CN117362428A - 一种a-fabp单克隆抗体2c6及其制备方法和用途 - Google Patents
一种a-fabp单克隆抗体2c6及其制备方法和用途 Download PDFInfo
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- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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Landscapes
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Abstract
本发明涉及一种抗A‑FABP单克隆抗体2C6及其制备方法和用途，具体公开了A‑FABP单克隆抗体2C6或其抗原结合片段，其具有如SEQ ID NO:1、SEQID NO:2和SEQ ID NO:3所示的三个重链互补决定区(CDR)，并具有如SEQ IDNO:10、SEQ ID NO:11和SEQ ID NO:12所示的三个轻链互补决定区。本发明提供了一种抗A‑FABP的单克隆抗体的制备方法、鉴定及应用，该单克隆抗体具有灵敏度高，特异性好等优点，可以广泛用于Western blot、ELISA、flowCytometry等不同手段检测A‑FABP，为研究人A‑FABP的功能提供了基础。
Description
技术领域
本发明涉及抗体领域，具体涉及一种A-FABP单克隆抗体2C6及其制备方法和用途。
背景技术
脂肪细胞型脂肪酸结合蛋白(adipocyte-type fatty acid binding protein,A-FABP)是载脂蛋白家族的成员之一，分子量为14.6KD，主要表达在成熟脂肪细胞和巨噬细胞中。A-FABP蛋白主要功能是作为游离脂肪酸分子的载体，在脂肪细胞中调节脂肪的储存和分解；在巨噬细胞中调节脂质的累积和促进多种炎性因子的表达，包括MCP-1、TNF-α、IL-6、IL-1β等。A-FABP蛋白可分泌到细胞外和血液中，促进炎症反应，同时与多种代谢疾病如肥胖、糖尿病、脂代谢紊乱、非酒精性脂肪肝炎、动脉粥样硬化等发生发展密切相关。而在动物实验中，小鼠移植A-FABP基因敲除鼠骨髓已被证明可以全面改善动脉粥样硬化，并且没有代谢副作用；同时A-FABP基因突变导致A-FABP表达水平降低的小鼠，具有较低的甘油三酯水平，降低了心血管疾病的风险，并且减少肥胖引起的2型糖尿病。有关研究还阐明了A-FABP蛋白在巨噬细胞中通过JNK/c-Jun/AP-1信号轴上调多种炎症因子表达的分子机制，并证实在发生缺血性脑卒中后，血液中和脑组织中的A-FABP蛋白表达水平都上升，促进炎性因子的表达，从而加剧脑卒中后神经炎症。因此，A-FABP可作为潜在的改善代谢性疾病和相关的心脑血管疾病的治疗靶点。
鉴于A-FABP蛋白在代谢类疾病及其心脑血管并发症中的重要作用，世界上多个制药公司和科研机构研发了数以百计的A-FABP蛋白抑制剂，目前为止绝大多数为小分子化合物。这些抑制剂尽管在体内实验中表现出很高的活性和良好的特异性，在多种动物疾病模型中也显示出良好的治疗效果，但是由于化学小分子类药物在体内靶点特异性低，副作用多，尤其是心脏毒性，目前还没有一个A-FABP蛋白小分子抑制剂能够进入临床试验阶段。美国哈佛大学曾在2015年研制了一个兔和鼠嵌合单抗，但是其对A-FABP蛋白的亲和力非常低，至今未进入临床试验。
作为生物学研究领域应用最广泛的工具，抗体产品的地位毋庸置疑。而抗体的特异性和应用范围长久以来困扰着抗体产业，也是整个生物学研究领域的极大危机。抗体产品糟糕的质量会直接导致实验结果的误差，以及研究结果无法被重复和再现。研究项目的进展往往会因为抗体带来的问题停滞不前，由此造成的损失也是惊人的。据2015年的数据统计，仅在美国，平均每年就有3.5亿美元被浪费在这些无效抗体上。而在世界范围内，每年竟有8亿美元被浪费，占到了全球科研抗体总开销的50％。
发明内容
本发明涉及一种抗A-FABP单克隆抗体及其制备方法和用途。用杂交瘤技术制备抗A-FABP单克隆抗体，可以用于ELISA和免疫印迹等生物学研究实验。
本发明一个方面提供了一种分离的抗A-FABP的抗体或其抗原结合片段，其具有如SEQ ID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的三个重链互补决定区(CDR)，并具有如SEQID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的三个轻链互补决定区。
本发明另个方面提供了一种分离的抗A-FABP的抗体或其抗原结合片段，其具有如SEQ ID No：1所示的重链可变区，如SEQ ID No:9所示的轻链可变区。
本发明再一个方面提供了一种核苷酸序列，其特征在于：其编码如前述的抗A-FABP的单克隆抗体或其抗原结合片段。
在本发明的技术方案中，所述的抗体为单克隆抗体。
本发明再一个方面提供了一种杂交瘤细胞株，所述杂交瘤细胞株已于2022年6月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号CGMCC No.45188，其能够产生如上所述的单克隆抗体或其抗原结合片段。
本发明再一个方面提供了一种重组载体，其特征在于：包含前述的核苷酸序列。
本发明再一个方面提供了一种宿主细胞，其特征在于：包含前述载体或载体组，优选地，所述宿主细胞是原核的或真核的，更优选的选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
本发明再一个方面提供了一种试剂盒，所述试剂盒包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了一种检测试剂，所述检测试剂包含如前述的抗体或其抗原结合片段。
本发明再一个方面提供了上述抗体或其抗原结合片段作为检测试剂的用途，所述试剂用于以下用途的试剂：酶联免疫吸附检测(ELISA)、免疫印迹(Western Blot)、流式细胞术(FACS)、免疫组织化学(IHC)检测或者免疫PCR。
在上述在免疫学检测中，抗体或其抗原结合片段可单独或与通过化学键偶联，静电吸附或者亲疏水性吸附，而连接缀合物包括辣根过氧化物酶(HRP)，碱性磷酸酶(AP)，生物素(Biotin)，异硫氰酸荧光素(FITC)，Cy3、Cy5、磁珠和琼脂糖等缀合物连接。
在本发明的技术方案中，检测试剂可用于非诊断的治疗目的检测。
本发明再一个方面提供了上述抗体或其抗原结合片段作为体外分离或纯化A-FABP的试剂的用途。
在本发明中，上述抗体或其抗原结合片段通过杂交瘤方法制备。
在本发明中，所述抗体或其抗原结合片段的重链和轻链可变区的核苷酸和氨基酸序列如下所示。
重链可变区氨基酸序列：QVQLKQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAFYYYGSRRFAYWGQGTLVTVSA
SEQ ID NO.1
其中，重链可变区CDR1氨基酸序列为：DTYMH SEQ ID NO.2
重链可变区CDR2氨基酸序列为：RIDPANGNTKYDPKFQG SEQ ID NO.3
重链可变区CDR3氨基酸序列为：YYYGSRRFAY SEQ ID NO.4
重链可变区FR1氨基酸序列为：QVQLKQSGAELVKPGASVKLSCTASGFNIK SEQ ID NO.5
重链可变区FR2氨基酸序列为：WVKQRPEQGLEWIG SEQ ID NO.6
重链可变区FR3氨基酸序列为：KATITADTSSNTAYLQLSSLTSEDTAVYYCAF SEQ IDNO.7
重链可变区FR4氨基酸序列为：WGQGTLVTVSA SEQ ID NO.8
轻链可变区氨基酸序列：
DIQMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK SEQ ID NO.9
其中，轻链可变区CDR1氨基酸序列为：KSSQSLLDSDGKTYLN SEQ ID NO.10
轻链可变区CDR2氨基酸序列为：LVSKLDS SEQ ID NO.11
轻链可变区CDR3氨基酸序列为：WQGTHFPWT SEQ ID NO.12
轻链可变区FR1氨基酸序列为：DIQMTQTPLTLSVTIGQPASISC SEQ ID NO.13
轻链可变区FR2氨基酸序列为：WLLQRPGQSPKRLIY SEQ ID NO.14
轻链可变区FR3氨基酸序列为：GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC
SEQ ID NO.15
轻链可变区FR4氨基酸序列为：FGGGTKLEIK SEQ ID NO.16
重链可变区基因全长357bp，编码氨基酸残基119个。重链可变区核苷酸序列如SEQID NO:17所示，重链可变区氨基酸序列如SEQ ID NO:1所示，重链CDR1氨基酸序列如SEQ IDNo:2所示，重链CDR 2氨基酸序列如SEQ ID No:3所示，重链CDR3氨基酸序列如SEQ ID No:4所示。
轻链可变区基因序列全长336bp，编码氨基酸残基112个。轻链可变区核苷酸序列如SEQ ID NO:18所示，轻链可变区氨基酸序列如SEQ ID NO:9，轻链CDR 1氨基酸序列如SEQID No:10所示，轻链CDR 2氨基酸序列如SEQ ID No:11所示，轻链CDR 3氨基酸序列如SEQID No:12所示。
关于生物材料保藏的说明
本发明涉及下列已在中国普通微生物菌种保藏管理中心(CGMCC，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)进行保藏的生物材料：
杂交瘤细胞株2C6，其保藏号为CGMCC No.45188，保藏日期为2022年6月16日，分类命名为杂交瘤细胞株(拉丁名Mus musculus)。
有益效果
本发明提供了一种抗A-FABP的单克隆抗体的制备方法、鉴定及应用，该单克隆抗体具有灵敏度高，特异性好等优点，适用于不同检测方法。本发明提供的单克隆抗体，可以广泛用于Western blot、ELISA、flowCytometry等不同手段检测A-FABP，为研究人A-FABP的功能提供了基础。
附图说明
图1为A-FABP蛋白单克隆抗体2C6特异性检验结果。
具体实施方式
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。
实施例1用人A-FABP蛋白免疫小鼠，筛选人A-FABP蛋白单克隆抗体
通过香港因诺诊断公司购买：人A-FABP蛋白(2000μg，Cat.No.41030)，蛋白纯度均>95％。
免疫雌性BALB/c小鼠(6周龄)。首次免疫使用弗氏完全佐剂进行抗原乳化，皮下6点注射，每只小鼠注射的抗原量为100ug。14天后，进行第二次免疫，使用弗氏不完全佐剂乳化抗原，皮下6点注射，每只小鼠注射的抗原量为100ug。14天后，进行第3次免疫，方法同二次免疫。14天后，对小鼠剪尾采集少量血液进行血清效价ELISA检测，选择抗体滴度最高(1:500000)的小鼠进行加强免疫，无需乳化，通过腹腔注射抗原蛋白，每只小鼠注射100μg。
第4次免疫后3～5天，处死小鼠取其脾细胞与SP2/0细胞融合，通过HAT培养基培养获得稳定的杂交瘤细胞。通过ELISA法筛选得到能分泌A-FABP抗体的杂交瘤细胞，通过有限稀释的方法进行亚克隆，筛选得到能分泌A-FABP抗体的单克隆杂交瘤细胞株2C6，通过逐级扩大培养，液氮冻存保种。杂交瘤细胞已在中国普通微生物菌种保藏管理中心(CGMCC，地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所)进行保藏的生物材料：杂交瘤细胞株2B8，其保藏号为CGMCC No.45188，保藏日期为2022年6月16日，分类命名为杂交瘤细胞株(拉丁名Mus musculus)。
制备和纯化腹水抗体：雌性BALB/c小鼠(8周龄)腹腔注射弗氏不完全佐剂，每只小鼠注射0.5mL，3～5天后腹腔注射处于对数生长期的杂交瘤细胞2C6，每只小鼠注射5×105个细胞(0.5mL)。10天后处死小鼠，获得腹水。5000rpm，4℃离心10min，去除沉淀，用10倍体积1×PBS溶液稀释腹水，混匀后过0.45μm滤膜。通过Protein G(Protein G Sepharose4Fast Flow,GE Healthcare)亲和纯化腹水，得到纯化的A-FABP抗体。
实施例2 A-FABP蛋白单克隆抗体2C6特异性检验
通过香港因诺诊断公司购买：人A-FABP蛋白(200μg，Cat.No.41030)、小鼠A-FABP蛋白(300μg，Cat.No.42030)、人E-FABP蛋白(100μg，Cat.No.41040)、小鼠E-FABP蛋白(100μg，Cat.No.41030)；通过北京义翘神州公司购买：人H-FABP蛋白(100μg，Cat.No.12476-HNAE)、小鼠H-FABP蛋白(100μg，Cat.No.51233-MNAE)，上述蛋白纯度均>95％。
通过纯化的小鼠或人A-FABP、H-FABP、E-FABP作为包被抗原，以ELISA法进一步检测的A-FABP蛋白单克隆抗体2C6特异性。发现克隆2C6与A-FABP特异性结合，但不与H-FABP或E-FABP结合。详见图1及表1。
表1.A-FABP蛋白单克隆抗体2C6特异性检验
实施例.3A-FABP蛋白单克隆抗体2C6腹水效价检测
2C6杂交瘤细胞腹膜内注射BALB/c小鼠制备腹水。腹腔注射克隆10天后，收集腹水并纯化。通过ELISA法检测抗体效价(包被抗原：A-FABP 1μg/ml)，发现亲和纯化的抗体2C6滴度达到1/200,000。详见表2。
表2.A-FABP蛋白单克隆抗体2C6腹水效价检验
| 1/1,000 | 1/2,000 | 1/20,000 | 1/200,000 | FT | NC | |
| 2C6 | 0.59 | 0.183 | 0.026 | 0.024 | 0.036 | 0.026 |
实施例4A-FABP蛋白单克隆抗体2C6抗体效价检测
进一步使用ELISA法检测，以不同浓度的A-FABP蛋白(包被抗原：A-FABP 1μg/ml、0.5μg/ml、0.05μg/ml、0.005μg/ml、0.0005μg/ml)检测单克隆抗体2C6的敏感性。发现抗体2C6对低浓度A-FABP蛋白敏感性较高。详见表3。
表3.A-FABP蛋白单克隆抗体2C6抗体效价检测
实施例5A-FABP蛋白单克隆抗体2C6基因测序
经免疫、融合及单克隆化、抗体纯化以及抗体特异性及效价鉴定之后，选取2C6单克隆抗体细胞株进行总RNA提取，并反转录成cDNA，然后以cDNA为模板PCR扩增抗体的重链可变区和轻链可变区。采用Invitrogen公司的TRIzol reagent试剂盒(15596-026)，按照其说明书进2C6单克隆抗体细胞株总RNA提取，接着采用Takara公司的5’RACE FULL试剂盒(D315)，以总RNA为模板，试剂盒中的随机引物进行反转录为第一链cDNA，然后重链以恒定区设计引物和试剂盒中的接头引物进行PCR扩增，轻链以恒定区设计引物和试剂盒中的接头引物进行PCR扩增。
琼脂糖凝胶回收试剂盒回收PCR片段进行TA克隆后挑单克隆进行PCR鉴定，鉴定正确菌株中选取部分样品送至Invitrogen测序。最终确定重链可变区核苷酸序列为SEQ IDNO:17，轻链可变区核苷酸序列为SEQ ID NO:18，重链可变区氨基酸序列为SEQ ID NO:1，轻链可变区氨基酸序列为SEQ ID NO:9，见表4。
表4、2C6抗体重链可变区和轻链可变区具体序列
2C6抗体重链可变区氨基酸序列如下(SEQ ID NO:1)：
QVQLKQSGAELVKPGASVKLSCTASGFNIKDTYMHWVKQRPEQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCAFYYYGSRRFAYWGQGTLVTVSA
其中，
重链可变区CDR1氨基酸序列为：DTYMH SEQ ID NO.2
重链可变区CDR2氨基酸序列为：RIDPANGNTKYDPKFQG SEQ ID NO.3
重链可变区CDR3氨基酸序列为：YYYGSRRFAY SEQ ID NO.4
重链可变区FR1氨基酸序列为：QVQLKQSGAELVKPGASVKLSCTASGFNIK SEQ ID NO.5
重链可变区FR2氨基酸序列为：WVKQRPEQGLEWIG SEQ ID NO.6
重链可变区FR3氨基酸序列为：KATITADTSSNTAYLQLSSLTSEDTAVYYCAF SEQ IDNO.7
重链可变区FR4氨基酸序列为：WGQGTLVTVSA SEQ ID NO.8
轻链可变区氨基酸序列：
DIQMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLLQRPGQSPKRLIYLVSKLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHFPWTFGGGTKLEIK SEQ ID NO.9
其中，轻链可变区CDR1氨基酸序列为：KSSQSLLDSDGKTYLN SEQ ID NO.10
轻链可变区CDR2氨基酸序列为：LVSKLDS SEQ ID NO.11
轻链可变区CDR3氨基酸序列为：WQGTHFPWT SEQ ID NO.12
轻链可变区FR1氨基酸序列为：DIQMTQTPLTLSVTIGQPASISC SEQ ID NO.13
轻链可变区FR2氨基酸序列为：WLLQRPGQSPKRLIY SEQ ID NO.14
轻链可变区FR3氨基酸序列为：GVPDRFTGSGSGTDFTLKISRVEAEDLGVYYC
SEQ ID NO.15
轻链可变区FR4氨基酸序列为：FGGGTKLEIK SEQ ID NO.16
2C6抗体重链可变区核苷酸序列如下(SEQ ID NO:17)：
CAGGTGCAGCTGAAGCAGTCTGGGGCAGAGCTTGTGAAGCCAGGGGCCTCAGTCAAGTTGTCCTGCACAGCTTCTGGCTTCAACATTAAAGACACCTATATGCACTGGGTGAAGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGAAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTATAACAGCAGACACATCCTCCAACACAGCCTACCTGCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTTTTTATTACTACGGTAGTAGGAGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAG
2C6抗体轻链可变区核苷酸序列如下(SEQ ID NO:18)：
GATATCCAGATGACACAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAAC
SEQUENCE LISTING
<110> 中国科学院深圳先进技术研究院
<120> 一种A-FABP单克隆抗体2C6及其制备方法和用途
<130> CP122010532C
<160> 18
<170> PatentIn version 3.3
<210> 1
<211> 119
<212> PRT
<213> 人工序列
<400> 1
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile
35 40 45
Gly Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe
50 55 60
Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr
65 70 75 80
Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Phe Tyr Tyr Tyr Gly Ser Arg Arg Phe Ala Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 2
<211> 5
<212> PRT
<213> 人工序列
<400> 2
Asp Thr Tyr Met His
1 5
<210> 3
<211> 17
<212> PRT
<213> 人工序列
<400> 3
Arg Ile Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys Phe Gln
1 5 10 15
Gly
<210> 4
<211> 10
<212> PRT
<213> 人工序列
<400> 4
Tyr Tyr Tyr Gly Ser Arg Arg Phe Ala Tyr
1 5 10
<210> 5
<211> 30
<212> PRT
<213> 人工序列
<400> 5
Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys
20 25 30
<210> 6
<211> 14
<212> PRT
<213> 人工序列
<400> 6
Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile Gly
1 5 10
<210> 7
<211> 32
<212> PRT
<213> 人工序列
<400> 7
Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr Leu Gln
1 5 10 15
Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala Phe
20 25 30
<210> 8
<211> 11
<212> PRT
<213> 人工序列
<400> 8
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
1 5 10
<210> 9
<211> 112
<212> PRT
<213> 人工序列
<400> 9
Asp Ile Gln Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser
20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser
35 40 45
Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly
85 90 95
Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110
<210> 10
<211> 16
<212> PRT
<213> 人工序列
<400> 10
Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr Leu Asn
1 5 10 15
<210> 11
<211> 7
<212> PRT
<213> 人工序列
<400> 11
Leu Val Ser Lys Leu Asp Ser
1 5
<210> 12
<211> 9
<212> PRT
<213> 人工序列
<400> 12
Trp Gln Gly Thr His Phe Pro Trp Thr
1 5
<210> 13
<211> 23
<212> PRT
<213> 人工序列
<400> 13
Asp Ile Gln Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys
20
<210> 14
<211> 15
<212> PRT
<213> 人工序列
<400> 14
Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr
1 5 10 15
<210> 15
<211> 32
<212> PRT
<213> 人工序列
<400> 15
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
1 5 10 15
Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys
20 25 30
<210> 16
<211> 10
<212> PRT
<213> 人工序列
<400> 16
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
1 5 10
<210> 17
<211> 358
<212> DNA
<213> 人工序列
<400> 17
caggtgcagc tgaagcagtc tggggcagag cttgtgaagc caggggcctc agtcaagttg 60
tcctgcacag cttctggctt caacattaaa gacacctata tgcactgggt gaagcagagg 120
cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtaa tactaaatat 180
gacccgaagt tccagggcaa ggccactata acagcagaca catcctccaa cacagcctac 240
ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc tttttattac 300
tacggtagta ggaggtttgc ttactggggc caagggactc tggtcactgt ctctgcag 358
<210> 18
<211> 337
<212> DNA
<213> 人工序列
<400> 18
gatatccaga tgacacagac tccactcact ttgtcggtta ccattggaca accagcctcc 60
atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata tttgaattgg 120
ttgttacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180
tctggagtcc ctgacaggtt cactggcagt ggatcaggga cagatttcac actgaaaatc 240
agcagagtgg aggctgagga tttgggagtt tattattgct ggcaaggtac acattttccg 300
tggacgttcg gtggaggcac caagctggaa atcaaac 337
Claims (10)
1.一种分离的抗A-FABP的单克隆抗体或其抗原结合片段，其特征在于：其具有如SEQID NO:2、SEQ ID NO:3和SEQ ID NO:4所示的三个重链互补决定区，并具有如SEQ ID NO:10、SEQ ID NO:11和SEQ ID NO:12所示的三个轻链互补决定区；
其中，重链互补决定区CDR1如SEQ ID NO:2所示；
重链互补决定区CDR2如SEQ ID NO:3所示；
重链互补决定区CDR3如SEQ ID NO:4所示；
轻链互补决定区CDR1如SEQ ID NO:10所示；
轻链互补决定区CDR2如SEQ ID NO:11所示；
轻链互补决定区CDR3如SEQ ID NO:12所示。
2.一种分离的抗A-FABP的单克隆抗体或其抗原结合片段，其特征在于：其具有如SEQID No:1所示的重链可变区，如SEQ ID No:9所示的轻链可变区。
3.一种核苷酸，其特征在于：其编码如权利要求1或2所述的单克隆抗体或其抗原结合片段。
4.一种重组载体，其特征在于：包含权利要求3所述的核苷酸序列。
5.一种宿主细胞，其特征在于：包含权利要求4所述的重组载体；
优选地，所述宿主细胞是原核的或真核的；
优选地，所述宿主细胞选自酵母细胞、哺乳动物细胞或适用于制备抗体或其抗原结合片段的其它细胞。
6.一种检测A-FABP用的试剂盒，其特征在于：所述试剂盒包含如权利要求1或2所述的单克隆抗体或其抗原结合片段。
7.一种检测A-FABP用的检测试剂，其特征在于：所述检测试剂包含如权利要求1或2所述的单克隆抗体或其抗原结合片段；
优选地，所述检测试剂用于酶联免疫吸附检测、免疫印迹、流式细胞术、免疫组织化学检测或者免疫PCR。
8.权利要求1或2所述的单克隆抗体或其抗原结合片段在制备检测A-FABP的试剂中的用途；
优选地，所述试剂用于以下用途的试剂：酶联免疫吸附检测、免疫印迹、流式细胞术、免疫组织化学检测或者免疫PCR。
9.权利要求1或2所述的单克隆抗体或其抗原结合片段在制备体外分离或纯化A-FABP的试剂中的用途。
10.一种杂交瘤细胞株，其特征在于，所述杂交瘤细胞株已于2022年6月16日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏号CGMCC No.45188，其能够产生如权利要求1或2所述的单克隆抗体或其抗原结合片段。
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