CN115925866A - 一种用于乳腺癌检测的单克隆抗体及其试剂盒 - Google Patents
一种用于乳腺癌检测的单克隆抗体及其试剂盒 Download PDFInfo
- Publication number
- CN115925866A CN115925866A CN202211405138.0A CN202211405138A CN115925866A CN 115925866 A CN115925866 A CN 115925866A CN 202211405138 A CN202211405138 A CN 202211405138A CN 115925866 A CN115925866 A CN 115925866A
- Authority
- CN
- China
- Prior art keywords
- cdr
- monoclonal antibody
- chain variable
- variable region
- epitope polypeptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 206010006187 Breast cancer Diseases 0.000 title claims abstract description 17
- 208000026310 Breast neoplasm Diseases 0.000 title claims abstract description 17
- 238000001514 detection method Methods 0.000 title claims abstract description 13
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 37
- 229920001184 polypeptide Polymers 0.000 claims abstract description 36
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 36
- 101000620365 Homo sapiens Protein TMEPAI Proteins 0.000 claims abstract description 22
- 102100022429 Protein TMEPAI Human genes 0.000 claims abstract description 22
- 230000027455 binding Effects 0.000 claims abstract description 5
- 239000000439 tumor marker Substances 0.000 claims abstract description 4
- 239000000427 antigen Substances 0.000 claims description 10
- 102000036639 antigens Human genes 0.000 claims description 10
- 108091007433 antigens Proteins 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 9
- 239000002773 nucleotide Substances 0.000 claims description 8
- 125000003729 nucleotide group Chemical group 0.000 claims description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 230000009870 specific binding Effects 0.000 claims description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 4
- 238000000034 method Methods 0.000 abstract description 10
- 238000012216 screening Methods 0.000 abstract description 7
- 238000013461 design Methods 0.000 abstract description 5
- 230000035945 sensitivity Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 9
- 206010003445 Ascites Diseases 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 230000003053 immunization Effects 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 238000002649 immunization Methods 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 210000004408 hybridoma Anatomy 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000007910 cell fusion Effects 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 206010006223 Breast discharge Diseases 0.000 description 1
- 206010006272 Breast mass Diseases 0.000 description 1
- 101001024441 Candida albicans (strain SC5314 / ATCC MYA-2876) Major facilitator superfamily multidrug transporter NAG3 Proteins 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101000662690 Homo sapiens Trafficking protein particle complex subunit 10 Proteins 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 102100037456 Trafficking protein particle complex subunit 10 Human genes 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- OBETXYAYXDNJHR-UHFFFAOYSA-N alpha-ethylcaproic acid Natural products CCCCC(CC)C(O)=O OBETXYAYXDNJHR-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 239000003098 androgen Substances 0.000 description 1
- 230000004791 biological behavior Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000010370 cell cloning Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000002038 chemiluminescence detection Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical class [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000004989 spleen cell Anatomy 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000012089 stop solution Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57415—Specifically defined cancers of breast
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- General Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Oncology (AREA)
- Hospice & Palliative Care (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明一方面公开了一种乳腺癌标志物PMEPA1的抗原表位多肽。本发明还公开了一种能特异性结合所述的PMEPA1抗原表位多肽的单克隆抗体。本发明还公开了一种用于乳腺癌检测的试剂盒。本发明基于筛选抗原性好、抗原表位多的序列设计两条彼此分开的抗原表位多肽，再此基础上筛选合适的单克隆抗体，以用于对PMEPA1蛋白的检测；该方法具有以下优点：1)可以提高筛选单克隆抗体的成功率；2)可以省略单克隆抗体的配对检验；3)可以提高单克隆抗体的特异性。本发明的试剂盒具有良好的特异性和敏感性，适用于PMEPA1蛋白的检测。
Description
技术领域
本发明属于生物技术领域，具体涉及一种用于乳腺癌检测的单克隆抗体及其试剂盒。
背景技术
乳腺癌是乳腺上皮细胞在多种致癌因子的作用下，发生增殖失控的现象。疾病早期常表现为乳房肿块、乳头溢液、腋窝淋巴结肿大等症状，晚期可因癌细胞发生远处转移，出现多器官病变，直接威胁患者的生命。
近年研究表明，乳腺癌的发生发展与蛋白的异常表达和信号通路的异常激活密切相关。有研究发现，在乳腺癌细胞中雄激素诱导的前列腺跨膜蛋白1(prostatetransmembrane protein,androgen induced 1，PMEPA1)基因较正常细胞出现5～10倍的过表达。目前已证实PMEPA1能通过参与调控多条信号通路,对恶性肿瘤细胞的增殖、侵袭、转移等多种生物学行为产生重要影响,并且对恶性肿瘤患者的预后起到一定的预测作用。
发明内容
为了弥补现有技术的不足，本发明的目的之一，提供一种乳腺癌标志物PMEPA1的抗原表位序列；本发明的目的之二，提供了一种针对PMEPA1抗原表位序列的单克隆抗体。
因此，本发明一方面公开了一种乳腺癌标志物PMEPA1的抗原表位多肽，所述的PMEPA1抗原表位多肽的氨基酸序列为：抗原表位多肽1：LMGVNSTAAAAAGQPNVSCTCNCKRSLFQSM；抗原表位多肽2：LGGPCPPSSNSGISATCYGSGGRMEGPPPTYS。
另一方面，本发明还公开了一种能特异性结合所述的PMEPA1抗原表位多肽的单克隆抗体，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的重链可变区的核苷酸序列如SEQ ID NO.1所示，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的轻链可变区的核苷酸序列如SEQ ID NO.2所示；所述的编码特异性结合抗原表位多肽2的单克隆抗体2的重链可变区的核苷酸序列如SEQ ID NO.3所示，所述的编码特异性结合抗原表位多肽2的单克隆抗体2的轻链可变区的核苷酸序列如SEQ ID NO.4所示。
优选地，本发明所述的单克隆抗体1的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：NSMN；
CDR-H2：EIRLKSNNYVTHYAESVKG；
CDR-H3：IYSPFAY；
所述的单克隆抗体1的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：PPSSEKASIDVSTIVS；
CDR-L2：SATYRYI；
CDR-L3：QQHYSSPST。
优选地，本发明所述的单克隆抗体1的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：ESGGGLVGSQPGGSMKLSCVASGFTIS；
FR-H2：WVRQSPEKGLDWVA；
FR-H3：RFTISRDDSKNSVYLQSMNNLRPEGSGIYYCTP；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体1的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSHKFDSTSVGDRVSITC；
FR-L2：WYQQKPGQSPKLLHY；
FR-L3：GVPDRFTASGSGTWFTFTISSVQAEDLAVYYC；
FR-L4：FGAGTCPTKLEL。
优选地，本发明所述的单克隆抗体2的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：DYYMY；
CDR-H2：ISNGGGTTYSPTVKG；
CDR-H3：HEHGYTPYVAY；
所述的单克隆抗体2的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：KSSQSLYSSNQKNYSWTLA；
CDR-L2：WASTRES；
CDR-L3：QQCYSYPRT。
优选地，本发明所述的单克隆抗体2的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：CEKLVESGGGLVQPGGTPLKLSCATSGFTFS；
FR-H2：WTPVRQTPEKRLEWVA；
FR-H3：RFTSRDNAKTLYLQSRKSEDTAMTPYYCAR；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体2的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSPSSTSLAVSVGEKVTMSC；
FR-L2：WYQQKPGQSPKLLIY；
FR-L3：GVPDRFTGSGSGTDFTTISSVKAEDLSWTAVYYC；
FR-L4：FGGGTKLEIK。
再一方面，本发明还公开了一种用于乳腺癌检测的试剂盒，所述试剂盒包括有效量的权利要求2所述的单克隆抗体1和单克隆抗体2；以及配套的检测试剂。
再一方面，本发明还公开了一种所述的PMEPA1抗原表位多肽在制备乳腺癌诊断试剂中的应用。
本发明基于筛选抗原性好、抗原表位多的序列(如实施例1所述)设计两条彼此分开的抗原表位多肽，再次基础上筛选合适的单克隆抗体，以用于对PMEPA1蛋白的检测。该方法具有以下优点：1)可以提高筛选单克隆抗体的成功率(避免使用全蛋白筛选时的无效抗体)；2)可以省略单克隆抗体的配对检验；3)可以提高单克隆抗体的特异性(抗原表位为高度特异性的抗原位点，能被多个单克隆抗体特异性结合)。
在筛选的单克隆抗体的基础上，形成了检测PMEPA1蛋白的试剂盒，该试剂盒具有良好的特异性和敏感性，适用于PMEPA1蛋白的检测。虽然本发明采用的检测方法为常规的ELISA方法，但是，本发明筛选的单克隆抗体同样可以应用于其他免疫学方法的检测，如胶体金检测、化学发光检测等等。
附图说明
图1PMEPA1蛋白跨膜区域预测分析结果。其中Inside表示胞内区，inside数值越大，表示该氨基酸位于胞内区的可能性越大；Outside表示胞外区，outside数值越大，表示该氨基酸位于胞外区的可能性越大；Transmembrane表示跨膜区，Transmembrane数值越大，表示该氨基酸在跨膜区的可能性越大。
图2PMEPA1蛋白二级结构预测分析结果。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市购。
实施例1：PMEPA1抗原表位多肽的设计与合成
从NCBI搜索PMEPA1，从中找到近年公开的PMEPA1序列，选择其中之一(NM_020182.5)作为本研究的目的蛋白，对该蛋白的跨膜区域(图1)和二级结构(图2)进行预测分析，选择其中两段作为PMEPA1的抗原表位多肽，并交公司合成多肽，以用于后续试验。其中抗原表位的设计遵循以下原则(参见中国发明专利2015106122558)：选择细胞膜蛋白表面区域或内部区域，避开跨膜区域；选择不形成a-helix的序列；两端的肽段比中间的排列合理；避免蛋白内部重复；避免同源性强的肽段。
经过设计，两个抗原表位多肽序列为：
抗原表位多肽1：LMGVNSTAAAAAGQPNVSCTCNCKRSLFQSM；
抗原表位多肽2：LGGPCPPSSNSGISATCYGSGGRMEGPPPTYS。
实施例2：抗PMEPA1抗原表位多肽单克隆抗体的制备
2.1动物免疫将实施例1制备的抗原表位多肽分别作为抗原，与等质量福氏完全佐剂充分乳化后背腹部皮下多点注射8周龄左右BALB/c雌性小鼠，0.2ml/只；间隔2周，取与一免等量抗原和等质量的福氏不完全佐剂充分乳化后，第二次腹腔注射0.2ml/只，过2周后用加倍剂量的抗原进行腹腔注射免疫，3天后取脾细胞进行细胞融合。
2.2细胞融合及杂交瘤细胞筛选采集小鼠脾脏研磨并分离脾细胞，使用PEG法将其与骨髓瘤Sp2/0细胞株融合，将融合后的细胞用HAT培养基重悬，均匀铺于96孔板内，每孔100μl，37℃，5％CO2培养。细胞培养箱中培养5天后，用HAT培养基换液一次，第10天用HT培养基换液，等到融合细胞覆盖孔底10～30％时。取培养上清，用PMEPA1抗原表位多肽包被的常规间接ELISA方法进行阳性克隆的检测。共获98个阳性孔，分别选择3个呈强阳性反应的细胞孔，进行3次有限稀释法细胞克隆，分别获得1株杂交瘤细胞，分别命名为1号(抗原表位多肽1免疫后筛选获得)和2号(抗原表位多肽2免疫后筛选获得)。经传代和多次冻存复苏后，细胞株均能良好生长，并稳定分泌抗体。经扩大培养后，用于腹水制备和液氮保存。
2.3单克隆抗体的腹水制备和纯化取8周龄左右BALB/c雌性小鼠经腹腔注射降植烷，0.3～0.5ml/只，7日后每只小鼠注射杂交瘤细胞1×106个。注射后7～10天可见小鼠腹部明显膨大，注射针头采取腹水，4℃8000rpm离心3min，收集上清液，即为单抗腹水。取1倍体积腹水，加入2倍体积醋酸盐缓冲液(0.06mol/L，pH值4.8)，混合均匀后在室温下边搅拌边加入辛酸(30μl/ml腹水)，4℃澄清2h小时，以4℃12000rpm离心20min，收集上清。再用50％饱和硫酸铵沉淀免疫球蛋白，4℃静置2小时，4℃3000rpm离心30min，收集沉淀。用2倍体积PBS溶解沉淀后用PBS透析过夜，即获得纯化的腹水抗体，置于-70℃保存备用。
2.4单克隆抗体分析提取杂交瘤细胞的总RNA并进行逆转录，将获得的cDNA保存于-15℃以下待用。设计特异性的巢式PCR引物，采用常规PCR方法扩增目的基因。其中，引物序列的设计按照文献(CN 111393525 B)进行。经过测序，单克隆抗体1(抗原表位多肽1免疫后制备)和单克隆抗体2(抗原表位多肽2免疫后制备)的可变区氨基酸序列信息见表1所示。
表1单克隆抗体氨基酸序列信息
实施例3：检测PMEPA1免疫学方法的建立及其试剂盒的制备
3.1试剂盒的制备为常规ELISA试剂盒制备方法，简述如下：
3.1.1酶标板制备使用单克隆抗体1包被酶标板(包被液为0.05M的碳酸盐缓冲液，pH9.6)，100ng/孔，0.1ml/孔，4℃包被过夜，洗涤3次后，使用含1％BSA的PBST封闭，37℃孵育2h，洗涤后存于-15℃以下保存备用；
3.1.2试剂盒检测方法使用样品稀释液(含1％ BSA的PBST缓冲液)将待检样品稀释50倍，混匀后加到包被的酶标板中，0.1ml/孔，37℃孵育1h；洗涤后，加入用样品稀释液1:10000倍稀释的HRP标记的单克隆抗体2(HRP标记为常规的改良高碘酸钠方法)，0.1ml/孔，37℃孵育30min；洗涤后，加入0.1ml TMB单组分显色液，37℃孵育10min；加入终止液(2M硫酸)，测定OD450nm值，当P/N值＞2.1时判为阳性，否则为阴性，其中P为样品OD450nm值，N为空白对照OD450nm值。
3.2试剂盒检测本研究从广东省某医院获取80名乳腺癌患者和80名非患者中的血液样本；使用3.1建立的试剂盒对其PMEPA1蛋白进行检测，结果显示，该试剂盒检测的80名乳腺癌患者均为阳性，80名非患者均为阴性，说明试剂盒具有良好的特异性和敏感性。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
Claims (8)
1.一种乳腺癌标志物PMEPA1的抗原表位多肽，其特征在于，所述的PMEPA1抗原表位多肽的氨基酸序列为：
抗原表位多肽1：LMGVNSTAAAAAGQPNVSCTCNCKRSLFQSM；
抗原表位多肽2：LGGPCPPSSNSGISATCYGSGGRMEGPPPTYS。
2.一种能特异性结合权利要求1所述的PMEPA1抗原表位多肽的单克隆抗体，其特征在于，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的重链可变区的核苷酸序列如SEQ ID NO.1所示，所述的编码特异性结合抗原表位多肽1的单克隆抗体1的轻链可变区的核苷酸序列如SEQ ID NO.2所示；所述的编码特异性结合抗原表位多肽2的单克隆抗体2的重链可变区的核苷酸序列如SEQ ID NO.3所示，所述的编码特异性结合抗原表位多肽2的单克隆抗体2的轻链可变区的核苷酸序列如SEQ ID NO.4所示。
3.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体1的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：NSMN；
CDR-H2：EIRLKSNNYVTHYAESVKG；
CDR-H3：IYSPFAY；
所述的单克隆抗体1的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：PPSSEKASIDVSTIVS；
CDR-L2：SATYRYI；
CDR-L3：QQHYSSPST。
4.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体1的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：ESGGGLVGSQPGGSMKLSCVASGFTIS；
FR-H2：WVRQSPEKGLDWVA；
FR-H3：RFTISRDDSKNSVYLQSMNNLRPEGSGIYYCTP；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体1的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSHKFDSTSVGDRVSITC；
FR-L2：WYQQKPGQSPKLLHY；
FR-L3：GVPDRFTASGSGTWFTFTISSVQAEDLAVYYC；
FR-L4：FGAGTCPTKLEL。
5.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体2的重链可变区序列包含CDR-H1、CDR-H2和CDR-H3，所述的重链可变区的CDR-H1、CDR-H2和CDR-H3的序列分别为：
CDR-H1：DYYMY；
CDR-H2：ISNGGGTTYSPTVKG；
CDR-H3：HEHGYTPYVAY；
所述的单克隆抗体2的轻链可变区序列包含CDR-L1、CDR-L2和CDR-L3，所述的轻链可变区的CDR-L1、CDR-L2和CDR-L3的序列分别为：
CDR-L1：KSSQSLYSSNQKNYSWTLA；
CDR-L2：WASTRES；
CDR-L3：QQCYSYPRT。
6.根据权利要求2所述的单克隆抗体，其特征在于，所述的单克隆抗体2的重链可变区序列还包含FR-H1、FR-H2、FR-H3和FR-H4，所述的重链可变区的FR-H1、FR-H2、FR-H3和FR-H4的序列分别为：
FR-H1：CEKLVESGGGLVQPGGTPLKLSCATSGFTFS；
FR-H2：WTPVRQTPEKRLEWVA；
FR-H3：RFTSRDNAKTLYLQSRKSEDTAMTPYYCAR；
FR-H4：WGQGTLVTVSA；
所述的单克隆抗体2的轻链可变区序列还包含FR-L1、FR-L2、FR-L3和FR-L4，所述的轻链可变区的FR-L1、FR-L2、FR-L3和FR-L4的序列分别为：
FR-L1：DIVMSQSPSSTSLAVSVGEKVTMSC；
FR-L2：WYQQKPGQSPKLLIY；
FR-L3：GVPDRFTGSGSGTDFTTISSVKAEDLSWTAVYYC；
FR-L4：FGGGTKLEIK。
7.一种用于乳腺癌检测的试剂盒，其特征在于，所述试剂盒包括有效量的权利要求2所述的单克隆抗体1和单克隆抗体2；以及配套的检测试剂。
8.一种如权利要求2所述的单克隆抗体在制备乳腺癌诊断试剂中的应用。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2022102292314 | 2022-03-09 | ||
| CN202210229231.4A CN114524872A (zh) | 2022-03-09 | 2022-03-09 | 一种用于乳腺癌检测的单克隆抗体及其试剂盒 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115925866A true CN115925866A (zh) | 2023-04-07 |
Family
ID=81626554
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210229231.4A Pending CN114524872A (zh) | 2022-03-09 | 2022-03-09 | 一种用于乳腺癌检测的单克隆抗体及其试剂盒 |
| CN202211405138.0A Pending CN115925866A (zh) | 2022-03-09 | 2022-11-10 | 一种用于乳腺癌检测的单克隆抗体及其试剂盒 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210229231.4A Pending CN114524872A (zh) | 2022-03-09 | 2022-03-09 | 一种用于乳腺癌检测的单克隆抗体及其试剂盒 |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN114524872A (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114720691B (zh) * | 2022-05-10 | 2022-12-09 | 深圳粒影生物科技有限公司 | 一种检测生物标志物的试剂盒及其制备方法和应用 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003095611A2 (en) * | 2002-05-10 | 2003-11-20 | Henry M. Jackson Foundation For The Advancement Of Military Medicine | Androgen-regulated pmepa1 and cancer |
| US20040092469A1 (en) * | 2000-01-28 | 2004-05-13 | Shiv Srivastava | Androgen-regulated PMEPA1 gene and polypeptides |
| CN105121665A (zh) * | 2013-04-26 | 2015-12-02 | 皇家飞利浦有限公司 | 使用多重细胞信号传导途径活性的治疗应答的医学预后和预测 |
| CN108025048A (zh) * | 2015-05-20 | 2018-05-11 | 博德研究所 | 共有的新抗原 |
| CN110678751A (zh) * | 2017-03-09 | 2020-01-10 | 克利拉生物技术有限公司 | 针对细胞衰老的生物标志物 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7834163B2 (en) * | 2003-06-26 | 2010-11-16 | Exonhit Therapeutics S.A. | Prostate specific genes and the use thereof as targets for prostate cancer therapy |
| EP2961853B1 (en) * | 2013-02-28 | 2018-09-19 | The Board of Regents of The University of Texas System | Methods for classifying a cancer as susceptible to tmepai-directed therapies and treating such cancers |
-
2022
- 2022-03-09 CN CN202210229231.4A patent/CN114524872A/zh active Pending
- 2022-11-10 CN CN202211405138.0A patent/CN115925866A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040092469A1 (en) * | 2000-01-28 | 2004-05-13 | Shiv Srivastava | Androgen-regulated PMEPA1 gene and polypeptides |
| WO2003095611A2 (en) * | 2002-05-10 | 2003-11-20 | Henry M. Jackson Foundation For The Advancement Of Military Medicine | Androgen-regulated pmepa1 and cancer |
| CN105121665A (zh) * | 2013-04-26 | 2015-12-02 | 皇家飞利浦有限公司 | 使用多重细胞信号传导途径活性的治疗应答的医学预后和预测 |
| CN108025048A (zh) * | 2015-05-20 | 2018-05-11 | 博德研究所 | 共有的新抗原 |
| CN110678751A (zh) * | 2017-03-09 | 2020-01-10 | 克利拉生物技术有限公司 | 针对细胞衰老的生物标志物 |
Non-Patent Citations (2)
| Title |
|---|
| ANWARUL HAQUE等: "PMEPA1/TMEPAI Is a Unique Tumorigenic Activator of AKT Promoting Proteasomal Degradation of PHLPP1 in Triple-Negative Breast Cancer Cells", 《CANCERS》, 30 September 2021 (2021-09-30), pages 13194934 * |
| 邹远康;张一萌;王婷;王杉;杨安钢;贾林涛;王磊;: "跨膜前列腺雄激素诱导蛋白1(PMEPA1)促进乳腺癌细胞迁移并维持乳腺癌细胞的间质样特性", 细胞与分子免疫学杂志, no. 02, 18 February 2016 (2016-02-18) * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114524872A (zh) | 2022-05-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US9823251B2 (en) | Anti-Uroplakin II antibodies systems and methods | |
| CN106999576A (zh) | 癌细胞特异性抗体、抗癌剂、及癌症的检查方法 | |
| JP6737592B2 (ja) | 抗活性型ｇｉｐ抗体 | |
| CN115724958B (zh) | 抗诺如病毒gⅱ基因组衣壳蛋白vp1的单克隆抗体及其应用 | |
| CN116355091A (zh) | 一种抗人神经丝轻链的单克隆抗体21d2-30d3及其产品和应用 | |
| CN106866820B (zh) | 一种用于捕获肿瘤细胞的抗人角蛋白18的单克隆抗体及其应用 | |
| CN115925866A (zh) | 一种用于乳腺癌检测的单克隆抗体及其试剂盒 | |
| CN110105449B (zh) | 一种特异性结合vegf的抗体及用途 | |
| EP3656794A1 (en) | Composition and methods for detecting cancer | |
| KR20120134547A (ko) | 항-ａｔｉｃ 자가면역항체를 포함하는 간암 진단 마커 및 이의 항원을 포함하는 간암 진단용 조성물 | |
| CN113203858B (zh) | 一种肿瘤检测试剂盒 | |
| CN110133278B (zh) | 一种用于检测人vegf蛋白表达水平的体外试剂盒 | |
| CN115746131A (zh) | 抗天然类风湿因子rf的单克隆抗体及其应用 | |
| CN110031616B (zh) | 一种辅助诊断疾病的检测试剂盒 | |
| JPWO2009044561A1 (ja) | 抗ｐｒｏＮＴ／ＮＭＮモノクローナル抗体 | |
| CN111303289B (zh) | 抗人Tn型糖基化MUC1抗体及其用途 | |
| CN111434686B (zh) | 一种抗人pbx1单克隆抗体，其制备方法及其在复发性流产临床诊断中的用途 | |
| CN110579610A (zh) | 用于检测t细胞活化的v域免疫抑制因子的试剂盒 | |
| CN110596369A (zh) | 一种用于检测人tim-3表达水平的试剂盒 | |
| CN110156891B (zh) | 一种高效、快速结合vegf的抗体及其用途 | |
| CN107709362B (zh) | Igf-1r抗体及其用于癌症诊断的用途 | |
| CN113203857B (zh) | 一种肿瘤诊断试剂盒 | |
| CN109776679B (zh) | 一种丝氨酸蛋白酶抑制因子spink1的抗体、其制备方法和应用 | |
| CA2821022A1 (en) | A monoclonal antibody against p28gank, the polypeptide used to prepare this antibody and their applications | |
| JP6586648B2 (ja) | 抗プログルカゴン抗体 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20231102 Address after: 2nd Floor, No. 846 Sanyuanli Avenue, Baiyun District, Guangzhou City, Guangdong Province, 510403 Applicant after: Guangdong Xingya Biotechnology Co.,Ltd. Address before: 510420 B75, 4th floor, building 2, No. 3, Dongping fufu Road, Yongping street, Baiyun District, Guangzhou, Guangdong Applicant before: Guangzhou nuocheng Biotechnology R & D Co.,Ltd. |