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CN112794900B - cBIN1 antibodies and uses thereof - Google Patents

cBIN1 antibodies and uses thereof Download PDF

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CN112794900B
CN112794900B CN202011626540.2A CN202011626540A CN112794900B CN 112794900 B CN112794900 B CN 112794900B CN 202011626540 A CN202011626540 A CN 202011626540A CN 112794900 B CN112794900 B CN 112794900B
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周杨钊
周康
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Second Xiangya Hospital of Central South University
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Abstract

本发明涉及免疫技术领域,尤其涉及cBIN1抗体及其应用。本发明提供的cBIN1抗体的重链的三个CDR区分别具有如SEQ ID NO:1、2和3所示的氨基酸序列;轻链的三个CDR区分别具有如SEQ ID NO:4、5和6所示的氨基酸序列。该抗体与抗原具有良好的结合能力,以其制备试剂能够实现对目标抗原的准确检测。The present invention relates to the field of immunization technology, in particular to cBIN1 antibody and application thereof. The three CDR regions of the heavy chain of the cBIN1 antibody provided by the present invention respectively have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3; the three CDR regions of the light chain respectively have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 3. The amino acid sequence shown in 6. The antibody has good binding ability with the antigen, and the reagent prepared by the antibody can accurately detect the target antigen.

Description

cBIN1抗体及其应用cBIN1 antibody and its application

技术领域technical field

本发明涉及免疫技术领域,尤其涉及cBIN1抗体及其应用。The present invention relates to the field of immunization technology, in particular to cBIN1 antibody and application thereof.

背景技术Background technique

心衰是多种原因导致的心脏结构和/或功能的异常改变,使心室收缩和/或舒张功能发生障碍,从而引起的一组复杂的临床综合征。心衰是各种心脏疾病的严重表现或晚期阶段,死亡率和再住院率居高不下。近几年各地的资料显示,我国心衰的患病率已显著增加至2%~3%,现症患者约1000万。Heart failure is a group of complex clinical syndromes caused by abnormal changes in cardiac structure and/or function caused by various reasons, resulting in dysfunction of ventricular systolic and/or diastolic function. Heart failure is a severe manifestation or late stage of various heart diseases, with high mortality and rehospitalization rates. In recent years, data from various places show that the prevalence of heart failure in my country has increased significantly to 2% to 3%, and there are about 10 million patients with the disease.

心衰心肌的核心病理改变是钙离子传输能力变弱,使心肌的收缩能力受限制。而导致心衰特征性的钙离子传输障碍的原因是兴奋-收缩(excitation-contraction)(E-C)失耦联与非同步的钙释放。生理条件下,膜电位去极化后诱发同步的钙释放,使钙离子穿过细胞膜,在肌浆网内触发更多的钙释放,即钙诱导的钙释放calcium-induced-calcium-release(CICR)过程。健康的和理想状态下的CICR过程依赖于位于心肌细胞之间结合部肌浆网膜上的兰尼碱受体(ryanodine receptors,RyRs)和横管(transverse-tubule,T-tubule)膜上的L型钙离子通道(L-type calcium channels,LTCCs)之间的紧密协作。横管内的LTCCs与结合部肌浆网膜上的RyRs形成了影响细胞浆外钙离子转运的二聚体复合物。心肌受各种原因导致的压力负荷增加而导致的病理性肥厚过程中,大量的横管结构发生错乱和重构,同时也伴随着这些二聚体结构的破坏,并最终导致非同步的CICR过程,并逐渐由肥厚失代偿发展为心衰。心衰时,除了横管的形态学上的一系列重构改变,受损的LTCC转移到横管,与RyRs形成二元复合体的过程也被扰乱,Ga2+转移能力下降,进而导致E-C失耦联与心脏功能下降。The core pathological change of the myocardium in heart failure is the weakened calcium ion transport capacity, which limits the contractility of the myocardium. The cause of the calcium transport disorder characteristic of heart failure is excitation-contraction (EC) decoupling and asynchronous calcium release. Under physiological conditions, depolarization of the membrane potential induces synchronized calcium release, allowing calcium ions to cross the cell membrane and trigger more calcium release within the sarcoplasmic reticulum, known as calcium-induced-calcium-release (CICR). )process. Healthy and ideal CICR processes rely on ryanodine receptors (RyRs) located on the sarcoplasmic membrane at the junction between cardiomyocytes and on the transverse-tubule (T-tubule) membrane. Tight collaboration between L-type calcium channels (LTCCs). The LTCCs in the transverse tube and the RyRs on the junctional sarcoplasmic omentum form a dimeric complex that affects the extracytoplasmic calcium transport. During the pathological hypertrophy of the myocardium due to increased pressure load from various reasons, a large number of transverse canal structures are disordered and remodeled, accompanied by the destruction of these dimer structures, and ultimately lead to the asynchronous CICR process. , and gradually developed from hypertrophic decompensation to heart failure. In heart failure, in addition to a series of remodeling changes in the morphology of the transverse canal, the damaged LTCC is transferred to the transverse canal, the process of forming binary complexes with RyRs is also disturbed, and the transfer ability of Ga 2+ is reduced, which in turn leads to EC. Decoupling and decreased cardiac function.

BIN蛋白(Bar蛋白编码基因)可分为AmphiphysinI、AmpII(又称Bin1)、Bin2、Bin3四种蛋白。作为含有BAR域的蛋白质超家族,Bin1包含一个特征由外显子1~9编码的N端条域(N-BAR)。有趣的是,Bin1的带有20个外显子的基因根据不同的拼接方案生成不同的Bin1蛋白而具有不同的细胞功能。心肌横管内心脏桥接整合因子1(cardiac bridgingintegrator 1,cBIN1)就是带有13,17两个外显子的BIN1蛋白亚型。BIN protein (Bar protein encoding gene) can be divided into four proteins: AmphiphysinI, AmpII (also known as Bin1), Bin2 and Bin3. As a superfamily of BAR domain-containing proteins, Bin1 contains a characteristic N-terminal strip domain (N-BAR) encoded by exons 1-9. Interestingly, the gene with 20 exons of Bin1 has different cellular functions according to different splicing schemes to generate different Bin1 proteins. Cardiac bridging integration factor 1 (cBIN1) in the transverse myocardial canal is a BIN1 protein isoform with two exons 13 and 17.

研究表明,cBIN1参与形成的横管微结构域具有促进和维持LTCC-RyR二元复合体形成的功能。现有的研究结果表明,拥有cBin1的微结构域可以通过以下4项机制促进有效的LTCC-RyR二元复合体的形成:1)促进LTCCs通过微结构域向前转运;2)促进已经转移到横管的LTCCs聚集成簇;3)在横管腔内形成保护性的细胞外钙离子的慢速弥散区,以控制LTCCs的转移动力;4)将胞浆内的RyRs重新召集会交界区肌浆网膜上,便于与LTCCs形成新的二元复合体。同时,cBin1微结构域通过续膜翻转和急性应激刺激下快速重组的能力,对维持心肌横管的稳态发挥了作用。与此同时,研究发现,在心衰相关刺激刺激下,cBin1微结构域会因为cBin1的转录减少而丢失,并伴随着二元复合体形成的减少和心肌收缩力的损伤。也就是说,当cBin1表达减少减少时,横管的微结构域结构会发生紊乱,进而降低心脏收缩能力和对β受体的反应。Studies have shown that the transverse tubular microdomain involved in the formation of cBIN1 has the function of promoting and maintaining the formation of the LTCC-RyR binary complex. Existing findings suggest that the microdomain possessing cBin1 can promote the formation of an efficient LTCC-RyR binary complex through the following four mechanisms: 1) promoting the forward transport of LTCCs through the microdomain; 2) promoting the transport of LTCCs to The LTCCs in the transverse tube aggregate into clusters; 3) form a protective slow diffusion zone of extracellular calcium ions in the transverse tube lumen to control the transfer kinetics of LTCCs; 4) reconvene the RyRs in the cytoplasm to the junctional muscle On the serosa, it is convenient to form a new binary complex with LTCCs. At the same time, the cBin1 microdomain plays a role in maintaining myocardial transverse canal homeostasis through the ability of continuous membrane turnover and rapid reorganization under acute stress stimulation. At the same time, the study found that the cBin1 microdomain is lost due to the reduction of cBin1 transcription, which is accompanied by the reduction of binary complex formation and the impairment of myocardial contractility under the stimulation of heart failure-related stimuli. That is, when cBin1 expression is reduced, the microdomain structure of the transverse tubules is disrupted, which in turn reduces cardiac contractility and responsiveness to beta receptors.

因此,cBIN1有望成为一种新型的心衰标记物。如果能够有效的检测心肌或血液内cBIN1的表达水平,可以客观的评价心脏结构完整性和功能状态,为心衰的评估,治疗提供客观可靠的依据,指导心衰的治疗。开发检测cBIN1的敏感抗体将有效促进对该标记物的检测和评估,为临床诊断和治疗心衰提供新的可靠的参考。Therefore, cBIN1 is expected to become a novel marker of heart failure. If the expression level of cBIN1 in the myocardium or blood can be effectively detected, the structural integrity and functional status of the heart can be objectively evaluated, providing an objective and reliable basis for the evaluation and treatment of heart failure, and guiding the treatment of heart failure. The development of a sensitive antibody to detect cBIN1 will effectively promote the detection and evaluation of this marker, and provide a new and reliable reference for clinical diagnosis and treatment of heart failure.

发明内容SUMMARY OF THE INVENTION

有鉴于此,本发明要解决的技术问题在于提供cBIN1抗体及其应用。In view of this, the technical problem to be solved by the present invention is to provide cBIN1 antibody and its application.

本发明提供的cBIN1抗体,其重链包含三个CDR区,其中至少一个CDR区的氨基酸序列具有如SEQ ID NO:1、2或3所示的氨基酸序列,或者与其具有至少80%序列同源性的序列;其轻链包含三个CDR区,其中至少一个CDR区的氨基酸序列具有如SEQ ID NO:4、5或6所示的氨基酸序列,或者与其具有至少80%序列同源性的序列。The cBIN1 antibody provided by the present invention, its heavy chain comprises three CDR regions, wherein the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO: 1, 2 or 3, or has at least 80% sequence homology with it Its light chain comprises three CDR regions, wherein the amino acid sequence of at least one CDR region has the amino acid sequence shown in SEQ ID NO: 4, 5 or 6, or a sequence with at least 80% sequence homology therewith .

在本发明中,所述cBIN1抗体重链的三个CDR区分别具有如SEQ ID NO:1、2和3所示的氨基酸序列;轻链的三个CDR区分别具有如SEQ ID NO:4、5和6所示的氨基酸序列。In the present invention, the three CDR regions of the cBIN1 antibody heavy chain have the amino acid sequences shown in SEQ ID NO: 1, 2 and 3 respectively; the three CDR regions of the light chain have the amino acid sequences shown in SEQ ID NO: 4, 2 and 3 respectively; Amino acid sequences shown in 5 and 6.

在本发明一些实施例中,所述cBIN1抗体重链的三个CDR区的氨基酸序列依次如SEQ ID NO:1、2和3所示;轻链的三个CDR区的氨基酸序列依次如SEQ ID NO:4、5和6所示。In some embodiments of the present invention, the amino acid sequences of the three CDR regions of the cBIN1 antibody heavy chain are shown in sequence as SEQ ID NOs: 1, 2 and 3; the amino acid sequences of the three CDR regions of the light chain are shown in sequence as SEQ ID NOs: NO: 4, 5 and 6 are shown.

其中,SEQ ID NO:1所示的序列为GFNIKDS;Wherein, the sequence shown in SEQ ID NO:1 is GFNIKDS;

SEQ ID NO:2所示的序列为DPEDDD;The sequence shown in SEQ ID NO:2 is DPEDDD;

SEQ ID NO:3所示的序列为STLVATPWFFDV;The sequence shown in SEQ ID NO:3 is STLVATPWFFDV;

SEQ ID NO:4所示的序列为SASQDINNYLN;The sequence shown in SEQ ID NO:4 is SASQDINNYLN;

SEQ ID NO:5所示的序列为YTSTLHS;The sequence shown in SEQ ID NO:5 is YTSTLHS;

SEQ ID NO:6所示的序列为QQYTHNLPWT。The sequence shown in SEQ ID NO: 6 is QQYTHNLPWT.

本发明提供的cBIN1抗体,其重链包含4个FR区,其中至少一个FR区的氨基酸序列具有如SEQ ID NO:9、10、11或12所示的氨基酸序列,或者与其具有至少80%序列同源性的序列;其轻链包含4个FR区,其中至少一个FR区的氨基酸序列具有如SEQ ID NO:13、14、15或16所示的氨基酸序列,或者与其具有至少80%序列同源性的序列。The cBIN1 antibody provided by the present invention, its heavy chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown in SEQ ID NO: 9, 10, 11 or 12, or has at least 80% of the sequence therewith Homologous sequence; its light chain comprises 4 FR regions, wherein the amino acid sequence of at least one FR region has the amino acid sequence shown in SEQ ID NO: 13, 14, 15 or 16, or has at least 80% sequence identity with it source sequence.

在本发明中,所述cBIN1抗体重链的4个FR区分别具有如SEQ ID NO:9、10、11或12所示的氨基酸序列;轻链的4个FR区分别具有如SEQ ID NO:13、14、15、16所示的氨基酸序列。In the present invention, the 4 FR regions of the cBIN1 antibody heavy chain respectively have the amino acid sequences shown in SEQ ID NO: 9, 10, 11 or 12; the 4 FR regions of the light chain respectively have the amino acid sequences shown in SEQ ID NO: 9, 10, 11 or 12; Amino acid sequences shown in 13, 14, 15, and 16.

在本发明一些实施例中,所述cBIN1抗体重链的4个FR区的氨基酸序列依次如SEQID NO:9、10、11或12所示;轻链的4个FR区的氨基酸序列如SEQ ID NO:13、14、15、16所示。In some embodiments of the present invention, the amino acid sequences of the 4 FR regions of the cBIN1 antibody heavy chain are shown in sequence as SEQ ID NO: 9, 10, 11 or 12; the amino acid sequences of the 4 FR regions of the light chain are shown in SEQ ID NO: 9, 10, 11 or 12. NO: 13, 14, 15, 16 are shown.

其中,SEQ ID NO:9所示的序列为EVQLQQSGAELVRPGASVKLSCTTS;Wherein, the sequence shown in SEQ ID NO:9 is EVQLQQSGAELVRPGASVKLSCTTS;

SEQ ID NO:10所示的序列为YMHWVKQRPEQGLEWIGRI;The sequence shown in SEQ ID NO:10 is YMHWVKQRPEQGLEWIGRI;

SEQ ID NO:11所示的序列为AVYAPKFQDRATMTADTSSNTAYLHLS SLTSDDTAVYYCTT;The sequence shown in SEQ ID NO: 11 is AVYAPKFQDRATMTADTSSNTAYLHLS SLTSDDTAVYYCTT;

SEQ ID NO:12所示的序列为WGTGTTVTVSS;The sequence shown in SEQ ID NO: 12 is WGTGTTVTVSS;

SEQ ID NO:13所示的序列为DIQMTQTTSSLSASLGDRVTISC;The sequence shown in SEQ ID NO: 13 is DIQMTQTTSSLSASLGDRVTISC;

SEQ ID NO:14所示的序列为WYQQKPDGTVKLLIY。The sequence shown in SEQ ID NO: 14 is WYQQKPDGTVKLLIY.

SEQ ID NO:15所示的序列为VPSRFSGSGSGTDYSLTITNLE PEDFATYYC;The sequence shown in SEQ ID NO: 15 is VPSRFSGSGSGTDYSLTITNLE PEDFATYYC;

SEQ ID NO:16所示的序列为FGGGTKLEIK。The sequence shown in SEQ ID NO: 16 is FGGGTKLEIK.

本发明中,所述具有至少80%序列同源性的序列为在原序列的基础上,经取代、缺失或添加一个或多个氨基酸获得的氨基酸序列中,所述多个为2个、3个、4个、5个、6个、7个、8个、9个、10个、11个、12个、13个、14个、15个、16个、17个、18个、19个、20个、21个、22个、23个、24个、25个、26个、27个、28个、29个、30个、31个、32个、33个、34个、35个、36个、37个、38个、39个、40个或41个。In the present invention, the sequence with at least 80% sequence homology is an amino acid sequence obtained by substitution, deletion or addition of one or more amino acids on the basis of the original sequence, and the multiple is 2 or 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41.

一些实施例中,本发明所述的cBIN1抗体的重链可变区具有如SEQ ID NO:7所示的氨基酸序列;其轻链可变区具有如SEQ ID NO:8所示的氨基酸序列。In some embodiments, the heavy chain variable region of the cBIN1 antibody of the present invention has the amino acid sequence shown in SEQ ID NO:7; the light chain variable region thereof has the amino acid sequence shown in SEQ ID NO:8.

本发明还提供了编码所述的抗体的核苷酸。The present invention also provides nucleotides encoding said antibodies.

本发明提供了编码所述抗体重链的核苷酸序列。The present invention provides nucleotide sequences encoding the antibody heavy chains.

本发明提供了编码所述抗体轻链的核苷酸序列。The present invention provides nucleotide sequences encoding the antibody light chains.

本发明中,所述编码抗体重链可变区的核苷酸具有与SEQ ID NO:17所示的核苷酸序列,或为其经取代、缺失或添加一个或多个核苷酸获得的、且与SEQ ID NO:17所示的核苷酸序列功能相同或相似的核苷酸序列。In the present invention, the nucleotides encoding the variable region of the antibody heavy chain have the nucleotide sequence shown in SEQ ID NO: 17, or are obtained by substitution, deletion or addition of one or more nucleotides , and a nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown in SEQ ID NO: 17.

在本发明的一些具体实施方案中,编码所述抗体重链可变区的核苷酸序列如SEQID NO:17所示或为SEQ ID NO:17的反向互补序列。In some specific embodiments of the present invention, the nucleotide sequence encoding the variable region of the heavy chain of the antibody is shown in SEQ ID NO: 17 or the reverse complement of SEQ ID NO: 17.

本发明中,所述编码抗体轻链可变区的核苷酸具有与SEQ ID NO:18所示的核苷酸序列,或为其经取代、缺失或添加一个或多个核苷酸获得的、且与SEQ ID NO:18所示的核苷酸序列功能相同或相似的核苷酸序列。In the present invention, the nucleotides encoding the variable region of the antibody light chain have the nucleotide sequence shown in SEQ ID NO: 18, or are obtained by substitution, deletion or addition of one or more nucleotides , and a nucleotide sequence that is functionally identical or similar to the nucleotide sequence shown in SEQ ID NO: 18.

在本发明的一些具体实施方案中,编码所述抗体轻链可变区的核苷酸序列如SEQID NO:18所示或为SEQ ID NO:18的反向互补序列。In some specific embodiments of the present invention, the nucleotide sequence encoding the variable region of the antibody light chain is shown in SEQ ID NO: 18 or the reverse complement of SEQ ID NO: 18.

本发明还提供了一种表达载体,其包括编码本发明所述cBIN1抗体的核苷酸。The present invention also provides an expression vector comprising nucleotides encoding the cBIN1 antibody of the present invention.

本发明还提供了转化或转染所述的表达载体的宿主细胞。The present invention also provides host cells transformed or transfected with the expression vector.

本发明所述的cBIN1抗体的制备方法,包括:培养本发明所述宿主细胞、诱导cBIN1抗体的表达。The preparation method of the cBIN1 antibody of the present invention includes: culturing the host cell of the present invention and inducing the expression of the cBIN1 antibody.

本发明还提供了经化学标记或生物标记的所述的cBIN1抗体。The present invention also provides the cBIN1 antibody chemically or biologically labeled.

本发明中,所述化学标记为同位素、免疫毒素和/或化学药物;所述生物标记为生物素、亲和素或酶标记。所述酶标记优选为辣根过氧化物酶或碱性磷酸酶。所述免疫毒素优选为黄曲霉毒素、白喉毒素、绿脓杆菌外毒素、蓖麻毒蛋白、相思子毒蛋白、槲寄生凝集素、蒴莲根毒素、PAP、造草素、白树毒素或丝瓜毒素。In the present invention, the chemical labels are isotopes, immunotoxins and/or chemical drugs; the biomarkers are biotin, avidin or enzyme labels. The enzyme label is preferably horseradish peroxidase or alkaline phosphatase. Said immunotoxin is preferably aflatoxin, diphtheria toxin, Pseudomonas aeruginosa exotoxin, ricin, acacia protein, mistletoe lectin, capsula root toxin, PAP, herbogen, gelonin or loofah toxin.

本发明还提供了所述cBIN1抗体与固体介质或半固体介质偶联制得的偶联物。本发明中所述固体介质或非固体介质选自胶体金、聚苯乙烯平板或珠粒。The present invention also provides a conjugate prepared by coupling the cBIN1 antibody with a solid medium or a semi-solid medium. The solid medium or non-solid medium in the present invention is selected from colloidal gold, polystyrene plate or beads.

本发明所述cBIN1抗体或所述的偶联物在制备检测cBIN1表达的产品中的应用。Application of the cBIN1 antibody or the conjugate of the present invention in the preparation of a product for detecting cBIN1 expression.

本发明还提供了一种试剂盒,其包括所述cBIN1抗体或所述的偶联物。The present invention also provides a kit comprising the cBIN1 antibody or the conjugate.

本发明所述的试剂盒中还包括ELISA检测试剂或者Western Blot检测试剂。The kit of the present invention also includes an ELISA detection reagent or a Western Blot detection reagent.

本发明还提供了一种cBIN1的检测方法,其采用本发明提供的试剂盒通过ELISA法或Western Blot法对样品中的cBIN1进行检测。所述样品为心肌样品或者血液样品。The present invention also provides a method for detecting cBIN1, which uses the kit provided by the present invention to detect cBIN1 in a sample by ELISA method or Western Blot method. The sample is a myocardial sample or a blood sample.

本发明还提供了心衰的诊断方法,即采用发明提供的试剂盒通过ELISA法或WesternBlot法对心肌或血液样品中的cBIN1进行检测。所述样品为心肌样品或者血液样品。The invention also provides a method for diagnosing heart failure, that is, using the kit provided by the invention to detect cBIN1 in myocardial or blood samples by ELISA method or WesternBlot method. The sample is a myocardial sample or a blood sample.

本发明提供的cBIN1抗体的重链的三个CDR区分别具有如SEQ ID NO:1、2和3所示的氨基酸序列;轻链的三个CDR区分别具有如SEQ ID NO:4、5和6所示的氨基酸序列。该抗体与抗原具有良好的结合能力,以其制备试剂能够实现对目标抗原的准确检测。The three CDR regions of the heavy chain of the cBIN1 antibody provided by the present invention have the amino acid sequences shown in SEQ ID NOs: 1, 2 and 3 respectively; the three CDR regions of the light chain respectively have the amino acid sequences shown in SEQ ID NOs: 4, 5 and 3 The amino acid sequence shown in 6. The antibody has good binding ability with the antigen, and the reagent prepared by the antibody can accurately detect the target antigen.

附图说明Description of drawings

图1示抗体与cBIN1 Isoform II2和cBIN1 Isoform II3的ELISA结合曲线;Figure 1 shows the ELISA binding curves of antibodies to cBIN1 Isoform II2 and cBIN1 Isoform II3;

图2示WB检测中抗体与蛋白结合效果。Figure 2 shows the binding effect of antibody and protein in WB detection.

具体实施方式Detailed ways

本发明提供了cBIN1抗体及其应用,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。The present invention provides cBIN1 antibody and its application, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications herein without departing from the content, spirit and scope of the present invention, so as to realize and apply the present invention. Invention technology.

除非特别指明,本发明中所使用的分子生物学实验方法和免疫检测法,基本上参照J.Sambrook等人,分子克隆:实验室手册,第2版,冷泉港实验室出版社,1989,以及F.M.Ausubel等人,精编分子生物学实验指南,第3版,John Wiley&Sons,Inc.,1995中所述的方法进行;限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓,实施例以举例方式描述本发明,且不意欲限制本发明所要求保护的范围。Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Ausubel et al., The Refined Molecular Biology Laboratory Guide, 3rd Edition, John Wiley & Sons, Inc., 1995, was performed as described; restriction enzymes were used according to the conditions recommended by the product manufacturer. Those skilled in the art appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

除非另有定义,本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。关于本领域的定义及术语,专业人员具体可参考Current Protocols in MolecularBiology(Ausubel)。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. For definitions and terms in the art, professionals can refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are the standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids.

所述“抗体”是指由能特异结合抗原的一种或多种多肽构成的蛋白质。抗体的一种形式构成了抗体的基本结构单元。这种形式是四聚物,它由两对完全相同的抗体链构成,每一对都有一个轻链和一个重链。在每对抗体链中,轻链和重链的可变区联合在一起共同负责结合抗原,而恒定区则负责抗体的效应器功能。The "antibody" refers to a protein composed of one or more polypeptides that can specifically bind to an antigen. A form of antibody constitutes the basic building block of an antibody. This form is a tetramer, which consists of two identical pairs of antibody chains, each pair having a light chain and a heavy chain. In each pair of antibody chains, the variable regions of the light and heavy chains are collectively responsible for antigen binding, while the constant regions are responsible for the antibody's effector functions.

所述抗体重链或轻链的“可变区”是该链的N端成熟区域。目前已知的抗体类型包括κ和λ轻链,以及α,γ(IgG1,IgG2,IgG3,IgG4),δ,ε和μ重链或它们的其它类型等价物。全长的免疫球蛋白“轻链”(大约25kDa或大约214个氨基酸)包含一个由NH2-末端上大约110个氨基酸形成的可变区,以及一个COOH-末端上的κ或λ恒定区。全长的免疫球蛋白“重链”(大约50kDa或大约446个氨基酸),同样包含一个可变区(大约116个氨基酸),以及重链恒定区之一,例如γ(大约330个氨基酸)。The "variable region" of the antibody heavy or light chain is the N-terminal mature region of the chain. Currently known antibody types include kappa and lambda light chains, as well as alpha, gamma (IgGl, IgG2, IgG3, IgG4), delta, epsilon and mu heavy chains or their equivalents of other classes. A full-length immunoglobulin "light chain" (about 25 kDa or about 214 amino acids) contains a variable region formed by about 110 amino acids on the NH2 -terminus, and a kappa or lambda constant region on the COOH-terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids), also contains a variable region (about 116 amino acids), and one of the heavy chain constant regions, such as gamma (about 330 amino acids).

“抗体”包括任何同型体的抗体或免疫球蛋白,或保持与抗原特异结合的抗体片段,包括但不限于Fab,Fv,scFv和Fd片段、嵌合抗体、人源化抗体、单链抗体以及包含抗体的抗原结合部分和非抗体蛋白质的融合蛋白质。抗体可以被标记和检测,例如,可以通过放射性同位素、能产生可检测物的酶、荧光蛋白质、生物素等等进行标记并被检测。抗体还可以结合于固相载体,包括但不限于聚苯乙烯平板或珠粒等等。"Antibody" includes antibodies or immunoglobulins of any isotype, or antibody fragments that retain specific binding to an antigen, including but not limited to Fab, Fv, scFv and Fd fragments, chimeric antibodies, humanized antibodies, single chain antibodies, and Fusion proteins comprising the antigen-binding portion of an antibody and a non-antibody protein. Antibodies can be labeled and detected, for example, by radioisotopes, detectable-producing enzymes, fluorescent proteins, biotin, and the like. Antibodies can also be bound to solid supports, including but not limited to polystyrene plates or beads, and the like.

本文使用的“CDR区”或“CDR”是指免疫球蛋白的重链和轻链的高变区,如Kabat etal.所定义(Kabat et al.,Sequences of proteins of immunological interest,5thEd.,U.S.Department of Health and Human Services,NIH,1991,以及以后版本)。存在三个重链CDR和三个轻链CDR。根据情况,本文所用术语CDR或CDRs是为了指示这些区域之一、或者这些区域的几个或者甚至全部,所述区域包含通过抗体对抗原或其识别表位的亲和力而负责结合的大部分氨基酸残基。"CDR region" or "CDR" as used herein refers to the hypervariable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy chain CDRs and three light chain CDRs. As the case may be, the term CDR or CDRs is used herein to denote one of these regions, or several or even all of these regions comprising the majority of the amino acid residues responsible for binding by the antibody's affinity for the antigen or its recognition epitope base.

本发明采用的试材皆为普通市售品,皆可于市场购得。下面结合实施例,进一步阐述本发明:The test materials used in the present invention are all common commercial products and can be purchased in the market. Below in conjunction with embodiment, the present invention is further elaborated:

实施例1:用于免疫的抗原的合成Example 1: Synthesis of Antigens for Immunization

本发明的抗原为人源Cardiac bridging integrator 1外显子13(cBIN1 exon13)对应蛋白序列LRKGPPVPPPPKHTPSKEVKQEQILSLFEDTFVPEISVTTPSQ,用于免疫的多肽吉尔生化公司通过郭化学方法合成,为提高抗原免疫原性,该多肽C端通过二硬脂酸酯(PEG4)偶联钥孔血蓝蛋白(KLH,keyhole limpet hemocyanin)。The antigen of the present invention is human Cardiac bridging integrator 1 exon 13 (cBIN1 exon13) corresponding to the protein sequence LRKGPPVPPPPKHTPSKEVKQEQILSLFEDTFVPEISVTTPSQ, and the polypeptide used for immunization is synthesized by Jill Biochemical Company by Guo chemical method. Stearate (PEG4) was coupled to keyhole limpet hemocyanin (KLH).

实施例2:抗人源cBIN1 exon13抗体的产生Example 2: Generation of anti-human cBIN1 exon13 antibodies

为了获得鼠源抗人cBIN1 exon13抗体,使用表1.1中免疫策略(表1)来免疫不同品系小鼠(Balb/c,上海灵畅生物;SJL,北京维通利华)。所使用的抗原如实施例1中所描述;佐剂包括:完全弗氏佐剂CFA(InvivoGen公司,货号vac-cfa-60)、IFA(InvivoGen公司,货号vac-ifa-60)。在终疫3天后将免疫小鼠的脾细胞与小鼠骨髓瘤细胞SP2/0使用聚乙二醇法进行融合,得到既能表达抗体又能在体外无限增殖的B细胞融合,并且在HAT选择培养基中培养。将融合后的杂交瘤细胞铺在96孔细胞培养板中,并且通过初级筛选挑选出阳性克隆进行2轮亚克隆。In order to obtain mouse-derived anti-human cBIN1 exon13 antibody, different strains of mice (Balb/c, Shanghai Lingchang Biology; SJL, Beijing Weitonglihua) were immunized using the immunization strategy in Table 1.1 (Table 1). The antigens used were as described in Example 1; adjuvants included: Complete Freund's Adjuvant CFA (InvivoGen, Cat. No. vac-cfa-60), IFA (InvivoGen, Cat. No. vac-ifa-60). After 3 days of final vaccination, the spleen cells of the immunized mice were fused with mouse myeloma cells SP2/0 using the polyethylene glycol method to obtain B cells that could express both antibodies and indefinitely proliferate in vitro. cultured in medium. The fused hybridoma cells were plated in 96-well cell culture plates, and positive clones were selected by primary screening for 2 rounds of subcloning.

表1.免疫策略Table 1. Immunization strategies

Figure BDA0002879339130000071
Figure BDA0002879339130000071

Figure BDA0002879339130000081
Figure BDA0002879339130000081

实施例3:抗人源cBIN1 exon13抗体可变区序列测定Example 3: Determination of variable region sequence of anti-human cBIN1 exon13 antibody

离心收集杂交瘤细胞,每5×106~10×106细胞加入1ml TRIzol和0.2ml氯仿,剧烈振荡15秒,室温放置3分钟,离心取水相加入0.5ml异丙醇,室温放置10分钟后收集沉淀,乙醇洗涤后干燥得到RNA。在冰浴离心管里面加入模板RNA和引物,使引物和模板正确配对后进行反转录过程,在进行PCR扩增。4个微量离心管中各加入dNTP/ddNTP混合物2.5μl,混合物37℃温浴5min,备用。在一个空的微量离心管中加入1pmol的PCR扩增双链DNA,10pmol测序引物,2μl 5×测序缓冲液,加双蒸水至总体积10μl,96℃加热8min,冰浴泠却1min,4℃10000g离心10s。加入2μl预冷的标记混合物(dCTP、dGTP、dTTP各0.75μmol/L),α-32P-dATP5μCi,1μl 0.1mol/L DDT,测序酶2U,加水至15μl,混匀后置冰上2min,标记新合成的DNA链。3.5μl标记反应混合物加入到准备好的4个微量离心管中,37℃温浴5min.每管各加入4μl终止液。样品在80℃的水浴中热变性5min,每一泳道加2μl加到测序胶上,电泳分离这些片段,收集序列信息。Hybridoma cells were collected by centrifugation, 1 ml of TRIzol and 0.2 ml of chloroform were added to each 5×10 6 to 10×10 6 cells, vigorously shaken for 15 seconds, and placed at room temperature for 3 minutes. The precipitate was collected, washed with ethanol and dried to obtain RNA. Add template RNA and primers to the ice-bath centrifuge tube to make the primer and template pair correctly and then perform reverse transcription and PCR amplification. 2.5 μl of dNTP/ddNTP mixture was added to each of the 4 microcentrifuge tubes, and the mixture was incubated at 37°C for 5 min for later use. In an empty microcentrifuge tube, add 1 pmol of PCR-amplified double-stranded DNA, 10 pmol of sequencing primers, and 2 μl of 5× sequencing buffer. Centrifuge at 10000g for 10s. Add 2 μl of pre-chilled labeling mixture (0.75 μmol/L each of dCTP, dGTP, and dTTP), 5 μCi of α-32P-dATP, 1 μl of 0.1 mol/L DDT, 2 U of sequencing enzyme, add water to 15 μl, mix well, put on ice for 2 min, and label Newly synthesized DNA strands. 3.5μl of the labeling reaction mixture was added to the prepared 4 microcentrifuge tubes and incubated at 37℃ for 5min. 4μl of stop solution was added to each tube. The samples were heat-denatured in a water bath at 80 °C for 5 min, and 2 μl was added to each lane on the sequencing gel. These fragments were separated by electrophoresis and sequence information was collected.

获得鼠源抗体(记为8G1-D7-F5)的VH和VL序列如下表所示。进一步,还使用Kabat等人描述的方法(Kabat等,Sequences of Proteins of Immunological Interest,第五版,Public Health Service,美国国立卫生研究院,贝塞斯达、马里兰州(1991),第647-669页),确定了鼠源单抗的CDR序列。The VH and VL sequences of the obtained murine antibody (denoted as 8G1-D7-F5) are shown in the table below. Further, the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), pp. 647-669) was also used. page), the CDR sequence of the mouse monoclonal antibody was determined.

表2.鼠源抗体的序列信息Table 2. Sequence information for murine antibodies

Figure BDA0002879339130000082
Figure BDA0002879339130000082

表3抗体序列Table 3 Antibody sequences

Figure BDA0002879339130000091
Figure BDA0002879339130000091

4:抗人源cBIN1 exon13抗体结合活性评价4: Evaluation of the binding activity of anti-human cBIN1 exon13 antibody

4.1抗体(8G1-D7-F5)在酶联免疫吸附(ELISA)中结合活性4.1 Antibody (8G1-D7-F5) binding activity in enzyme-linked immunosorbent assay (ELISA)

按以10μg/ml起始浓度,三倍稀释,11个检测点稀释抗体。分别用吉凯基因化学有限公司重组表达的蛋白cBIN1 Isoform II2(cBIN1 exon1-13&18-20)和cBIN1 IsoformII3(cBIN1 exon 1-12&18-20)作为包被抗原,0.1μg/well包板。将稀释好的抗体加入Elisa板,每孔100ul。室温孵育1h,洗板。二抗按1:10000稀释,每孔加100ul,室温孵育1h,洗板。TMBA液与B液按1:1混合,每孔100ul室温孵育5min显色。显色结束后每孔加入50ul 2%H2SO4终止反应,OD450读数。GraphPadPrism 6分析数据,计算抗体结合cBIN1 Isoform II2和cBIN1 Isoform II3的EC50值。抗体结合曲线如图1所示,抗体和两种蛋白的结合EC50如表3所示。Dilute the antibody at 10 μg/ml starting concentration, three-fold dilution, and 11 detection points. The proteins cBIN1 Isoform II2 (cBIN1 exon1-13&18-20) and cBIN1 IsoformII3 (cBIN1 exon 1-12&18-20) recombinantly expressed by Jikai Gene Chemical Co., Ltd. were used as coating antigens, and 0.1 μg/well was used to coat plates. Add diluted antibody to Elisa plate, 100ul per well. Incubate for 1 h at room temperature and wash the plate. Dilute the secondary antibody at 1:10000, add 100ul to each well, incubate at room temperature for 1h, and wash the plate. TMBA solution and B solution were mixed at 1:1, and 100ul of each well was incubated at room temperature for 5min for color development. After color development, 50ul of 2% H2SO4 was added to each well to stop the reaction, and the OD450 was read. Data were analyzed with GraphPad Prism 6 and EC50 values for antibody binding to cBIN1 Isoform II2 and cBIN1 Isoform II3 were calculated. The antibody binding curve is shown in Figure 1, and the binding EC50 of the antibody and the two proteins is shown in Table 3.

表3.抗体与抗原的ESLIA结合EC50(μg/ml)Table 3. ESLIA binding EC50 (μg/ml) of antibody to antigen

抗体Antibody cBIN1IsoformII2cBIN1IsoformII2 cBIN1IsoformII3cBIN1IsoformII3 8G1-D7-F58G1-D7-F5 0.0580.058 不结合not combined 阳性对照抗体positive control antibody 0.6140.614

抗体8G1-D7-F5高亲和力结合含有exon13的cBIN1 Isoform II2蛋白,且不结合缺失exon13的cBIN1 Isoform II3蛋白,说明抗体8G1-D7-F5对于cBIN的结合在其exon13对应的区域。抗体8G1-D7-F5在酶联免疫吸附中的结合活性比阳性对照抗体(Novus,NBP2-21689)高11-12倍。Antibody 8G1-D7-F5 binds to cBIN1 Isoform II2 protein containing exon13 with high affinity, and does not bind to cBIN1 Isoform II3 protein lacking exon13, indicating that antibody 8G1-D7-F5 binds to cBIN in the region corresponding to exon13. The binding activity of antibody 8G1-D7-F5 in ELISA was 11-12 times higher than that of the positive control antibody (Novus, NBP2-21689).

4.2抗体在免疫印迹实验中(WB)结合活性4.2 Antibody Binding Activity in Western Blot Experiments (WB)

WB中使用吉凯基因化学有限公司重组表达的蛋白cBIN1 Isoform II2(cBIN1exon 1-13&18-20),在120mA电流下,蛋白在12%SDS-PAGE胶上电泳1小时。电泳结束后,使用转移电泳装置,在4℃、300mA恒流条件下电转150min,将蛋白转移到PVDF膜上。分别使用抗体1F3-D11-B和阳性对照抗体(Novus,NBP2-21689)在10μg/ml浓度下进行一抗孵育,与封闭好的PVDF膜室温孵育2小时,清洗后与二抗孵育,室温下孵育PVDF膜1.5小时,彻底清洗后显影。结果如图2。如图2所示,抗体8G1-D7-F5在WB中高亲和力结合含有exon13的cBIN1Isoform II2蛋白,且抗体8G1-D7-F5的结合量显著高于阳性对照抗体(Novus,NBP2-21689)。The protein cBIN1 Isoform II2 (cBIN1exon 1-13&18-20) recombinantly expressed by Jikai Gene Chemical Co., Ltd. was used in WB, and the protein was electrophoresed on a 12% SDS-PAGE gel for 1 hour at a current of 120 mA. After electrophoresis, the protein was transferred to PVDF membrane by electrophoresis at 4°C and 300mA constant current for 150min using a transfer electrophoresis device. The antibody 1F3-D11-B and the positive control antibody (Novus, NBP2-21689) were used to incubate the primary antibody at a concentration of 10 μg/ml, and incubated with the blocked PVDF membrane for 2 hours at room temperature. After washing, incubated with the secondary antibody at room temperature. Incubate PVDF membranes for 1.5 hours, wash thoroughly and develop. The results are shown in Figure 2. As shown in Figure 2, the antibody 8G1-D7-F5 binds the cBIN1Isoform II2 protein containing exon13 with high affinity in WB, and the binding amount of the antibody 8G1-D7-F5 is significantly higher than that of the positive control antibody (Novus, NBP2-21689).

以上仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, some improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as It is the protection scope of the present invention.

序列表sequence listing

<110> 中南大学湘雅二医院<110> The Second Xiangya Hospital of Central South University

<120> cBIN1抗体及其应用<120> cBIN1 antibody and its application

<130> MP2037306<130> MP2037306

<160> 18<160> 18

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 1<400> 1

Gly Phe Asn Ile Lys Asp SerGly Phe Asn Ile Lys Asp Ser

1 51 5

<210> 2<210> 2

<211> 6<211> 6

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 2<400> 2

Asp Pro Glu Asp Asp AspAsp Pro Glu Asp Asp Asp

1 51 5

<210> 3<210> 3

<211> 12<211> 12

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 3<400> 3

Ser Thr Leu Val Ala Thr Pro Trp Phe Phe Asp ValSer Thr Leu Val Ala Thr Pro Trp Phe Phe Asp Val

1 5 101 5 10

<210> 4<210> 4

<211> 11<211> 11

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 4<400> 4

Ser Ala Ser Gln Asp Ile Asn Asn Tyr Leu AsnSer Ala Ser Gln Asp Ile Asn Asn Tyr Leu Asn

1 5 101 5 10

<210> 5<210> 5

<211> 7<211> 7

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 5<400> 5

Tyr Thr Ser Thr Leu His SerTyr Thr Ser Thr Leu His Ser

1 51 5

<210> 6<210> 6

<211> 10<211> 10

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 6<400> 6

Gln Gln Tyr Thr His Asn Leu Pro Trp ThrGln Gln Tyr Thr His Asn Leu Pro Trp Thr

1 5 101 5 10

<210> 7<210> 7

<211> 121<211> 121

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 7<400> 7

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly AlaGlu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 151 5 10 15

Ser Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp SerSer Val Lys Leu Ser Cys Thr Thr Ser Gly Phe Asn Ile Lys Asp Ser

20 25 30 20 25 30

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp IleTyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45 35 40 45

Gly Arg Ile Asp Pro Glu Asp Asp Asp Ala Val Tyr Ala Pro Lys PheGly Arg Ile Asp Pro Glu Asp Asp Asp Asp Ala Val Tyr Ala Pro Lys Phe

50 55 60 50 55 60

Gln Asp Arg Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala TyrGln Asp Arg Ala Thr Met Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr

65 70 75 8065 70 75 80

Leu His Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr CysLeu His Leu Ser Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95 85 90 95

Thr Thr Ser Thr Leu Val Ala Thr Pro Trp Phe Phe Asp Val Trp GlyThr Thr Ser Thr Leu Val Ala Thr Pro Trp Phe Phe Asp Val Trp Gly

100 105 110 100 105 110

Thr Gly Thr Thr Val Thr Val Ser SerThr Gly Thr Thr Val Thr Val Ser Ser

115 120 115 120

<210> 8<210> 8

<211> 108<211> 108

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 8<400> 8

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu GlyAsp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 151 5 10 15

Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Asp Ile Asn Asn TyrAsp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Asp Ile Asn Asn Tyr

20 25 30 20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu IleLeu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45 35 40 45

Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser GlyTyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60 50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr Asn Leu Glu ProSer Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr Asn Leu Glu Pro

65 70 75 8065 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr His Asn Leu ProGlu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Thr His Asn Leu Pro

85 90 95 85 90 95

Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysTrp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 100 105

<210> 9<210> 9

<211> 25<211> 25

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 9<400> 9

Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly AlaGlu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala

1 5 10 151 5 10 15

Ser Val Lys Leu Ser Cys Thr Thr SerSer Val Lys Leu Ser Cys Thr Thr Ser

20 25 20 25

<210> 10<210> 10

<211> 19<211> 19

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 10<400> 10

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp IleTyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

1 5 10 151 5 10 15

Gly Arg IleGly Arg Ile

<210> 11<210> 11

<211> 41<211> 41

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 11<400> 11

Ala Val Tyr Ala Pro Lys Phe Gln Asp Arg Ala Thr Met Thr Ala AspAla Val Tyr Ala Pro Lys Phe Gln Asp Arg Ala Thr Met Thr Ala Asp

1 5 10 151 5 10 15

Thr Ser Ser Asn Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser AspThr Ser Ser Asn Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Asp

20 25 30 20 25 30

Asp Thr Ala Val Tyr Tyr Cys Thr ThrAsp Thr Ala Val Tyr Tyr Cys Thr Thr

35 40 35 40

<210> 12<210> 12

<211> 11<211> 11

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 12<400> 12

Trp Gly Thr Gly Thr Thr Val Thr Val Ser SerTrp Gly Thr Gly Thr Thr Val Thr Val Ser Ser

1 5 101 5 10

<210> 13<210> 13

<211> 23<211> 23

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 13<400> 13

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu GlyAsp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 151 5 10 15

Asp Arg Val Thr Ile Ser CysAsp Arg Val Thr Ile Ser Cys

20 20

<210> 14<210> 14

<211> 15<211> 15

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 14<400> 14

Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile TyrTrp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr

1 5 10 151 5 10 15

<210> 15<210> 15

<211> 32<211> 32

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 15<400> 15

Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr SerGly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser

1 5 10 151 5 10 15

Leu Thr Ile Thr Asn Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr CysLeu Thr Ile Thr Asn Leu Glu Pro Glu Asp Phe Ala Thr Tyr Tyr Cys

20 25 30 20 25 30

<210> 16<210> 16

<211> 10<211> 10

<212> PRT<212> PRT

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 16<400> 16

Phe Gly Gly Gly Thr Lys Leu Glu Ile LysPhe Gly Gly Gly Thr Lys Leu Glu Ile Lys

1 5 101 5 10

<210> 17<210> 17

<211> 363<211> 363

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 17<400> 17

gaggttcagc tgcagcagtc tggggcagag cttgtgaggc caggggcctc agtcaagttg 60gaggttcagc tgcagcagtc tggggcagag cttgtgaggc caggggcctc agtcaagttg 60

tcctgcacaa cttctggctt caacattaaa gactcctata tgcactgggt gaagcagagg 120tcctgcacaa cttctggctt caacattaaa gactcctata tgcactgggt gaagcagagg 120

cctgaacagg gcctggagtg gattggaagg attgatcctg aggatgatga tgctgtatac 180cctgaacagg gcctggagtg gattggaagg attgatcctg aggatgatga tgctgtatac 180

gccccgaagt tccaggacag ggccactatg actgcagaca catcctccaa cacagcctat 240gccccgaagt tccaggacag ggccactatg actgcagaca catcctccaa cacagcctat 240

ctgcacctca gcagcctgac atctgacgac actgccgtct attactgtac tacatctacg 300ctgcacctca gcagcctgac atctgacgac actgccgtct attactgtac tacatctacg 300

ctagtagcca cgccctggtt cttcgatgtc tggggcacag ggaccacggt caccgtctcc 360ctagtagcca cgccctggtt cttcgatgtc tggggcacag ggaccacggt caccgtctcc 360

tca 363tca 363

<210> 18<210> 18

<211> 324<211> 324

<212> DNA<212> DNA

<213> 人工序列(Artificial Sequence)<213> Artificial Sequence

<400> 18<400> 18

gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60gatatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gtgcaagtca ggacattaac aattacttaa actggtatca gcagaaacca 120atcagttgca gtgcaagtca ggacattaac aattacttaa actggtatca gcagaaacca 120

gatggcactg ttaaactcct gatttattac acatcaactt tacactcagg agtcccgtca 180gatggcactg ttaaactcct gatttattac acatcaactt tacactcagg agtcccgtca 180

aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcaccaa cctggagcct 240aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcaccaa cctggagcct 240

gaagattttg ccacttacta ttgtcaacag tatactcata accttccgtg gacgttcggt 300gaagattttg ccacttacta ttgtcaacag tatactcata accttccgtg gacgttcggt 300

ggaggcacca agctggaaat caaa 324ggaggcacca agctggaaat caaa 324

Claims (10)

  1. An antibody cBIN1, characterized in that,
    the amino acid sequences of three CDR regions of the heavy chain are respectively shown in SEQ ID NO. 1, 2 and 3;
    the amino acid sequences of the three CDR regions of the light chain are respectively shown in SEQ ID NO. 4, 5 and 6.
  2. 2. The cBIN1 antibody according to claim 1,
    the heavy chain variable region has an amino acid sequence shown as SEQ ID NO. 7;
    the light chain variable region has an amino acid sequence shown in SEQ ID NO. 8.
  3. 3. A nucleic acid encoding the antibody of claim 1 or 2.
  4. 4. An expression vector comprising the nucleic acid of claim 3.
  5. 5. A host cell transformed or transfected with the expression vector of claim 4.
  6. 6. The method of preparing the cBIN1 antibody of claim 1 or 2, comprising: culturing the host cell of claim 5 to induce expression of the cBIN1 antibody.
  7. 7. The cBIN1 antibody of claim 1 or 2, chemically or biologically labeled.
  8. 8. A conjugate prepared by conjugating the cBIN1 antibody of claim 1, 2 or 7 to a solid or semi-solid medium.
  9. 9. Use of a cBIN1 antibody according to claim 1, 2 or 7 or a conjugate according to claim 8 in the preparation of a product for detecting cBIN1 expression.
  10. 10. A kit comprising the cBIN1 antibody of claim 1, 2 or 7 or the conjugate of claim 8.
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US6162601A (en) * 1997-08-08 2000-12-19 Incyte Pharmaceuticals, Inc. Human pinin splice variant
CN1878795A (en) * 2002-12-02 2006-12-13 阿布格尼克斯公司 Antibodies directed to phospholipase A2 and uses thereof
WO2012087437A2 (en) * 2010-11-09 2012-06-28 Sarcotein Diagnostics Llc Bin1 expression as a marker of skeletal muscle mass and neurological conditions
WO2013049666A1 (en) * 2011-09-30 2013-04-04 Sarcotein Diagnostics, Llc Bin1 expression as a marker of cancer
WO2013053741A1 (en) * 2011-10-14 2013-04-18 Centre National De La Recherche Scientifique (Cnrs) Anti-spla2-v antibodies and uses thereof
WO2018035710A1 (en) * 2016-08-23 2018-03-01 Akeso Biopharma, Inc. Anti-ctla4 antibodies

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6162601A (en) * 1997-08-08 2000-12-19 Incyte Pharmaceuticals, Inc. Human pinin splice variant
CN1878795A (en) * 2002-12-02 2006-12-13 阿布格尼克斯公司 Antibodies directed to phospholipase A2 and uses thereof
WO2012087437A2 (en) * 2010-11-09 2012-06-28 Sarcotein Diagnostics Llc Bin1 expression as a marker of skeletal muscle mass and neurological conditions
WO2013049666A1 (en) * 2011-09-30 2013-04-04 Sarcotein Diagnostics, Llc Bin1 expression as a marker of cancer
WO2013053741A1 (en) * 2011-10-14 2013-04-18 Centre National De La Recherche Scientifique (Cnrs) Anti-spla2-v antibodies and uses thereof
WO2018035710A1 (en) * 2016-08-23 2018-03-01 Akeso Biopharma, Inc. Anti-ctla4 antibodies

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