CN110981956B - Monoclonal antibody for identifying cell membrane mammaglobin A and application - Google Patents
Monoclonal antibody for identifying cell membrane mammaglobin A and application Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and discloses a monoclonal antibody for identifying cell membrane mammaglobin A and application thereof. The antibody comprises a variable region heavy chain variable region and a variable region light chain variable region; the gene sequence of the heavy chain variable region is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, the gene sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4. The antibody of the invention has recognition ability in different genotype breast cancer cells and has typical cell membrane binding characteristics.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a monoclonal antibody for identifying cell membrane mammaglobin A and application thereof.
Background
Breast cancer is a high-grade tumor worldwide and is also the leading type of tumor that causes death in women. In addition, breast cancer has high heterogeneity, basic genotypes of the breast cancer include four types of Luminal A, Luminal B, Her2 and Sanyin, and no molecular marker can be widely used in different types of breast cancer. HER2/neu is a breast cancer marker which is clinically more classical at present. However, HER2/neu is only highly expressed in approximately 20% of breast cancer patients, and most breast cancer patients do not benefit from HER 2/neu-based studies.
The search for a broader spectrum of breast cancer markers and their recognition ligands is a significant challenge in the art.
Disclosure of Invention
The invention aims to provide a monoclonal antibody for identifying cell membrane mammaglobin A and application thereof. The antibody has recognition capability in different genotype breast cancer cells and has typical cell membrane binding characteristics.
The above purpose of the invention is realized by the following technical scheme:
embodiments of the first aspect of the invention provide a monoclonal antibody recognizing cell membrane mammaglobin a, the antibody comprising a variable region heavy chain variable region and a light chain variable region; the gene sequence of the heavy chain variable region is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, the gene sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
Further, the site of binding to cell membrane mammaglobin a is from the 42 th amino acid to the 51 th amino acid of the N-terminal of the cell membrane mammaglobin a molecule.
The embodiment of the second aspect of the invention provides an application of a monoclonal antibody, wherein the monoclonal antibody is the monoclonal antibody, and the monoclonal antibody is used for preparing an antibody coupling drug for targeting breast cancer cells.
The beneficial effects of the invention include: the monoclonal antibody provided by the invention can effectively recognize the Mam-A on the cell membrane of the breast cancer, and is completely different from the immune epitope combined by the commercial antibody which recognizes the Mam-A on the cell membrane and is reported in the past; in addition, compared with the monoclonal antibody for identifying the cell membrane Mam-A reported in the past, the monoclonal antibody provided by the invention has obviously higher affinity to the Mam-A and has obviously higher diagnostic efficacy on clinical breast cancer specimens.
Drawings
FIG. 1 is a diagram illustrating screening of Mam-A monoclonal antibodies recognizing cell membranes according to an embodiment of the present invention.
FIG. 2 is a graph of surface plasmon resonance analysis of antigen-antibody interactions in accordance with an embodiment of the present invention.
FIG. 3 shows the sensitivity of the chip for detecting antibodies of clinical breast cancer according to an embodiment of the present invention.
FIG. 4 shows an example of a phage immunization pre-blocking assay.
FIG. 5 shows an alignment of polypeptide p733 with Mam-A sequences in accordance with an embodiment of the invention.
FIG. 6 shows a polypeptide immune pre-blocking assay in accordance with an embodiment of the present invention.
FIG. 7 shows the targeted binding of mAb785 antibody conjugated nanoparticle to breast cancer cells in an embodiment of the invention.
Detailed Description
In the following description, different "one embodiment" or "an embodiment" may not necessarily refer to the same embodiment, in order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art. Various embodiments may be replaced or combined, and other embodiments may be obtained according to the embodiments without creative efforts for those skilled in the art. A monoclonal antibody recognizing cell membrane mammaglobin a, said antibody comprising a variable region heavy chain variable region and a light chain variable region; the gene sequence of the heavy chain variable region is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, the gene sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
In some embodiments of the invention, the site of binding to cell membrane mammaglobin a is from amino acids 42 to 51 of the N-terminus of the cell membrane mammaglobin a molecule.
The monoclonal antibody is used for preparing an antibody conjugate medicament targeting breast cancer cells.
A monoclonal antibody for recognizing cell membrane mammaglobin A is secreted by hybridoma cell strain mAb 785. The mAb785 hybridoma is obtained by amplifying a Mam-A gene sequence through PCR, inserting the sequence into a pBV/GST carrier through the processes of enzyme digestion, connection and the like for expression, extracting and purifying to obtain gene recombinant human Mam-A protein, immunizing a mouse, separating splenocytes from the immunized mouse with better serum antibody titer, fusing the splenocytes with SP20 cells through a conventional method to obtain a series of hybridoma cells secreting anti-Mam-A monoclonal antibodies, and further performing series of screening, wherein the hybridoma cells are currently stored in military medical research institutes.
(1) The screening of the mAb785 monoclonal antibody is mainly realized by the following steps:
1) removing the signal peptide sequence of the first 19 amino acids from the N-terminal, and cloning the human Mam-A protein to express 4 segments A, B, C, D in a manner that adjacent segments are overlapped, wherein the A segment comprises 20-52 amino acid sequences, the B segment comprises 43-70 amino acid sequences, the C segment comprises 56-82 amino acid sequences, and the D segment comprises 67-93 amino acid sequences;
2) respectively coating an Elisa plate by using the Mam-A protein and the 4 fragments, reacting with the prepared series of anti-Mam-A monoclonal antibodies, and screening monoclonal antibodies respectively targeting different sections of the Mam-A to obtain 6 monoclonal antibodies for identifying different sites; meanwhile, a commercial antibody mAb304 which recognizes the cell membrane Mam-A and is reported in the past is used as a control;
3) respectively carrying out immunofluorescence staining on the 6 screened antibodies and the commercialized antibody 304 by using a breast cancer cell ZR75.1 of cytoplasm expressing Green Fluorescent Protein (GFP), observing staining characteristics by laser confocal, and screening to obtain two monoclonal antibodies for identifying typical cell membrane staining characteristics;
4) detecting the affinity performance of the two newly screened monoclonal antibodies for recognizing the cell membrane Mam-A and the commercialized 304 antibody and the Mam-A by using Biocore analysis, and further screening and determining the antibody with better affinity;
5) the sensitivity and specificity of the three antibodies are respectively evaluated by using a tissue chip, and monoclonal antibodies with high sensitivity and good specificity to breast cancer are screened. And comparing the antibodies with the highest detection rate on the breast cancer specimen and the lowest false positive rate on the non-breast cancer specimen.
(2) The variable region of monoclonal antibody mAb785 comprises a heavy chain variable region and a light chain variable region; the gene sequence of the heavy chain variable region is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2; the gene sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
(3) The monoclonal antibody is used for immunofluorescence staining of a breast cancer cell strain ZR75.1, and typical cell membrane staining characteristics can be observed; the fluorescent staining reagent can also be used for immunofluorescence staining of breast cancer cells of other three genotypes, including MCF-7(LuminalB), SKBR3(HER2 high expression) and MDA-MB-231 (triple negative) cell strains;
(4) the immune epitope sequence recognized by the monoclonal antibody is positioned at the amino acid sites of N42-N51 in the Mam-A protein, and the binding function of the immune epitope sequence can be blocked by the polypeptide containing the amino acid;
(5) the application of the monoclonal antibody in the aspects of breast cancer histological diagnosis and antibody conjugate drug development is also within the protection scope of the invention, and the monoclonal antibody can be specifically used for immunohistochemical detection of breast cancer specimens and preparation of antibody conjugate drugs.
The monoclonal antibody provided by the invention can effectively recognize the Mam-A on the cell membrane of the breast cancer, and is completely different from the immune epitope combined by the commercial antibody which recognizes the Mam-A on the cell membrane and is reported in the past; in addition, compared with the monoclonal antibody for identifying the cell membrane Mam-A reported in the past, the monoclonal antibody provided by the invention has obviously higher affinity to the Mam-A and has obviously higher diagnostic efficacy on clinical breast cancer specimens.
In connection with the figures 1-7,
FIG. 1 is a diagram illustrating screening of Mam-A monoclonal antibodies recognizing cell membranes according to an embodiment of the present invention.
Cell membrane dye Dil, 6 monoclonal antibodies recognizing different sections of Mam-A provided by the invention and the previously reported immune fluorescent staining of the commercial monoclonal antibody mAb304 recognizing cell membrane Mam-A and breast cancer cells ZR75.1 (ZR75.1 expresses green fluorescent protein GFP in cytoplasm).
FIG. 2 is a graph of surface plasmon resonance analysis of antigen-antibody interactions in accordance with an embodiment of the present invention.
a, analyzing the interaction between the monoclonal antibody mAb785 screened by the invention and the Mam-A protein; a', analysis of the interaction of mAb785 with targeting polypeptide a; b, analyzing the interaction between the monoclonal antibody mAb656 screened by the invention and the Mam-A protein; b', analysis of the interaction of mAb785 with targeting polypeptide C; c, analyzing the interaction between the monoclonal antibody mAb304 of the same type and the Mam-A protein reported previously; c', interaction analysis of mAb304 with targeting polypeptide D.
FIG. 3 shows the sensitivity of the chip for detecting antibodies of clinical breast cancer according to an embodiment of the present invention.
a, immunohistochemically staining representative results of breast cancer and a tissue chip beside the cancer with different antibodies; b, detecting rates of different antibodies on breast cancer specimens and tissue specimens beside the cancer; and c, carrying out immunohistochemical detection on the breast cancer sample and the paracancer sample by using different antibodies to obtain the expression level thermograph of the Mam-A.
FIG. 4 shows an example of a phage immunization pre-blocking assay.
a, phage 731 and phage 734 are used in the immune pre-blocking experiment of mAb785 antibody, and excessive two phages cannot completely block the binding of mAb785 to breast cancer specimen, indicating that polypeptides on the surface of two phages may not be bound to the functional region of antibody; b, the phage 734 is used for an immune pre-blocking experiment of the mAb785 antibody, and excessive phage completely blocks the binding of the antibody mAb785 to a breast cancer sample, indicating that the polypeptide on the surface of the phage binds to the functional region of the antibody.
FIG. 5 shows an alignment of polypeptide p733 with Mam-A sequences in accordance with an embodiment of the invention.
The first 10 amino acids in the phage 733 surface polypeptide p733 are highly similar to the N42-51 amino acid sequence in the Mam-A sequence, only the amino acids at positions 2, 6 and 9 are different, and the amino acid structures at the three positions are similar and are possibly interchangeable amino acids; and the polypeptide p733-3R is obtained after the amino acids at the positions 2, 6 and 9 in the p733 are replaced by the amino acids at the corresponding positions of Mam-A.
FIG. 6 shows a polypeptide immune pre-blocking assay in accordance with an embodiment of the present invention. a, the number of the first-time contact points is less than the number of the second-time contact points,
the polypeptides p733 and p733-3R both completely blocked the binding of mAb785 antibody to Mam-A, indicating that the 3 pairs of different amino acids in p733 and p733-3R are interchangeable amino acids; b, polypeptide p733-3RC obtained by removing two tail amino acids PV from polypeptide p733-3R-2It also completely blocked mAb785 binding to Mam-A, indicating that the terminal PV two amino acids are not an essential part of the mAb785 recognition sequence; c, at p733-3RC-2Polypeptide p733-3RC obtained by removing two front amino acids YK on the basis- 2N-2The inability to completely block mAb785 binding to Mam-A indicates that YK is an essential component of the mAb785 recognition sequence.
FIG. 7 shows the targeted binding of mAb785 antibody conjugated nanoparticle to breast cancer cells in an embodiment of the invention.
Under the same co-incubation conditions, mAb785 antibody-conjugated nanoparticles (mAb 785-nanomedicine particles) bound to breast cancer cells in large numbers, while no antibody-conjugated nanomedicine particles bound to breast cancer cells in small numbers.
Example 1: monoclonal antibody screening for recognizing cell membrane Mam-A
(1) Clonal expression of the Mam-a protein with different fragments: designing PCR primers according to the sequence of each fragment, adding Xho I and Xba I at two ends respectively, obtaining genes of the Mam-A protein (8-93AA), the A fragment (20-52AA), the B fragment (43-70AA), the C fragment (56-82AA) and the D fragment (67-93AA) section through PCR amplification, carrying out enzyme digestion on the purified products through Xho I and Xba I, inserting the products into a pBVIL1 vector (Mam-A protein, A fragment, B fragment and D fragment) or a PBV220 vector (C fragment), transforming HB101, selecting positive clones, selecting a target gene to obtain high expression, sequencing and identifying correct strains, carrying out induced expression, carrying out inclusion body extraction according to the third edition relevant chapters of molecular cloning experimental guidance of Sambroob and the like, and purifying by using a Ni column.
(2) Screening of anti-Mam-A monoclonal antibodies recognizing different epitopes: the Mam-A protein and the 4 fragments expressed above were coated on an Elisa plate, and then a series of Mam-A monoclonal antibodies were reacted with each Elisa to examine the reaction characteristics of the different antibodies. As shown in Table 1 below, the 19 anti-Mam-A monoclonal antibodies exhibited 6 different reactivity characteristics with different sections of Mam-A (a: reactivity with Mam-A protein and A fragment; B: reactivity with Mam-A, A, fragment B, fragment C: reactivity with Mam-A and B fragments; d: reactivity with Mam-A protein, fragment B and fragment C; e: reactivity with Mam-A protein and fragment C; and f: reactivity with Mam-A protein only). mAb304 is a reported commercial control antibody recognizing cell membrane Mam-A. The preparation, characterization and screening of anti-Mam-a monoclonal antibodies are shown in table 1.
TABLE 1 preparation, characterization and screening of anti-Mam-A monoclonal antibodies
All antibodies were reactive with full-length Mam-A
(3) Screening of monoclonal antibodies recognizing cell membrane Mam-A: the breast cancer cell line ZR75.1 expressing GFP cytoplasm is used for immunofluorescence staining of the 6 screened anti-Mam-A monoclonal antibodies recognizing different sections and ZR75.1 cells, and the staining characteristics of different antibodies are observed by laser confocal with cell membrane dye Dil and a previously reported monoclonal antibody 304 recognizing cell membrane Mam-A as controls. FIG. 1 shows the immunofluorescence staining profile of cell membrane dye Dil, representative monoclonal antibodies of the invention that recognize different segments of Mam-A, and a commercial mAb304 control antibody. Obviously, monoclonal antibody mAb656 and mAb785 have typical cell membrane staining characteristics.
(4) Identification of the affinity properties of different monoclonal antibodies recognizing the cell membrane Mam-A: the affinity of the two monoclonal antibodies mAb656 recognizing cell membrane Mam-A and mAb785 binding to antigen was analyzed by surface plasmon resonance. Analyzer model Biacore 3000TMThe instrument (GE Healthcare, USA) uses a sensing chip as a CM5 chip from GE. As shown in FIG. 2, the results of the interaction between mAb656, mAb785 and the previously reported commercial antibody mAb304 recognizing cell membrane Mam-A and the full-length Mam-A and the target fragment, respectively, indicate that mAb785 has the best affinity for Mam-A and the target fragment.
(5) Sensitivity and specificity evaluation of different monoclonal antibodies recognizing the cell membrane Mam-a for breast cancer diagnosis: shanghai Zhanghao chip company bought breast cancer organization chip: comprises 16 cases of breast cancer specimens and paracarcinoma tissue specimens; non-breast cancer tumor chips: comprises 12 tumor specimens and tissues beside the cancer; normal tissue specimens: contains 19 normal tissue specimens. Immunohistochemical staining was performed using mAb656, mAb785 and commercial mAb304 antibody and tissue chips, respectively, to analyze the sensitivity and specificity of the different antibodies for breast cancer detection. As shown in the following tables 2 and 3,
TABLE 2 immunoreaction of different antibodies with tumor specimens of non-breast cancer
TABLE 3 immunoreaction of different antibodies with normal tissue chips
Example 2: immune epitope identification recognized by mAb785 antibody
(1) Phage display screening polypeptides with affinity for mAb 785: the 12 peptide phage display peptide library was purchased from BioLabs (cat # 0411702). Elisa plates were coated with 5ug/mL mAb785 antibody. Adding 100uL of the amplified phage library into the Elisa plate, incubating for 0.5h at 37 ℃, and washing with PBS for multiple times to remove unbound phage; the combined phage is eluted by eluent and amplified; the amplified phage library is used as a new enrichment library to repeat the enrichment process. After 3 rounds of enrichment, 20 amplified phage plaques are selected for sequencing to obtain polypeptide sequences carried by the phage, and the occurrence frequency of different polypeptides is calculated. Table 3 below shows the enriched polypeptides obtained by 3 rounds of enrichment sequencing and the frequency of appearance. The polypeptides 731, 733, 734 occur most frequently and are considered to have a higher affinity for the antibody mAb 785. The enriched polypeptides displayed by phage and the frequency of appearance are shown in Table 4.
TABLE 4 phage display of enriched polypeptides and frequency of appearance
(2) Immune pre-blocking experiments identified polypeptides that bound to mAb785 functional region: amplifying the 731, 733 and 734 phages, then mixing the excess phages with 100uL of 0.5ug/mL mAb785 antibody respectively, incubating for 0.5h at 37 ℃, blocking the antibody functional region through the affinity polypeptide on the phages, performing immunohistochemical staining on a breast cancer specimen by using the blocked antibody solution, and comparing with the unblocked antibody, staining negatively by the antibody solution with the completely blocked functional region, and staining positively by the antibody with the unblocked functional region. The results are shown in fig. 4, only 733 phages successfully blocked antibody function, suggesting that the 733 polypeptide carried by them might be the protein sequence targeted for binding by mAb 785.
(3) Determination of the immunoepitope bound by the mAb785 antibody to Mam-A: the first 10 amino acids (aa) in the 733 polypeptide were found to be highly similar to N42-N51 aa of the Mam-A protein by alignment with the Mam-A protein sequence, as shown in FIG. 5, differing only by amino acids 2, 6, 9. Panel b shows that structural comparisons of the amino acids that differ show a high degree of similarity, possibly alternative amino acids. Subsequently, a polypeptide p733 was synthesized, replacing the polypeptide p733-3R of 3 different amino acids. Chemically synthesizing polypeptides p733 and p733-3R, and performing immune pre-blocking experiments on the two polypeptides and the mAb785 antibody respectively, wherein the blocked antibody is used for immunohistochemical staining of a breast cancer section. The results are shown in FIG. 6, and p733-3R can completely block the binding of mAb785 antibody to Mam-A, indicating that the 3 different amino acids are indeed alternative amino acids. Further, it was speculated that the terminal two amino acids P, V may not be useful for binding of the polypeptide to an antibody, and that the synthetic polypeptide p733-3R-2 was removed, and the results of the immune pre-blocking experiments showed that the p733-3R-2 polypeptide also completely blocked the binding of mAb785 to Mam-A. And the p733-3R-2 is completely identical with the N42-51 amino acid sequence of the Mam-A protein, which indicates that N42-N51 is the immune epitope recognized by the mAb785 antibody.
Example 3: application of mAb785 in antibody coupling drugs
(1) Dissolving PLGA (with the molecular weight of 100000,) in dichloromethane to prepare a 6% solution, adding fluorescein FITC as a drug substitute, and dissolving in dichloromethane to obtain an oil phase solution;
(2) adding 1mL of the oil phase solution into 10mL of 2.5% PVA water phase solution, and performing ultrasonic emulsification for 1 minute by using a 4% maximum power of a SCIENTZ-IID ultrasonic instrument;
(3) slowly stirring the emulsion at room temperature for more than 4 hours to completely volatilize the organic solvent as much as possible;
(4) centrifuging at 12000rpm to collect nanoparticles, washing with sterile water for three times to obtain nanoparticles;
(5) dissolving about 40ug of nanoparticles in 1mL NHS (50mM)/EDC (100mM) mixture, incubating at room temperature for 30min, and activating the surface carboxyl groups of the nanoparticles;
(6) centrifugation at 12000rpm for 5 min to remove excess NHS/EDC mixture, PBS wash three times, and use 1mLPBS heavy suspension;
(7) adding 1mL of mAb785 antibody 50uL with a concentration of 5mg/mL, and incubating overnight at 4 ℃;
(8) centrifuging at 12000rpm to remove redundant antibodies, washing with PBS for three times, and reselecting 1mL sterile PBS to obtain antibody-coupled nano-drug particles;
(9) inoculating breast cancer cells MCF-7 and ZR75.1 on a 12-hole culture plate, growing until 50-60% of the cells are combined, respectively adding 10uL of the prepared antibody coupling nanoparticles or antibody-free nano-drug particles, and incubating for 2 hours at 37 ℃;
(10) unbound nanoparticles were removed and washed three times with PBS;
(11) DAPI is used for staining cell nucleuses, and the target binding condition of the antibody-conjugated drug nano-drug particles and tumor cells is observed under a fluorescence microscope. As shown in fig. 7, a large number of antibody-conjugated nano-drug particles were bound to tumor cells, and few antibody-free nano-drug particles were bound to tumor cells, indicating that the mAb 785-based antibody-conjugated drug had a good breast cancer targeting function.
It should be noted that the above embodiments can be freely combined as necessary. The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> military medical research institute of military science institute of people's liberation force of China
<120> monoclonal antibody for identifying cell membrane mammaglobin A and application
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<160>4
<170>PatentIn version 3.3
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<211>132
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>2
Met Asn Phe Gly Val Ser Leu Ile Phe Leu Val Leu Val Leu Lys Gly
1 5 10 15
Val Gln Cys Glu Val Lys Leu Val Glu SerGly Gly Gly Leu Val Lys
20 25 30
Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu
35 40 45
Ser Asn His Gly Met Ser Trp Val Arg Gln Thr Ser Glu Lys Arg Leu
50 55 60
Glu Trp Val Ala Ser Ile Ser Ile Arg Gly Asn Thr Tyr Tyr Leu Asp
65 70 75 80
Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Arg Asn Ile
85 90 95
Leu Tyr Leu Gln Met Ser Gly Leu Arg Ser Glu Asp Thr Ala Ile Tyr
100 105 110
Tyr Cys Thr Tyr Phe Thr Ser Val Val Trp Gly Gln Gly Thr Thr Leu
115 120 125
Thr Val Ser Ser
130
<210>3
<211>393
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>3
atgagtcctg cccagttcct gtttctgtta gtgctctgga ttcgggaaac caacggtgat 60
gttgtgctga cccagactcc actcactttg tcggttacca ttggacaacc agcctccatc 120
tcttgcaagt caagtcagag cctcttagat agtgatggaa agacattttt gaattggttg 180
tttcagaggc caggccagtc tccaaagcgc ctaatctatc tggtgtctaa actggactct 240
ggagtccctg acaggttcac tggcagtgga tcagggacag attttacact gaaaatcagc 300
agagtggagg ctgacgattt gggagtttat tattgctggc aaggtacaca ttttcctcag 360
acgttcggtg gaggcaccaa gctggaaatc aaa 393
<210>4
<211>131
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>4
Met Ser Pro Ala Gln Phe Leu Phe Leu Leu Val Leu Trp Ile Arg Glu
1 5 10 15
Thr Asn Gly Asp Val Val Leu Thr Gln Thr Pro Leu Thr Leu Ser Val
20 25 30
Thr Ile Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu
35 40 45
Leu Asp Ser Asp Gly Lys Thr Phe Leu Asn Trp Leu Phe Gln Arg Pro
50 55 60
Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser
65 70 75 80
Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
85 90 95
Leu Lys Ile Ser Arg Val Glu Ala Asp Asp Leu Gly Val Tyr Tyr Cys
100 105 110
Trp Gln Gly Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu
115 120 125
Glu Ile Lys
130
Claims (3)
1. A monoclonal antibody recognizing cell membrane mammaglobin a, comprising a variable region heavy chain variable region and a variable region light chain variable region; the gene sequence of the heavy chain variable region is shown as SEQ ID NO.1, the amino acid sequence is shown as SEQ ID NO.2, the gene sequence of the light chain variable region is shown as SEQ ID NO.3, and the amino acid sequence is shown as SEQ ID NO. 4.
2. The monoclonal antibody of claim 1, wherein the site of binding to cell membrane mammaglobin a is from amino acid 42 to amino acid 51 of the N-terminus of the cell membrane mammaglobin a molecule.
3. The use of a monoclonal antibody according to claim 1 or 2 for the preparation of an antibody conjugate for targeting breast cancer cells.
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| CN1766634A (en) * | 2005-06-13 | 2006-05-03 | 上海晶泰生物技术有限公司 | Method for detecting mammaglobin and kit therefor |
| CN102296119B (en) * | 2011-09-08 | 2013-08-07 | 苏州友林生物科技有限公司 | Detection method and kit for milk globulin mRNA in circulatory blood |
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