CN107266568A - Anti- FOXP3 protein monoclonal antibodies and application thereof - Google Patents

Abstract

The invention relates to the field of biotechnology, and discloses a hybridoma cell strain (preservation number is CGMCC No. 13823) and monoclonal antibody UMAB248 produced by the hybridoma cell strain. The present invention also relates to the application of monoclonal antibody UMAB248 in the preparation of immunoassay tools for detecting FOXP3 protein, the immunohistochemical kit containing monoclonal antibody UMAB248, and the preparation of monoclonal antibody UMAB248 in the kit for marking tumors Applications. The monoclonal antibody of the present invention can specifically bind to the FOXP3 protein without cross-reaction with other proteins in the cell, thus significantly improving the specificity, accuracy and reliability of the FOXP3 protein immune detection.

Description

Anti-FOXP3 protein monoclonal antibody and use thereof

technical field

The invention relates to the field of biotechnology, in particular to a monoclonal antibody UMAB248 that can specifically bind to FOXP3 protein, a cell line producing the monoclonal antibody, and a method and use of using the antibody for diagnosis.

Background technique

Forkhead-like transcription factor 3 (fork head box protein 3, FOXP3) is a member of the vertebrate forkhead-like transcription factor family, which was first reported by Brunkow et al. in 2001. It was confirmed as a specific marker of CD4+CD25+ regulatory T cells (regulatory T cell, Treg) in 2003. Highly expressed in CD4+CD25+Treg cells of the "active" mechanism (a tolerance mechanism mediated by a group of functionally suppressed cells) in the immune system, FOXP3+CD4+CD25+Treg cells in the tumor immune escape mechanism High expression of FOXP3 has been detected in melanoma, pancreatic cancer, gastric cancer, liver cancer, breast cancer, lung cancer and other tumors. And some studies have confirmed that the level of FOXP3 expression can be used as a very meaningful index to evaluate the treatment effect and whether there is recurrence or metastasis.

Clinically, immunohistochemistry (IHC) pathological experiments are commonly used to detect the expression of proteins in tumor cells. However, the core of IHC experiments is monoclonal antibodies that specifically bind proteins, and their performance directly determines the sensitivity and specificity of the entire detection. . Therefore, it is of great significance to develop a monoclonal antibody against FOXP3 protein with high binding specificity for IHC detection of FOXP3 expression level.

Contents of the invention

In view of this, the object of the present invention is to provide a monoclonal antibody binding to FOXP3 protein with high specificity and its application in the preparation of immunoassay tools for detecting FOXP3 protein.

The present invention provides a hybridoma cell strain, which is preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short), with a preservation date of April 6, 2017 and a preservation number of CGMCC No.13823.

The present invention also provides a monoclonal antibody UMAB248 specifically binding to the FOXP3 protein, which is produced by the above-mentioned hybridoma cell line.

The preparation method of the monoclonal antibody of the present invention is as follows:

(1) Construction of recombinant expression vector: according to the nucleotide sequence of FOXP3 ORF (the nucleotide sequence of FOXP3 ORF is shown in SEQ ID NO.1, 1296bp; the amino acid sequence of FOXP3 is shown in SEQ ID NO.2)

Design primers for PCR amplification of FOXP3 ORF 1bp to 1296bp sequence, introduce restriction endonuclease sites SgfI and MluI on both sides of the gene, insert expression vector pET23a-N-His, and construct FOXP3 amino acid sequence from 1st to 1st Recombinant expression plasmid pET23a-rFOXP3 at position 431; upstream amplification primer sequence, SEQ ID NO.3: CAC GCGATCGC ATGCCCAACCCCAGGCCTG downstream amplification primer sequence SEQ ID NO.4: ACCG ACGCGT TCAGGGGCCAGGTGTAGGGT

(2) Expression and purification of FOXP3 recombinant protein: transform the FOXP3 recombinant expression plasmid into E. Coli cells, lyse and centrifuge to obtain the supernatant, and purify with nickel column affinity chromatography to obtain purified FOXP3 recombinant protein;

(3) Screening and preparation of monoclonal antibodies: the above-mentioned purified FOXP3 recombinant protein was used to immunize Balb/c mice, the spleen cells of the mice were fused with SP2/0 cells, monoclonals were obtained by limiting dilution, and positive hybridization was screened by ELISA Tumor cells were obtained to obtain a hybridoma cell line that can secrete specific antibodies against FOXP3, which was named UMAB248, and the subtype was identified as IgG1; antibodies were prepared in serum-free medium, and FOXP3 monoclonal antibody UMAB248 was obtained by purification with affinity chromatography. The sensitivity and specificity of the monoclonal antibody were verified by Western Blot and immunohistochemical experiments respectively.

The invention also provides the application of the monoclonal antibody UMAB248 in the preparation of an immunodetection tool for detecting the FOXP3 protein.

Specifically, the immunoassay tool is a kit, chip or test paper.

In a specific embodiment, the present invention provides an immunohistochemical detection kit, including the above-mentioned monoclonal antibody UMAB248, which can detect the expression of FOXP3 in tissue cells.

The present invention also provides the application of the above-mentioned monoclonal antibody in the preparation of a kit for marking tumors. The tumor specifically refers to a tumor in which the proliferation of tumor cells is closely related to the expression of FOXP3, including but not limited to melanoma, pancreatic cancer, gastric cancer, liver cancer, breast cancer, and lung cancer.

Compared with the prior art, the present invention provides a hybridoma cell line (the preservation number is CGMCC No. 13823), and the monoclonal antibody UMAB248 produced by the hybridoma cell line. The present invention also provides the application of monoclonal antibody UMAB248 in the preparation of immunoassay tools for detecting FOXP3 protein, the immunohistochemical kit containing monoclonal antibody UMAB248, and the preparation of monoclonal antibody UMAB248 in the preparation of a kit for marking tumors in the application. The monoclonal antibody of the present invention can specifically bind to FOXP3 protein, truly reflect the expression level of FOXP3 protein in tumor cells, and can be applied to the detection of FOXP3 protein expression level in melanoma, pancreatic cancer, gastric cancer, liver cancer, breast cancer, and lung cancer.

deposit information

The classification and designation of the hybridoma cell line UMAB248 used for preservation is: mouse anti-human forkhead box protein P3 (FOXP3) monoclonal hybridoma cell line;

The full name of the depository unit: General Microbiology Center of China Committee for the Collection of Microbial Cultures;

Depository unit abbreviation: CGMCC;

Address of Preservation Unit: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing;

Deposit date: April 6, 2017;

Deposit number: CGMCC No.13823.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the design of the cloning site in Example 1, in which the shading part is the ORF region;

Figure 2 - Figure 8 shows the immunohistochemical results of formalin-fixed, paraffin-embedded breast, colon, gastric cancer, liver cancer, lung, lymph, and melanoma tissues in Example 4 (the primary antibody is the FOXP3 monoclonal antibody UMAB248) ;

Figure 9 shows the results of OriGene protein chip identification in Example 5 (the primary antibody is FOXP3 monoclonal antibody UMAB248, 1:100; the secondary antibody is DyLight649-conjugated AffiniPure Fragment Goat-anti-Mouse IgG, 1:400).

detailed description

The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.

Embodiment 1, the construction of FOXP3 recombinant expression plasmid

Using the plasmid RC217580 (including FOXP3ORF 4080bp) obtained from Aurui Dongyuan Biotechnology Co., Ltd. of the United States as a template, two primers were designed and introduced into restriction sites SgfI and MluI respectively, and cloned into the expression vector pET23a-N-His to establish FOXP3 Recombinant expression plasmid. The design of the cloning site is shown in Figure 1.

Embodiment 2, expression and purification of FOXP3 recombinant protein

1. Transform E.coli cells: Thaw 100ul competent cells on ice, add plasmid DNA and mix gently, heat shock at 42°C for 90s after ice bath for 30 minutes, and then continue to ice bath for 1-2 minutes. Add 500ul of fresh anti-antibiotic-free LB medium into the ultra-clean bench, incubate on a shaker at 37°C for 45min, and then take an appropriate amount of bacterial solution and evenly spread it on a plate containing antibiotics, and place the culture plate upside down in a constant temperature incubator at 37°C for overnight cultivation.

2. Cell lysis: Pick a single clone in a fresh medium, culture at 37°C and 200 rpm until the OD value reaches 0.4-0.6, add IPTG (final concentration 1 mM) to induce culture for 7 hours. The cells were collected by centrifugation, and then the cells were resuspended in lysis buffer, ultrasonically disrupted for 20 min, and then centrifuged at 12000 rpm for 20 min at 4°C to collect the supernatant. Take a small amount of supernatant for WB identification with anti-His antibody.

3. Nickel column affinity chromatography column purification: equilibrate the nickel column with buffer, filter the supernatant with a 0.45 μm filter membrane, load the sample and collect the effluent, rinse with buffer to remove unbound protein, and finally use The eluate of imidazole was eluted, collected and identified by SDS-PAGE, and the eluted proteins that met the requirements were combined and added with 10% glycerol, and the purified recombinant FOXP3 protein was identified by SDS-PAGE.

Example 3, Preparation and Screening of FOXP3 Monoclonal Antibody

The recombinantly produced purified FOXP3 protein fragment was used to immunize Balb/c mice (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) according to standard methods. The specific method is as follows:

1. Animal immunization: The purified FOXP3 antigen was emulsified with complete Freund's adjuvant, and immunized 6-8 week-old Balb/c mice by subcutaneous or intraperitoneal injection. For the second immunization, it was emulsified with incomplete Freund's adjuvant, and the immunization dose was 50 μg per mouse. After the two immunizations, the tail blood was taken to measure the serum titer by ELISA gradient dilution; according to the results, it was determined whether to boost the immunization, and the mouse with the highest antibody titer was selected for cell fusion.

2. Cell fusion: myeloma cells use sp2/0 derived from Balb/c, and they are in the logarithmic growth phase during fusion; the spleen of immunized mice is taken to make a single-cell suspension of lymphocytes; mouse spleen lymphocytes and myeloma Cells were mixed at a ratio of 1:5-1:10, 1 mL of 50% PEG (PH 8.0) at 37°C was added dropwise, incomplete medium and the rest of the stop solution were added, the supernatant was discarded by centrifugation, and HAT medium was added to suspend and mix evenly. To 50mL, put into 3.5cm Petri dishes, put in a wet box, and place in a 37°C, 5% _CO2 constant temperature incubator for cultivation.

3. Screening and cloning: select cell clones within 7-10 days of fusion, and use FOXP3 to purify the recombinant protein for ELISA test. Label the cell line number. Perform limiting dilution on the cells in the positive wells, measure the ELISA value 5-6 days after each limiting dilution, and pick the monoclonal wells with higher OD280 positive values for limiting dilution until the ELISA test results for the entire plate of the 96-well plate are positive. Pick the monoclonal clone with high positive value. Its corresponding fusion plate cell line is UMAB248.

4. Preparation and purification of ascites monoclonal antibody: male Balb/c mice aged 10-12 weeks were intraperitoneally injected with 0.5ml pristine, and one week later, each mouse was intraperitoneally injected with monoclonal cells washed and resuspended in PBS with a 1mL syringe Suspension, the amount of cells used is 5×10 ⁶ /mouse, and 2 mice are used for each antibody strain. Ascites was collected after mouse ascites accumulated, and the supernatant was obtained by centrifugation. The ascites was purified by affinity chromatography, and the corresponding column material was selected according to the antibody subtype. The monoclonal antibody produced by the cell line UMAB248 was IgG1, and protein G was used for purification. The concentration of the purified monoclonal antibody was measured, detected by WB, aliquoted, and frozen at -20°C.

Example 4, Immunohistochemical detection of monoclonal antibody UMAB248 as primary antibody

(1) Experimental method:

1. Formalin-fixed human lymph node tissue and human tonsil tissue blocks were taken for paraffin embedding, and sliced with a Finesse tissue slicer with a tissue thickness of 6 μm.

2. Dewaxing and hydration: Analytical pure xylene 3×10min, absolute ethanol 3×10min, 95% ethanol 5min, 85% ethanol 5min, 75% ethanol 5min, deionized water soaking 3min×3 times

3. Add antigen retrieval solution (0.01M, pH 6.0 sodium citrate buffer solution) to autoclave for 3 minutes. When the temperature of the autoclave drops to about 90°C, turn on the autoclave, take out the specimen, and cool it down to room temperature naturally. Soak in deionized water for 3min x 3 times.

4. Use 3% hydrogen peroxide to inactivate tissue endogenous peroxidase, and let it stand at room temperature for 10 minutes. Soak in deionized water for 5min x 3 times.

5. Add blocking solution (PBS+5% skimmed milk powder+5% normal goat serum), and incubate at 37°C for 60min.

6. Remove the blocking solution, do not rinse, add FOXP3 monoclonal antibody (UMAB248), dilution ratio: 1:150, use blocking solution for dilution. Place in a humid box and incubate at 37°C for 60min. Wash twice with PBST (0.1% Tween-20), 5 min each time. Wash once with PBST (0.02% Tween-20), 5 min each time.

7. Add reagent 1 of Polink-kit 2 (Catlog No. D37-15) dropwise, and incubate at 37°C for 10-20 minutes. Wash 3 times with PBS, 5 min each time. Add reagent 2 of the Polink-2 kit (Catlog No. D37-15) dropwise, incubate at 37°C for 10-20 minutes, and wash with PBS 3 times, 5 minutes each time.

8. Apply DAB solution (Zhongshan Jinqiao ZLI-9019) to develop color, and develop color for 3-10 minutes. Wash with distilled water.

9. Counterstain the nuclei with hematoxylin for 2 minutes, rinse with distilled water, and differentiate with 1% hydrochloric acid. Rinse with distilled water 3 times and let stand at room temperature for 1 min.

10. Dehydration and transparency: 75% ethanol for 5 min, 100% ethanol for 5 min x 3 times, 85% ethanol for 5 min, 95% ethanol for 5 min, 100% ethanol for 3 x 5 min, xylene for 3 x 5 min, and neutral gum for sealing.

11. Microscopic examination, see Figure 2-7.

(2) Experimental results:

From the results in Figure 2-Figure 8, it can be seen that some specific nuclear staining of infiltrating lymphocytes can be seen in breast, colon, gastric cancer, liver cancer, lung, lymphatic, and melanoma tissues. The results are consistent with the localization and tissue expression specificity of FOXP3, indicating that the monoclonal antibody UMAB248 can be used to detect the level of FOXP3 protein by immunohistochemistry.

Example 5, Specific detection of monoclonal antibody UMAB248

The OriGene High Density Protein Chip contains 10,000 HEK293T cell protein overexpression lysates, each protein lysate has two copies of replicates on the chip. Protein lysates were blotted on nitrocellulose membranes. The location of each protein lysate can be accurately positioned through the Excel file. The protein on the protein chip is divided into 40 sub-matrixes, and there are some references on each sub-matrix. Through the reference, the protein content on each chip spot can be quantified, the repeatability of each immune reaction data can be monitored, and the direction of positive signals can be located. . The following is the experimental method for UMAB248 antibody identification experiment using OriGene protein (OriGene Cat PA100001) chip:

1. Put a protein chip in a 50mL centrifuge tube, soak the chip with 40mL deionized water, place on a shaker, and mix at room temperature for 30 minutes. Discard the deionized water and equilibrate the chip with 10 mL of PBST. Incubate at room temperature for 10 minutes.

2. Add 40 mL of 5% skimmed milk (diluted with PBST) to a 50 mL centrifuge tube, place on a shaker, and block at room temperature for 30 minutes.

3. Dilute the primary antibody UMAB248 with blocking solution (5% skimmed milk), and the dilution ratio is listed as 1:100.

4. Paste the clean parafilm on the laboratory bench, and drop 250-300 μL of primary antibody on the parafilm.

5. Pull the protein chip out of the blocking solution, put the NC membrane side of the protein chip down, touch the antibody from one side of the chip, and slide it down slowly. Depending on the surface tension of the liquid, the antibody will slowly infiltrate the NC membrane of the chip until the whole Zhang NC membranes were soaked in the primary antibody solution. Avoid generating air bubbles during the whole operation. Move the chip to a 4°C environment, let it stand, and incubate with the primary antibody overnight. Cover the chip with a Petri dish lid with a wet paper towel attached to it to prevent antibody evaporation from prolonged incubation.

6. The next day, move the chip to a 50mL centrifuge tube, rinse the chip twice with PBST to remove excess antibody. The chip was washed with 40 mL of PBST (0.1% Tween-20), placed on a shaker, mixed evenly, and washed three times, 5 min each time.

7. Dilute the secondary antibody DyLight649-conjugated AffiniPureFragment Goat-anti-Mouse IgG with blocking solution (5% skimmed milk), and the dilution ratio is 1:400.

8. Follow steps 4 and 5 above to incubate with the secondary antibody. Incubate for 1 hour at room temperature. Cover the chip with aluminum foil to prevent signal bleaching.

9. Follow step 6 above to wash the chip with PBST.

10. Rinse the chip with deionized water to remove residual salt and denaturant.

11. Dry the chip at room temperature to ensure that the chip is completely dry.

12. Use a chip scanner to read the fluorescent signal.

13. Determine the direction of the chip and the position of the positive signal according to BSA-Cy3 and BSA-Cy5.

14. Find out the corresponding protein lysate ID according to the positive signal site, and find the corresponding protein name, NCBI accession number, protein ID, protein size and other information according to the lysate database information.

The results are shown in Figure 9. Monoclonal antibody UMAB248 can specifically recognize FOXP3 protein on the OriGene protein chip, indicating that monoclonal antibody UMAB248 has better specificity.

SEQUENCE LISTING

<110> Wuxi Aorui Dongyuan Biotechnology Co., Ltd.

<120> Anti-FOXP3 protein monoclonal antibody and use thereof

<210> 1

<211>2961

<212>DNA

<213> Artificial sequence

<400> 1

ORIGIN

1 ATGCCCAACC CCAGGCCTGG CAAGCCTCTCG GCCCCTTCCT TGGCCCTTGG CCCATCCCCA

61 GGAGCCTCGC CCAGCTGGAG GGCTGCACCC AAAGCCTCAG ACCTGCTGGG GGCCCGGGGC

121 CCAGGGGGAA CCTTCCAGGG CCGAGATCTT CGAGGCGGGG CCCATGCCTC CTCTTCTTCC

181 TTGAACCCCA TGCCACCATC GCAGCTGCAG CTGCCCACAC TGCCCCTAGT CATGGTGGCA

241 CCCTCCGGGG CACGGCTGGG CCCCTTGCCC CACTTACAGG CACTCCTCCA GGACAGGCCA

301 CATTTCATGC ACCAGCTCTC AACGGTGGAT GCCCACGCCC GGACCCCCTGT GCTGCAGGTG

361 CACCCCCTGG AGAGCCCAGC CATGATCAGC CTCACACCAC CCACCACCGC CACTGGGGTC

421 TTCTCCCTCA AGGCCCGGCC TGGCCTCCCA CCTGGGATCA ACGTGGCCAG CCTGGAATGG

481 GTGTCCAGGG AGCCGGCACT GCTCTGCACC TTCCCAAAATC CCAGTGCACC CAGGAAGGAC

541 AGCACCCTTT CGGCTGTGCC CCAGAGCTCC TACCCACTGC TGGCAAATGG TGTCTGCAAG

601 TGGCCCGGAT GTGAGAAGGT CTTCGAAGAG CCAGAGGACT TCCTCAAGCA CTGCCAGGCG

661 GACCATCTTC TGGATGAGAA GGGCAGGGCA CAATGTCTCC TCCAGAGAGA GATGGTACAG

721 TCTCTGGAGC AGCAGCTGGT GCTGGAGAAG GAGAAGCTGA GTGCCATGCA GGCCCACCTG

781 GCTGGGAAAA TGGCACTGAC CAAGGCTTCA TCTGTGGCAT CATCCGACAA GGGCTCCTGC

841 TGCATCGTAG CTGCTGGCAG CCAAGGCCCT GTCGTCCCAG CCTGGTCTGG CCCCCGGGAG

901 GCCCCTGACA GCCTGTTTGC TGTCCGGAGG CACCTGTGGG GTAGCCATGG AAACAGCACA

961 TTCCCAGAGT TCCTCCACAA CATGGACTAC TTCAAAGTTCC ACAACATGCG ACCCCCTTTC

1021 ACCTACGCCA CGCTCATCCG CTGGGCCATC CTGGAGGCTC CAGAGAAGCA GCGGACACTC

1081 AATGAGATCT ACCACTGGTT CACACGCATG TTTGCCTTCT TCAGAAACCA TCCTGCCACC

1141 TGGAAGAACG CCATCCGCCA CAACCTGAGT CTGCACAAGT GCTTTGTGCG GGTGGAGAGC

1201 GAGAAGGGGG CTGTGTGGAC CGTGGATGAG CTGGAGTTCC GCAAGAAACG GAGCCAGAGG

1261 CCCAGCAGGT GTTCCAACCC TACACCTGGC CCCTGA

//

<210> 2

<211>986

<212> PRT

<213> Artificial sequence

<400> 2

ORIGIN

1 MPNPRPGKPS APSLALGPSP GASSPWRAAP KASDLLGARG PGGTFQGRDL RGGAHASSSS

61 LNPMPPSQLQ LPTLPLVMVA PSGARLGPLP HLQALLQDRP HFMHQLSTVD AHARTPVLQV

121 HPLESPAMIS LTPPTTATGV FSLKARPGLP PGINVASLEW VSREPALLCT FPNPSAPRKD

181 STLSAVPQSS YPLLANGVCK WPGCEKVFEE PEDFLKHCQA DHLLDEKGRA QCLLQREMVQ

241 SLEQQLVLEK EKLSAMQAHL AGKMALTKAS SVASSDKGSC CIVAAGSQGP VVPAWSGPRE

301 APDSLFAVRR HLWGSHGNST FPEFLHNMDY FKFHNMRPPF TYATLIRWAI LEAPEKQRTL

361 NEIYHWFTRM FAFFRNHPAT WKNAIRHNLS LHKCFVRVES EKGAVWTVDE LEFRKKRSQR

421 PSRCSNPTPGP

Claims (7)

1. a kind of monoclonal antibody UMAB248 of specific binding FOXP3 albumen, is CGMCC No.13823's by deposit number Hybridoma cell strain is produced.

2. a kind of hybridoma cell strain, its deposit number is CGMCC No.13823.

3. monoclonal antibody as claimed in claim 1 is being prepared for detecting answering in the immune detection instrument of FOXP3 albumen With.

4. application according to claim 3, the immune detection instrument is kit, chip or test paper.

5. a kind of immunologic combined detection reagent kit, including the monoclonal antibody described in claim 1.

6. application of the monoclonal antibody as claimed in claim 1 in the kit for tagged tissue cell is prepared.

7. application according to claim 6, the histocyte is melanoma, cancer of pancreas, stomach cancer, liver cancer, breast cancer, lung Cancer.