小为19kD的REG1A重组蛋白。
6、REG1A重组蛋白的鉴定——鼠抗HIS标签单克隆抗体作为一抗的Western blot分析
将纯化后的REG1A重组蛋白进行SDS-PAGE后，将分离的蛋白从凝胶转至PVDF膜上，用5％脱脂奶粉室温封闭1h，后加入1：500稀释的鼠抗HIS标签单克隆抗体，4℃过夜。第二天TBST洗膜3次，加入辣根过氧化物酶(HRP)标记的羊抗鼠IgG抗体，37℃孵育1h，后PBST洗膜3次，进行ECL显色。显色结果(图2)显示在15-25kD范围内有大小约19kD的阳性单一条带，而转入空载体质粒的菌株无条带，表明REG1A重组蛋白成功表达。
实施例2、制备REG1A单克隆抗体。
1、制备融合蛋白REG1A的单克隆抗体
动物免疫：首次免疫取上述制备的带有组氨酸标签的REG1A重组蛋白，PBS稀释至合适比例，与完全福氏佐剂，以1：1的比例充分混合乳化。经颈背部、腹股沟皮下多点注射的方式，100mg/只，免疫小鼠3只，小鼠取6-8周的BALB/C鼠。两周后进行第二次免疫，取稀释的REG1A重组蛋白与不完全佐剂，以1：1的比例充分混合乳化，注射方式同上。两周后进行第三次免疫，方法同第二次免疫。三次免疫结束后取血清，其中即含有REG1A重组蛋白的多克隆抗体，经间接ELISA法或放免法可检测其效价。
2、细胞融合
①小鼠骨髓瘤细胞准备：SP2/0小鼠骨髓瘤细胞复苏，在含有8-氮杂鸟苷酸(20ug/mL)的无血清1640培养基中至少培养三代，以保持其HGPRT型。
②小鼠饲养细胞准备：细胞融合前1-2天，取6-8周龄BALC/B小鼠，腹腔注射不含血清的1640培养基，收集腹腔灌洗液，1000rpm离心5min，弃上清。加入HAT培养基重悬，将细胞稀释至1000个/ml，以100ul/孔将饲养细胞铺至96孔板中。
③小鼠脾细胞准备：细胞融合前3-4天对小鼠进行加强免疫，PBS稀释后的REG1A重组蛋白，腹腔注射，100mg/只。免疫当天取小鼠脾脏，制备脾细胞悬液，计数备用。
取对数生长期的小鼠骨髓瘤细胞SP2/0与脾细胞悬液，按4：1的比例将其与脾细胞混合，1000rpm离心5min，弃上清。轻轻弹匀试管底部的细胞沉淀。将试管置于37℃蒸馏水中，沿管壁缓慢加入1ml聚乙二醇融合剂，边加边缓慢转动混匀，1min内加完。后在3min内由慢到快加入3ml 37℃预热的1640培养基(未加血清)。重复上述步骤5min内加入20ml 37℃预热的1640培养基(未加血清)，37℃水浴中静置10min。后1500rpm离心5min，弃上清，HAT培养基重悬，以100ul/孔加入到含有饲养细胞的96孔板中。培养箱中培养7-8天后观察细胞生长情况，之后2-3天进行一次半换液。
3、阳性杂交瘤细胞的筛选
当观察到有明显集落生成，且集落未融合前进行筛选。用REG1A重组蛋白为抗原，以0.1ug/ml浓度包被，取杂交瘤上清液100ul/孔进行间接ELISA检测，再在原培养板中不加HAT培养基，100ul/孔。
4、阳性杂交瘤的亚克隆
取阳性杂交瘤中OD值较高的孔，现转移至24孔板中扩大培养。用有限稀释的方法进行3-4次亚克隆，选取持续阳性、OD值高、形态好的单细胞亚克隆作为定植株，进行扩大培养和冻存保种。
5、制备单克隆抗体腹水
用体内诱生法制备单克隆抗体腹水，选取8周左右的BALB/C小鼠，向其腹腔内注射0.5ml石蜡油。将扩大培养的杂交瘤，吸去上清，将细胞重悬计数，以2×106个/mL，取0.5mL注射入小鼠腹腔。7天后收集腹水，即为REG1A单克隆抗体。
结果显示，A2、A4、A5、A6杂交瘤细胞株均可产生REG1A单克隆抗体。如图3所示，经单克隆抗体亚型鉴定分析，各REG1A单克隆抗体的重链均为IgG1型，且轻链为均为λ型。
6、以原核表达的无组氨酸标签的REG1A蛋白作为抗原，与A2、A4、A5、A6杂交瘤细胞株的REG1A单克隆抗体分别进行Western blot分析，结果如图4所示，所有细胞株的单克隆抗体均能与REG1A蛋白特异性结合，其中A6杂交瘤细胞株产生的抗体特异性相对较强，由此成功获得高特异性的REG1A单克隆抗体。
7、将A6细胞株杂交瘤扩大培养后腹腔注射入小鼠体内，收集小鼠腹水即得到REG1A单克隆抗体，用间接ELISA法进行效价检测，如图5所示，所得抗体的有效稀释倍速在128000以上。
8、将A6细胞株杂交瘤的REG1A单克隆抗体进行测序，结果如下：
(1)重链
氨基酸序列(SEQ ID NO:1)：
SGPELVKPGASVKISCKASGYTFTDYNTHWVKQSHGKSLEWIGHIFPYNGGNVYNQKFKIKATLTVDISSSTAYMELRSLTSEDSAVYYCARRGPGTGYFDVWGAGTTVTVSS。
具体可变区信息如图6所示。
核酸序列(SEQ ID NO:3)：
tcaggacctgagctggtgaaacctggggcctcagtgaagatctcctgcaaggcttctggctacacattcactgactataacacacactgggtgaagcagagccatggcaagagccttgagtggattggacatatttttccttacaatggtggtaatgtctacaaccagaagttcaagatcaaggccacattgactgtagacatttcctccagcacagcctacatggaactccgcagcctgacatctgaagactctgcagtctattactgtgcaagaaggggacctgggacggggtacttcgatgtctggggcgcagggaccacggtcaccgtctcctcag。
(2)轻链
氨基酸序列(SEQ ID NO:2)：
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSGSGSGTDYSLTISNLDQEDIATYFCQQGHTLPRTFGGGTKLEIK。
具体可变区信息如图7所示。
核酸序列(SEQ ID NO:4)：
gatatccagatgacacagactacatcctccctgtctgcctctctgggagacagagtcaccatcagttgcagggcaagtcaggacattagcaattatttaaactggtatcagcagaaaccagatggaactgttaaactcctgatctactacacatcaagattacactcaggagtcccatcaaggttcagtggcagtgggtctggaacagattattctctcaccattagcaacctggaccaagaagatattgccacttacttttgccaacagggtcatacacttcctcggacgttcggtggaggcaccaagctggaaatcaaac。
注：在具体实施时，可根据实际情况在抗体的重链或轻链上增设标记，如荧光标记、酶标记、放射性标记等。
实施例3、REG1A单克隆抗体的Western blot分析
现有相关研究已验证糖尿病患者血清中REG1A水平升高，以4例糖尿病患者血清为抗原，采用实施例2所制备的REG1A单克隆抗体作为一抗孵育显色后，结果如图8所示，REG1A单克隆抗体能够特异性识别血清中大小约19kD的REG1A蛋白。
实施例4、REG1A单克隆抗体的免疫组化分析
正常情况下REG1A在十二指肠组织中表达丰富。取正常的人十二指肠和肾组织的蜡片(注：上述蜡片为合法来源且符合伦理准则规定)，经脱蜡、抗原修复后，以10％BSA室温封闭1h，PBS洗涤3次，加1：500稀释的实施例2所制备的REG1A单克隆抗体，4℃过夜。第二天PBS洗涤3次，加入辣根过氧化物酶(HRP)标记的兔抗鼠IgG抗体，室温孵育1h。PBS洗涤3次，后DAB显色，苏木素复染，封片剂封片。免疫组化结果显示，十二指肠上皮细胞包浆中可见棕色染色(图9)，肾组织中未见，说明实施例2所制备的REG1A单克隆抗体可特异性识别正常组织中的REG1A蛋白。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
序列表
<110> 东南大学附属中大医院
<120> 再生蛋白REG1A单克隆抗体及其应用
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Asn Gly Gly Asn Val Tyr Asn Gln Lys Phe Lys Ile Lys Ala Thr Leu
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Claims (8)
1.一种再生蛋白REG1A单克隆抗体，所述抗体包括重链和轻链，其特征是，所述抗体具有以下技术特征：
所述重链包括重链CDR1、重链CDR2以及重链CDR3；重链CDR1的氨基酸序列为VKQSH；重链CDR2的氨基酸序列为GGNVYNQKFKIKATLT；重链CDR3的氨基酸序列为PGTGYFDV；
所述轻链包括轻链CDR1、轻链CDR2以及轻链CDR3；轻链CDR1的氨基酸序列为RASQDISNYLN；轻链CDR2的氨基酸序列为YTSRLHS；轻链CDR3的氨基酸序列为QQGHTLPRT。
2. 根据权利要求1所述的再生蛋白REG1A单克隆抗体，其特征是，所述重链的氨基酸序列如SEQ ID NO:1所示；所述轻链的氨基酸序列如SEQ ID NO:2所示。
3.根据权利要求1或2所述的再生蛋白REG1A单克隆抗体，其特征是，所述抗体的重链或轻链上具有标记，所述标记包括荧光标记、酶标记、以及放射性标记。
4.编码权利要求1或2所述再生蛋白REG1A单克隆抗体的核酸。
5. 根据权利要求4所述的核酸，其特征是，所述核酸包括编码重链的核酸且序列如SEQID NO:3所示，以及编码轻链的核酸且序列如SEQ ID NO:4所示。
6.权利要求1或2所述再生蛋白REG1A单克隆抗体用于制备试剂盒的用途。
7.根据权利要求6所述的用途，其特征是，所述试剂盒为针对再生蛋白REG1A的检测试剂盒或诊断试剂盒。
8.权利要求4或5所述核酸用于制备再生蛋白REG1A单克隆抗体的用途。
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