CN113583132B - anti-PR protein monoclonal antibody and preparation method and application thereof - Google Patents
anti-PR protein monoclonal antibody and preparation method and application thereof Download PDFInfo
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
- G01N33/743—Steroid hormones
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- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
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- C07K—PEPTIDES
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- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/23—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a GST-tag
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- 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
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones
- G01N2333/723—Steroid/thyroid hormone superfamily, e.g. GR, EcR, androgen receptor, oestrogen receptor
Abstract
The invention relates to a monoclonal antibody capable of recognizing human PR antigen, a preparation method thereof and application thereof in immunoassay. The invention provides a rabbit monoclonal antibody for resisting PR protein, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 6. The antibody has high specificity and sensitivity, can specifically recognize cells expressing PR protein, and is suitable for immunological detection, especially for immunohistochemical detection.
Description
Technical Field
The invention relates to the field of biomedical engineering, in particular to an anti-PR protein monoclonal antibody and a preparation method and application thereof.
Background
PR (progestogen receptor) is a protein molecule, an intracellular nuclear receptor belonging to the superfamily of steroid hormones, which binds to the corresponding hormone to cause a specific reaction and further form a hormone-receptor complex, allowing the hormone to exert its biological effect. The progestagen receptor has two isoforms, PR-a and PR-B, which are composed of three domains: the C-terminal domain binds primarily to progestin, the central domain is the DNA binding domain, and the N-terminal domain is currently of unknown structure and may be involved in a variety of responses.
PR is a dual-functional protein that can be used both as a transcription factor in a ligand-dependent manner to directly interact with nuclear DNA to regulate transcription and as a non-transcription factor to regulate extra-nuclear signaling pathways. Plays a key role in mediating and regulating the functions of ovary, uterus and mammary gland and reproductive activity. Expressed in normal and benign and malignant endometrial and mammary epithelial cells.
Studies have demonstrated that the expression levels of ER, PR are closely related to overall survival, tumor-free survival, recurrence-free survival, progression-free survival and clinical efficacy in breast cancer patients. Studies have shown that PR status is a predictive indicator independent of ER status, particularly for pre-menopausal breast cancer patients. PR is often used with ER and HER-2 as a routine immunohistochemical test item for breast cancer patients.
Disclosure of Invention
The inventor provides an anti-PR protein monoclonal antibody, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 6.
Furthermore, the DNA sequence of the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO.3, and the DNA sequence of the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID NO. 4.
Further, the monoclonal antibody specifically recognizes the PR protein.
Further, the monoclonal antibody is a rabbit monoclonal antibody.
The inventor also provides a preparation method of the anti-PR protein monoclonal antibody, an antigen for immunizing rabbits is recombinant protein, and the recombinant protein is expressed by escherichia coli in a recombinant mode; selecting a protein fragment at sites 306-455, wherein the protein fragment is an amino acid sequence shown in SEQ ID NO. 1; optimizing into a gene segment suitable for being expressed in escherichia coli, wherein the gene segment is a nucleotide sequence shown as SEQ ID NO. 2; and cloning the gene fragment to pGEX-4T-AB1 vector.
Further, the recombinant protein comprises a GST protein tag and a histidine protein tag.
The inventor also provides the application of the monoclonal antibody in PR protein immunoassay.
Further, the immunodetection includes immunohistochemistry, immunoblotting and enzyme-linked immunoassay.
The inventor finally provides a PR protein immunohistochemical detection reagent, which contains the monoclonal antibody as an effective component.
Different from the prior art, the invention has the following beneficial technical effects: the technical scheme provides an anti-PR protein rabbit monoclonal antibody, wherein the amino acid sequence of a heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 6. The antibody has high specificity and sensitivity, can specifically recognize cells expressing PR protein, and is suitable for immunological detection, especially for immunohistochemical detection.
Drawings
FIG. 1 is a graph of purified pectin from PR recombinant peptide fragments fused with histidine tag in example 1.
FIG. 2 is a comparison of immunohistochemical staining results for breast cancer; wherein the left part is rabbit monoclonal antibody PR (14B 3), and the right part is commercially available PR.
FIG. 3 is a graph comparing the results of immunohistochemical staining of endometrium; wherein the left part is rabbit monoclonal antibody PR (14B 3), and the right part is commercially available PR.
Detailed Description
To explain technical contents, structural features, and objects and effects of the technical solutions in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
Example 1 preparation of recombinant PR protein fragments
1. Gene optimization and synthesis
PR selects protein fragments of sites 306-455 according to the protein sequence with accession number P06401 in the Uniprot database, and the protein fragments are the amino acid sequence shown in SEQ ID NO. 1; optimizing the gene fragment suitable for expression in E.coli Rosetta (DE 3) and having a nucleotide sequence shown in SEQ ID NO. 2; and cloning the gene fragment to pGEX-4T-AB1 vector.
Coli Rosetta (DE 3) with expression vectors, selecting clones on the plate for inoculation, and performing PCR identification of bacterial liquid. And selecting the clone with positive PCR result for sequencing analysis, and using the clone with completely correct sequence.
The selection of different antigens for immunization makes it possible to prepare antibodies with different binding characteristics, the molecules present simultaneously a plurality of variants due to variable cleavage, and finally the recognition ability and pattern of different antibodies on antigen-expressing cells are different. PR molecules are analyzed according to a published sequence, a region which is suitable for soluble expression and has good immunogenicity is selected for recombinant expression according to the structure, antigenicity, hydrophilicity and hydrophobicity of constituent amino acids and a secondary structure on cells, a gene sequence of sites 306-455 of PR is selected for codon optimization, and the molecular weight is about 50kDa. PR recombinant peptide segments are obtained by using prokaryotic expression gene sequences through sequence optimization design. The recombinant immunogen consists of antigenic PR recombinant peptide and protein tags for recombinant protein purification, wherein the protein tags are GST and HIS.
2. Protein expression and purification
The overnight strain cultured by a single colony is transferred to 100mL LB culture medium according to the proportion of 1. The recombinant protein is provided with a histidine tag, and affinity purification of the protein is performed by using a nickel column. Elution was carried out with 150mmol/L imidazole and SDS PAGE separation was carried out.
FIG. 1 is a graph of purified pectin of recombinant PR protein fused with histidine tag. The final purified protein has the concentration of 3mg/mL and the purity of 80 percent, and can be used for the requirements of animal immunization and antibody screening and identification.
Example 2 Single B cell sorting of Rabbit anti-human PR monoclonal antibody
1. Immune and ELISA assays
The recombinant PR protein of example 1 was emulsified with Freund's complete adjuvant and 6 rabbits were selected for immunization at a dose of 300. Mu.g/rabbit. The booster was administered once on days 7, 21, 42, 63, respectively, and the antigen was emulsified with Freund's incomplete adjuvant at a dose of 150. Mu.g/mouse. On day 95, the ballistic immunization was carried out, and the antigen was mixed with physiological saline in a dose of 300. Mu.g/mouse. And performing serum ELISA titer detection after the third, fourth and fifth immunizations, wherein serum of the fourth immunizations of 4 rabbits is subjected to IHC detection, and selecting 1 rabbit for subsequent monoclonal antibody screening according to the result (namely, the positive corresponding to the antibody is used for detecting the picture, and immunohistochemical staining can be observed on the corresponding detection site in the positive picture).
2. Spleen cell isolation and B lymphocyte sorting
Monoclonal antibody preparation was performed on selected rabbits. Spleens were harvested 3 days after the ballistic immunization, rabbit spleens were placed in RMPI basal medium containing 100U/ml penicillin and 100ug/ml streptomycin, minced with a surgical blade and ground into 100 μm cell mesh. The resulting cell suspension was filtered to remove large cell masses and tissue envelopes, centrifuged at 400g for 5 minutes, and the supernatant was removed to retain the spleen cell mass. The spleen cell pellet was resuspended in hypotonic solution and erythrocytes were lysed and centrifuged again at 400g for 5min, retaining the spleen cells. Spleen cells were resuspended in RMPI basal medium containing 100U/ml penicillin and 100. Mu.g/ml streptomycin, centrifuged at 400g for 5 minutes, and the resulting spleen cells were resuspended in complete medium (RMPI basal medium containing 10% fetal bovine serum, 100U/ml penicillin, and 100. Mu.g/ml streptomycin).
Specific steps for sorting B lymphocytes are described in china patent 201910125091.4, "method for efficiently isolating single antigen-specific B lymphocytes from spleen cells".
About 2000 single B cell clones were sorted and cultured, positive clones specifically recognizing the recombinant PR protein of example 1 were screened by ELISA, and 50 supernatants from high to low were selected for IHC verification based on ELISA data.
3. B cell culture positive clone detection
IHC of single B cell culture supernatants using multiple tumor and normal tissue chips containing PR positive and negative, among which 13 clone culture supernatants gave better IHC results to prepare LEM supernatants, which were IHC verified to identify clones with excellent sensitivity and specificity (14B 3).
The sensitivity and specificity screening criteria were: the photograph was examined with a positive test corresponding to the antibody, where immunohistochemical staining was observed at the corresponding detection site, but no immunohistochemical staining was observed at the non-detection site. Compared to the control antibody, the staining intensity reached even higher than the control antibody.
4. Determination of affinity constant
PR antigen polypeptide was coated at a concentration of 2. Mu.g/ml, 100. Mu.L/well, overnight at 4 ℃ and washed 3 times with PBS-T. Add 200. Mu.l of blocking solution per well for blocking at 37 ℃ for 2h, wash with PBS-T for 3 times. The purified (14B 3) monoclonal antibody was purified from 1:200 began a 2-fold gradient dilution, and finally 1 well was blank, incubated at 37 ℃ for 1h and washed 3 times with PBS-T. HRP-labeled goat anti-mouse secondary antibody 1:20000 dilution, 100. Mu.L per well, incubation at 37 ℃ for 1h, PBS-T wash 3 times. 0.1% of TMB (Sigma) and 0.03% of H by adding 100. Mu.l per well 2 O 2 The reaction solution was developed in the citric acid-phosphoric acid buffer for 10min, and 50. Mu.L of a 0.5M sulfuric acid solution was added thereto to terminate the reaction. And measuring the light absorption value with the wavelength of 450nm by using a microplate reader. Drawing a curve of the OD value corresponding to the dilution factor of the antibody, and finding out the dilution factor A corresponding to the 'platform OD value' of more than or equal to 1/2. The affinity constant was calculated to be 1.82X 10 using the following formula 10 。
5. Clone of encoding rabbit monoclonal antibody gene (14B 3) and construction of rabbit monoclonal antibody expression plasmid
The positive cloned cells are collected, lysed, and the RNA is extracted and reverse transcribed into cDNA. Naturally paired rabbit monoclonal antibody light and heavy chain variable region genes (SEQ ID No.5 and SEQ ID No. 6) were amplified from the corresponding positively cloned cDNA by PCR and sequenced to determine the sequence.
And (3) respectively constructing rabbit monoclonal antibody expression vector plasmids by using the determined naturally paired light chain variable region gene sequences and heavy chain variable region gene sequences of the rabbit monoclonal antibody.
Example 2 was completed by entrusted martian ebutake biotechnology limited by fuzhou new biotechnology development limited, wherein immunohistochemical screening was performed by fuzhou new biotechnology development limited.
Example 3 PR Rabbit monoclonal antibody expression
1. Plasmid amplification and extraction
Taking out a tube (100 μ l) of allelochemicals (DH 5 α), inserting into ice, and ice-cooling for 5-10min; adding 5 μ l plasmid, shaking gently, and placing on ice for 30min; shaking gently, placing into 42 deg.C water bath for 90s for heat shock, rapidly placing back into ice, and standing for 5min; respectively adding 800 μ l LB culture medium (injection: without antibiotic) into the above materials in a clean bench, mixing gently, fixing on a shaking table, shaking at 37 deg.C for 1h; taking 50-100 mul of the conversion mixed solution from a clean bench, respectively dripping the conversion mixed solution into a marked solid LB plate culture dish containing Amp, and uniformly coating the solution by using a glass coating rod (sterilization); the culture medium is placed in an incubator at 37 ℃ for 30min, the bacteria liquid on the surface completely permeates into the culture medium, and then the culture medium is placed in the incubator at 37 ℃ for overnight culture after being inverted. Single colonies were picked with a pipette tip and injected into 4ml LB medium (containing 2uL 200mg/ml Amp), shaking cultured at 37 ℃ for 169h, 37 ℃ and 220rpm.
Adding 1ml of the bacterial liquid into 100ml of LB culture medium (containing 50 mu l of 200mg/ml Amp), and performing shake culture at 37 ℃ for 16h; plasmid extraction was performed using the SanPrep endotoxin removal Mass DNA miniprep kit (manufacturer's) according to the instructions.
2. Transfection
The 293F cell concentration was adjusted to 2.5-3X 106 viable cells/ml with Expi293FTM Expression Medium and cultured overnight.
1) Counting by using a blood cell counting plate, wherein the concentration of living cells is about 4.5-5.5 multiplied by 106 viable cells/ml, and the number of the living cells needs to meet the requirement of an expression system;
2) Diluting the cell concentration to 3X 106 viable cells/ml with Expi293FTM Expression Medium;
3) Adding the plasmid DNA into Opti-MENTM I reduced Serum Medium, gently blowing, reversing and mixing;
4) Gently inverting Expifeactine (TM) 293 Reagent 4-5 times, mixing Expifeactine (TM) 293 Reagent and Opti-MENTM I reduced Serum Medium, gently blowing and inverting for 2-3 times, and standing at room temperature for 5min;
5) Mixing the solutions obtained in the steps 3) and 4), gently beating and reversing for 2-3 times, and uniformly mixing;
6) Placing the solution in the step 5) at room temperature for 10-20min;
7) Slowly sucking the mixed solution into the cell culture solution, and gently shaking the triangular flask;
8)8％CO 2 shake culturing at 37 deg.C for 5-7 days;
9) After 18-22 hours, expifeacylamine (TM) 293 Transfecton Enhancer1 and Expifeacylamine (TM) 293 Transfecton Enhancer2 were added (note: mix well before use) and gently shake well and continue culturing.
3. Purification of monoclonal antibodies
Antibodies were purified from the supernatant using HiTrap rProtein A FF affinity chromatography as described. Purity was confirmed by SDS-PAGE and concentration was measured by Bradford method. Purified antibody was stored at-20 ℃.
Example 4 immunohistochemical tissue chip staining and identification
Chip preparation process
HE sections were first stained for each sample to identify tumor sites. The tissue chip was produced using a fully automatic tissue chip machine of 3 DHISTECH. And putting the prepared tissue chip wax block into a wax block manufacturing mould, putting the mould into an oven at 68 ℃ for 10 minutes to melt the tissue core and the wax of the receptor wax block into a whole, then lightly taking the mould out of the oven, cooling the paraffin in a semi-molten state for about 30 minutes at room temperature, freezing the mould in a refrigerator at-20 ℃ for 6 minutes, taking the tissue chip wax block out of the mould, and slicing or putting the tissue chip wax block into the refrigerator at 4 ℃ for storage for later use. And (3) trimming the tissue chip, then carrying out continuous slicing, determining the thickness to be 3 mu m, floating the continuous slice in 40% alcohol, naturally unfolding the slices, transferring the separated slices into warm water at 50 ℃ for 30 seconds, mounting the slices by using a glass slide treated by polylysine, baking the prepared tissue chip in an oven at 68 ℃ for 2 hours, taking out, cooling at room temperature, and storing in a refrigerator at-4 ℃.
IHC staining and analysis
Conventional xylene dewaxing was performed 3 times for 6 minutes each, 100%, 95%, 85% gradient ethanol hydration for 3 minutes each, and finally tap water rinse. Antigen retrieval was performed and the sections were then placed in a wet box and washed 3 x 3 min with PBS. Dropwise addition of 3% of 2 O 2 Incubate for 10min and wash with PBS for 3 x 3 min. Spin-drying the slices, dripping primary antibody diluted in a proper proportion (the dilution proportion of the antibody is designed according to the concentration of the antibody in the primary dilution) and incubating for 1 hour at room temperature (25 ℃), washing for 3X 3 minutes by PBS, dripping secondary antibody and incubating for 15-30 minutes at room temperature, washing for 3X 3 minutes by PBS, throwing off the PBS, and developing for 3-10 minutes by using a freshly prepared DAB developing solution. Hematoxylin counterstain for 25 seconds, PBS bluing for 30 seconds. Dehydration was carried out in sequence according to an alcohol gradient of 85% (3 min) -95% (3 min) -100% (3 min), finally xylene was cleared for 3 min, and neutral gum was mounted.
The immunohistochemical staining results were divided into: positive and negative. Positive expression must be on a specific antigenic site in cells and tissues to be considered positive. Under the condition that the tissue staining distribution is clear and the cell positioning is accurate, the staining result is further divided according to the difference of staining intensity, which is as follows:
1. the sample is weakly positive; marked "+";
2. the sample is moderately positive; marked "+";
3. the sample is highly positive; marked as "+ + +".
4. The sample was negative and marked "-".
Data statistics
1. Tumor tissue chip detection results:
the antibody PR (14B 3) and the commercially available antibody PR (1E 2) were simultaneously detected in 17 breast cancer tumor groups, and the detection results were compared.
The immunohistochemical results of PR were counted. The whole experimental process adopts a double-blind design, and the statistical results are as follows:
the result shows that the rabbit monoclonal antibody PR (14B 3) has accurate staining positioning, clear staining, no non-specific staining and clean background. In the immunohistochemical detection, the positive rate was comparable to that of the commercially available antibody, but the positive intensity was higher than that of the commercially available antibody. In 1 case of breast cancer, the control PR antibody showed significant background staining, whereas the PR antibody of the invention showed clear background and no non-specific staining. The specificity is higher, and the false positive result is effectively avoided.
FIG. 2 is a comparison of the immunohistochemical staining results of breast cancer (left is rabbit monoclonal antibody PR of the present invention, right is commercially available 1E 2).
2. Detection results of the normal tissue chip:
the normal tissue chip comprises 30 normal tissue samples, and the normal tissue samples are mainly selected from fresh and timely fixed operation specimens; each tissue included 3 different case samples. The 30 normal tissues include: brain, heart, cerebellum, esophagus, adrenal gland, stomach, ovary, small intestine, pancreas, colorectal, parathyroid, liver, pituitary, salivary gland, testis, kidney, thyroid, prostate, breast, uterus, spleen, bladder, tonsil, skeletal muscle, thymus (young child), skin, bone marrow, peripheral nerve, lung, mesothelial cells.
The rabbit monoclonal antibody PR (25C 4) and the commercially available PR antibody (commercially available 1E 2) are synchronously detected on a normal tissue chip, and the negative and positive detection results are consistent, which shows that the specificity of the antibody in the normal tissue is equivalent to that of the commercially available antibody.
FIG. 3 is a comparison of the results of combined immunohistological staining of endometrium (left rabbit mAb PR, right commercial PR).
It should be noted that, in this document, relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or terminal that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or terminal. Without further limitation, an element defined by the phrases "comprising 8230; \8230;" or "comprising 8230; \8230;" does not exclude additional elements from existing in a process, method, article, or terminal device that comprises the element. Further, herein, "greater than," "less than," "more than," and the like are understood to exclude the present numbers; the terms "above", "below", "within" and the like are to be understood as including the number.
It should be noted that, although the above embodiments have been described herein, the invention is not limited thereto. Therefore, based on the innovative concepts of the present invention, the technical solutions of the present invention can be directly or indirectly applied to other related technical fields by making changes and modifications to the embodiments described herein, or by using equivalent structures or equivalent processes performed in the content of the present specification and the attached drawings, which are included in the scope of the present invention.
Sequence listing
<110> Fuzhou mai New Biotechnology development Co., ltd
<120> anti-PR protein monoclonal antibody, preparation method and application thereof
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tcatcgcggt tcaaaggcag tggatctggg acagaattca ttctcaccat cagcggcgtg 300
gagtgtgccg atgctgccac ttactactgt caacagggtt ttagtagtaa tgttgataat 360
cttttcggcg gagggaccga ggtggtggtc agaggtgatc cagttgcacc tactgtcctc 420
atcttcccac cagctgctga tcaggtggca actggaacag tcaccatcgt gtgtgtggcg 480
aataaatact ttcccgatgt caccgtcacc tgggaggtgg atggcaccac ccaaacaact 540
ggcatcgaga acagtaaaac accgcagaat tctgcagatt gtacctacaa cctcagcagc 600
actctgacac tgaccagcac acagtacaac agccacaaag agtacacctg caaggtgacc 660
cagggcacga cctcagtcgt ccagagcttc aataggggtg actgttag 708
<210> 5
<211> 165
<212> PRT
<213> Artificial sequence (Artificial)
<400> 5
Met Glu Thr Gly Leu Arg Trp Leu Leu Leu Val Ala Val Leu Lys Gly
1 5 10 15
Val Gln Cys Gln Ser Val Glu Glu Ser Gly Gly Arg Leu Val Thr Pro
20 25 30
Gly Thr Ala Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser
35 40 45
Thr Tyr Tyr Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
50 55 60
Trp Ile Gly Ser Ile Ser Asn Gly Asp Thr Thr Tyr Tyr Ala Ser Trp
65 70 75 80
Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser Thr Thr Val Asp Leu
85 90 95
Ser Ile Thr Ser Pro Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Gly
100 105 110
Arg Ala Ile Tyr Gly Ala Cys Asp Leu Trp Gly Arg Gly Thr Leu Val
115 120 125
Thr Val Ser Ser Arg Gln Pro Lys Ala Pro Ser Val Phe Pro Leu Ala
130 135 140
Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr Leu Gly Cys Leu
145 150 155 160
Val Lys Gly Tyr Leu
165
<210> 6
<211> 235
<212> PRT
<213> Artificial sequence (Artificial)
<400> 6
Met Asp Thr Arg Ala Pro Thr Gln Leu Leu Gly Leu Leu Leu Leu Trp
1 5 10 15
Leu Pro Gly Ala Thr Phe Ala Ile Asp Met Thr Gln Thr Pro Ala Ser
20 25 30
Val Glu Val Ala Val Gly Asp Thr Val Thr Ile Lys Cys Gln Ala Ser
35 40 45
Gln Asn Ile Ser Asn Leu Leu Ala Trp Tyr Gln Gln Lys Ser Gly Gln
50 55 60
Pro Pro Lys Leu Leu Ile Tyr Leu Ala Ser Thr Leu Ala Ser Gly Val
65 70 75 80
Ser Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Phe Ile Leu Thr
85 90 95
Ile Ser Gly Val Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
Gly Phe Ser Ser Asn Val Asp Asn Leu Phe Gly Gly Gly Thr Glu Val
115 120 125
Val Val Arg Gly Asp Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro
130 135 140
Ala Ala Asp Gln Val Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala
145 150 155 160
Asn Lys Tyr Phe Pro Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr
165 170 175
Thr Gln Thr Thr Gly Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala
180 185 190
Asp Cys Thr Tyr Asn Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln
195 200 205
Tyr Asn Ser His Lys Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr
210 215 220
Ser Val Val Gln Ser Phe Asn Arg Gly Asp Cys
225 230 235
Claims (5)
1. The monoclonal antibody for resisting PR protein is characterized in that the amino acid sequence of the heavy chain variable region of the monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 5; the amino acid sequence of the monoclonal antibody light chain variable region is the amino acid sequence shown in SEQ ID NO. 6.
2. The monoclonal antibody according to claim 1, wherein the DNA sequence encoding the heavy chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No.3, and the DNA sequence encoding the light chain variable region of the monoclonal antibody is the nucleotide sequence shown in SEQ ID No. 4.
3. The monoclonal antibody of claim 1, wherein the monoclonal antibody specifically recognizes a PR protein.
4. The monoclonal antibody of claim 1, wherein the monoclonal antibody is a rabbit monoclonal antibody.
5. An immunohistochemical detection reagent for PR protein, which comprises the anti-PR protein monoclonal antibody according to any one of claims 1 to 4 as an active ingredient.
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| CN114874327B (en) * | 2022-06-21 | 2023-04-04 | 图凌(杭州)生物医药有限公司 | Monoclonal antibody for detecting progestational hormone receptor and preparation method and application thereof |
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