Monoclonal antibody for specific detection of MMP12 protein and its application

Abstract

The present invention belongs to the field of biological detection, and discloses a monoclonal antibody for specific detection of MMP12 protein and its application. The monoclonal antibody for specific detection of MMP12 protein comprises a heavy chain variable region as shown in SEQ ID NO.7 and a light chain variable region as shown in SEQ ID NO.8. The monoclonal antibody for specific detection of MMP12 protein provided by the present invention has good biological activity, strong specificity, high titer, high sensitivity, and no cross-reactivity with other antigens, and has broad application prospects. The present invention also discloses a kit for specific detection of MMP12 protein, comprising the above-mentioned monoclonal antibody for specific detection of MMP12 protein, which can be used for specific detection of MMP12 protein in serum or whole blood, and can be used as an auxiliary diagnostic means for cancers with high expression of MMP12 protein, and has the advantages of simple detection method, high accuracy and low cost.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a monoclonal antibody for specifically detecting MMP12 protein and application thereof. Background Art

[0002] Matrix metalloproteinases (MMPs) are proteolytic enzymes that hydrolyze the extracellular matrix and can degrade almost all protein components in the extracellular matrix (ECM). The induction of MMP activity contributes to the disintegration of intercellular connections and the degradation of ECM, and plays a role in many biological processes, such as fetal development, tissue remodeling and growth, wound repair, tissue defense mechanisms and immune responses, all of which require proteolytic activity. However, in pathological tissues, this function of MMPs directly helps tumor cells overcome physical limitations, destroy local tissue structures to allow tumor growth, and destroy the basement membrane to promote its invasion and metastasis.

[0003] MMP12 is overexpressed in cervical scraping cells of cervical precursor lesions and contributes to the invasion and metastasis of cervical cancer cells. MMP12 shows significantly higher expression in serum specimens of patients with colon cancer compared with healthy control serum groups. In addition, MMP12 polymorphisms are associated with a high risk of disseminated colorectal cancer (CRC) and are also important prognostic markers for overall survival and tumor recurrence in patients with hepatocellular carcinoma (HCC) after liver resection, non-invasive oral cancer, breast cancer, and esophageal cancer. Functionally, increased expression of MMP12 promotes gastric cancer cell migration and invasion through p38 MAPK-dependent NF-κB activation. Myeloid cell-autonomous defects induced by MMP12 lead to aberrant myelopoiesis, immunosuppression, and tumorigenesis in LAC. MMP-12 promotes glioma invasiveness by interacting with tenascin-C.

[0004] These studies have shown that MMP12 protein may be related to the occurrence and progression of a variety of malignant tumors and is expected to become a new tumor marker. Therefore, a kit for the specific detection of MMP12 protein can achieve simple, rapid, and sensitive nonspecific detection of a variety of cancers, as a supplementary means for early detection of cancer in medicine. Currently, there is no product on the market that detects tumors for MMP12 protein. Summary of the invention

[0005] In order to overcome the deficiencies of the prior art, one of the purposes of the present invention is to provide a monoclonal antibody for specific detection of MMP12 protein, which has strong specificity and affinity, high sensitivity, and can be applied to Western blot, ELISA, and immunofluorescence detection.

[0006] The second purpose of the present invention is to provide the application of the monoclonal antibody.

[0007] One of the purposes of the present invention is achieved by the following technical solution:

[0008] A monoclonal antibody for specifically detecting MMP12 protein comprises a heavy chain variable region as shown in SEQ ID NO.7 and a light chain variable region as shown in SEQ ID NO.8.

[0009] Preferably, the heavy chain variable region of the monoclonal antibody specifically detecting MMP12 protein comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO.7.

[0010] Preferably, the light chain variable region of the monoclonal antibody specifically detecting MMP12 protein comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity with SEQ ID NO.8.

[0011] The second object of the present invention is achieved by adopting the following technical solution:

[0012] The monoclonal antibody for specifically detecting MMP12 protein is used in preparing a kit for specifically detecting MMP12 protein.

[0013] Furthermore, the kit for specifically detecting MMP12 protein includes a magnetic particle component, an acridinium ester labeling component, an acridinium ester luminescent substrate solution, and an MMP12 protein calibrator.

[0014] Preferably, the preparation method of the magnetic particle component is as follows:

[0015] (1) Activate the carboxyl groups on the surface of carboxyl magnetic beads using EDC Solution and NHS Solution;

[0016] (2) mixing the monoclonal antibody with the carboxyl magnetic beads activated in step (1), and coupling at 25° C. for 2 hours to obtain a monoclonal antibody specifically detecting MMP12 protein coated with carboxyl magnetic beads;

[0017] (3) Block the sites on the surface of carboxyl magnetic beads that are not bound to antibodies using a blocking solution;

[0018] (4) The magnetic beads prepared in step (3) are dispersed and cleaned by ultrasound, and diluted with magnetic bead storage solution for later use.

[0019] Preferably, the magnetic bead storage solution in step (4) is composed of: 6% Tris-HCl buffer, 0.2% Tween-20, 0.85% NaCl, 2% BSA, 0.2% Proclin-300, and the rest is ultrapure water.

[0020] Preferably, the preparation method of the acridinium ester labeling component is:

[0021] The goat anti-mouse IgG antibody and the acridinium ester working solution were mixed and reacted at room temperature in the dark for 1 hour; the labeling termination buffer was added and mixed at room temperature; and the acridinium ester-labeled goat anti-mouse IgG antibody was purified by a desalting column to obtain the acridinium ester-labeled goat anti-mouse IgG antibody.

[0022] Preferably, the acridinium ester labeling termination buffer is composed of a mixed aqueous solution of lysine and NaHCO3, wherein the concentration of the lysine is 0.55 mol / L and the concentration of the NaHCO3 is 0.2 mol / L.

[0023] Preferably, the acridinium ester luminescent substrate solution is a 0.05% H2O2 and 100 mmol / L NaOH solution.

[0024] Preferably, the sample detected by the kit is whole blood or serum.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The monoclonal antibody for specific detection of MMP12 protein provided by the present invention has good biological activity, strong specificity, high titer, high sensitivity, and no cross-reactivity with other antigens, and has broad application prospects. The present invention also discloses a kit for specific detection of MMP12 protein, comprising the above-mentioned monoclonal antibody for specific detection of MMP12 protein, which can be used for specific detection of MMP12 protein in serum or whole blood, and can be used as an auxiliary diagnostic means for cancers with high expression of MMP12 protein, and has the advantages of simple detection method, high accuracy and low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic diagram of the structure of the recombinant vector for producing MMP12 protein in the present invention;

[0028] Figure 2 The electrophoresis diagrams of proteins before and after purification of ascites containing MMP12-2 monoclonal antibody are shown;

[0029] Figure 3 This is a diagram of the specific identification results of the monoclonal antibody;

[0030] Figure 4 This is the label curve of the kit for specific detection of MMP12 protein. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific implementation methods. It should be noted that, under the premise of no conflict, the various embodiments or technical features described below can be arbitrarily combined to form a new embodiment.

[0032] Example 1

[0033] Preparation of MMP12 protein:

[0034] (1) Construction of expression vector: First, the codon preference of E. coli expression was optimized based on the amino acid sequence of MMP12 protein (SEQ ID NO.1) using the ExpOptimizer online codon optimization tool of Newpro Biotechnology. NdeI and XhoI restriction sites were added to both ends of the target fragment, and the gene fragment was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The sequence is shown in SEQ ID NO.2.

[0035] SEQ ID NO.1

[0036] MKFLLILLLQATASGALPLNSSTSLEKNNVLFGERYLEKFYGLEINKLPVTKMKYSGNLMKEKIQEMQHFLGLKVTGQLDTSTLEMMHAPRCGVPDVHHFREMPGGPVWRKHYITYR INNYTPDMNREDVDYAIRKAFQVWSNVTPLKFSKINTGMADILVVFARGAHGDFHAFDGKGGILAHAFGPGSGIGGDAHFDEDEFWTTHSGGTNLFLTAVHEIGHSLGLGHSSDPKAV MFPTYKYVDINTFRLSADDIRGIQSLYGDPKENQRLPNPDNSEEPALCDPNLSFDAVTTVGNKIFFFKDRFFWLKVSERPKTSVNLISSLWPTLPSGIEAAYEIEARNQVFLFKDDKY WLISNLRPEPNYPKSIHSFGFPNFVKKIDAAVFNPRFYRTYFFVDNQYWRYDERRQMMDPGYPKLITKNFQGIGPKIDAVFYSKNKYYYFFQGSNQFEYDFLLQRITKTLKSNSWFGC

[0037] SEQ ID NO.2

[0038]

[0039] NdeI-MMP12-F: CGGCAGCCATATGATGAAATTCCTGCTG (SEQ ID NO.3)

[0040] XhoI-MMP12-R:GGTGGTGCTCGAGACAGCCGAA (SEQ ID NO.4)

[0041] The total RNA of NCI-H1395 cells was extracted by Trizol method, and the target fragment MMP12 was obtained by PCR amplification using its reverse transcribed cDNA as a template. The pET-28a(+) expression vector plasmid was double-digested with NdeI and XhoI, and the two fragments were connected using a seamless cloning kit at 16°C for 6 hours after gel recovery. The recombinant vector structure is as follows Figure 1 As shown. The ligation product was added to the competent cells of E. coli DH5α, ice bathed for 30 minutes, reacted at 42℃ for 1min 30s, ice bathed for 2min, and 1mL LB liquid medium was added, incubated at 250rpm and 37℃ for 5h. The bacteria were collected by centrifugation at 4000rpm, 800μL of supernatant was removed and resuspended, coated with kanamycin-resistant LB plates, and cultured at 37℃ overnight. A single colony was picked into 5mL LB liquid medium, cultured at 250rpm and 37℃ for 12h. After the positive clones were identified by colony PCR, sequencing analysis was performed. The positive plasmid with correct sequencing was named pET28a+MMP12, and the E. coli with recombinant MMP12 gene was named DH5α-MMP12.

[0042] (2) Induced expression and purification of MMP12 protein: The DH5α-MMP12 bacteria constructed in step (1) were cultured in 5 mL LB medium at 37°C overnight, inoculated with 2% of fresh TB medium, cultured at 37°C until OD600 reached 0.8, and then induced at 16°C for 20 h. Finally, the bacteria were collected by centrifugation at 8000 rpm for 20 min. The protein was extracted using a bacterial total protein extraction kit (purchased from Sangon Biotech Co., Ltd., catalog number C600596), and Ni was added. 2+ Purification by resin affinity chromatography.

[0043] Example 2

[0044] Produce mouse anti-MMP12 protein monoclonal antibody:

[0045] (1) Immunization of mice: The MMP12 protein prepared in Example 1 was used as an immunogen, emulsified with an equal volume of complete Freund's adjuvant (CFA), and 20 μL / mouse was used to immunize 30 female BALB / c mice aged 6-8 weeks, and injected at four locations on the back of the mice. The second immunization was performed two weeks after the first immunization, and each subsequent immunization was separated by two weeks. In the subsequent subcutaneous injection, the protein was emulsified with an equal volume of incomplete Freund's adjuvant (IFA). After the third injection, the serum was collected and the mouse serum antibody titer was analyzed using ELISA to evaluate the immune effect. The serum titer was qualified when it reached 1:5000 or above, and booster immunization was performed three days before cell fusion.

[0046] (2) Hybridoma cells: Mice were killed by carbon dioxide gas, and the spleen was removed aseptically, squeezed and ground in a plate to prepare a spleen cell suspension. Splenocytes were selected by CD3ε microbeads and anti-mouse IgM microbeads, and fused with Sp2 / 0 myeloma cells using PEG-1500 according to conventional methods. The cells were then inoculated in a 96-well plate with hypoxanthine-aminopterin-thymidine (HAT) medium. When the cell clones to be fused grew to cover 1 / 2 of the bottom of the plate well, the supernatant of the hybridoma cell culture well was detected by indirect ELISA. A total of 10 hybridoma cell lines that stably secreted monoclonal antibodies against human MMP12 protein were obtained, and the clone numbers were named MMP12-1 to MMP12-10. After three subcloning and ELISA screenings, the hybridoma cell with the strongest sensitivity and specificity, clone number MMP12-2, was selected, and the antibody secreted by it was called MMP12-2 monoclonal antibody.

[0047] (3) Antibody preparation: 1×10 6 The hybridoma cells of MMP12-2 were inoculated into the peritoneal cavity of BALB / c female mice sensitized with paraffin oil. The hybridoma cells were grown in the peritoneal cavity of the mice to produce monoclonal antibodies. After 10 days, the ascites was collected and centrifuged. The monoclonal antibodies were purified using the Protein A / G purification system of Thermo Company. The purity of the monoclonal antibodies was analyzed by Coomassie Brilliant Blue staining after 15% SDS-PAGE electrophoresis. The results are shown in Figure 2 In the figure, M is a marker, 1 is the ascites containing MMP12-2 monoclonal antibody before purification, and 2 is the ascites containing MMP12-2 after purification. Lane 1 has many miscellaneous bands, and lane 2 mainly has two bands of the heavy chain and light chain of the antibody, indicating that the purity of the antibody is significantly improved after purification. The concentration of the antibody was detected by the BCA method and adjusted to 10 mg / mL for standby use.

[0048] Example 3

[0049] Preparation of a specific MMP12 protein detection kit:

[0050] Preparation of magnetic microparticle components: The carboxyl magnetic bead coupling kit was purchased from Sangon Biotech Co., Ltd., catalog number D601013-0001. Take 100μL of magnetic bead resuspension solution into a clean centrifuge tube, remove the supernatant by magnetic separation, and resuspend with 500μL MEST Solution. Add freshly prepared 100μL EDC Solution and 100μL NHS Solution, vortex to fully suspend the magnetic beads, activate the carboxyl groups on the surface of the magnetic beads, and activate at 25°C for 45 minutes. After activation, place on a magnetic stand for separation for 10 seconds and remove the supernatant. Add 100μL MMP12-2 monoclonal antibody, mix gently, and couple at 25°C for 2 hours. Add 500μL Locking Solution to resuspend the magnetic beads, and react overnight at 4°C on a side-swinging shaker to block the unbound sites on the surface of the magnetic beads. The magnetic beads were dispersed by ultrasound, the supernatant was removed by magnetic force, and the beads were washed three times. Magnetic bead storage solution (6% Tris, 0.2% Tween-20, 0.85% NaCl, 2% BSA, 0.2% Proclin-300, the solvent was ultrapure water) was added to dilute the magnetic beads to a concentration of 0.23 mg / mL for later use.

[0051] Preparation of acridinium ester labeled components: Take 10 μL diluted acridinium ester mother solution (2.5 mg / mL) (purchased from Dalian Meilun Biotechnology Co., Ltd., catalog number MT0013-2) and add 90 μL anhydrous DMSO to make acridinium ester working solution (0.25 mg / mL). Use 0.2M NaHCO3 solution to dilute 50 μg of goat anti-mouse IgG antibody (purchased from Abcam, catalog number ab6708) to 300 μL, add 10 μL acridinium ester working solution. Wrap with tin foil and label at room temperature for 1 hour. Add 100 μL labeling termination buffer (0.55 mol / L lysine, 0.2 mol / L NaHCO3 solution) and mix at room temperature for 30 minutes. Purify with a desalting column, collect acridinium ester labeled goat anti-mouse IgG antibody and detect the concentration.

[0052] Acridinium ester luminescent substrate solution: Luminescent substrate solution A is 0.05% H2O2, and luminescent substrate solution B is 100mmol / L NaOH.

[0053] MMP12 protein calibrator: The MMP12 protein prepared in Example 1 was diluted to 0, 1, 5, 10, 50, 100, 200, 500, and 1000 pg / mL respectively with antigen diluent (1×PBS containing 1% BSA).

[0054] The magnetic particle component, the acridinium ester labeling component, the acridinium ester luminescent substrate solution and the MMP12 protein calibrator together constitute a kit for specifically detecting the MMP12 protein.

[0055] Test Example 1

[0056] MMP12-2 monoclonal antibody sequence:

[0057] The gene sequence encoding the antibody variable region was amplified using universal primers: forward 5'-AAGACAGCTATCGCGATT-3' (SEQ ID NO.5) and reverse 5'-GCCCCCTTATTAGCGTTT-3' (SEQ ID NO.6). The sequencing results were searched and analyzed using DNAMAN and the database to obtain the amino acid sequence encoding the MMP12-2 monoclonal antibody. The heavy chain variable region sequence is shown in SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.8.

[0058] SEQ ID NO.7

[0059] EVQLQQSGCELIRPGVSVEMSCKASAYSWTSGYKEDWVKQKPANVLEWIGYINYDTE FKKASLTSDRTNSTAYMELSSLTSEDSAVYYCTRNPEGDMDFDYNYILWGQVTQAGVTS

[0060] SEQ ID NO.8

[0061] DIVMNTSCSSLAVTAGQEVTVSCKSSQNNWYQQKPGQSPKLLVYRSFLTGAGVPDRFI GSGSGTDYSATITTVNAEDLADYFCQLYGTARPASLQTAPFAVGTKLEVR

[0062] Test Example 2

[0063] Specificity identification of MMP12-2 monoclonal antibody:

[0064] The MMP12 recombinant protein and BSA protein were mixed with the loading buffer in a ratio of 3:1 and denatured, and then SDS-PAGE electrophoresis was performed. The gel was stained and decolorized to observe the electrophoresis results. The decolorized gel was semi-dry transferred to a nitrocellulose membrane, and the membrane was blocked with blocking solution at 37°C for 60 minutes, and the monoclonal antibody prepared in Example 2 was added for reaction. The membrane was washed to remove unbound antibodies. Anti-mouse IgG secondary antibody labeled with alkaline phosphatase was added and shaken at 37°C for 40 minutes. The membrane was washed to remove unbound secondary antibody. BCIP / NBT color development solution was used for color development. The results are shown in Figure 2. Figure 3 As shown, it shows that the MMP12-2 monoclonal antibody has good specificity.

[0065] Test Example 3

[0066] Determination of the potency of MMP12-2 monoclonal antibody:

[0067] Take 100 μL MMP12 protein, coat with coating buffer at 4°C overnight, wash the plate with PBST, and block with 5% skim milk for 2h. Discard the liquid in the well, wash the plate 3 times with PBST, take the monoclonal antibody prepared in Example 2 and dilute it with PBS gradient and add it to the well of the ELISA plate, use the non-immunized mouse serum dilution as a blank control, react for 45min, wash the plate and add 100 μL HRP enzyme-labeled goat anti-mouse IgG antibody (1:5000 dilution) and incubate for 45min. Discard the liquid in the well, wash the plate with PBST, add 100 μL / well of TMB colorimetric solution, color for 7min in dark, add 2mol / L sulfuric acid to terminate the reaction, place in an ELISA instrument, and measure its OD value at a wavelength of 450nm. Use non-immunized mouse serum (1:1000 dilution) as a blank control. The results are shown in Table 1.

[0068] Table 1

[0069] Dilution multiple 1000 4000 16000 64000 256000 1024000 4096000 Blank control Absorbance 3.42 2.73 1.94 1.31 1.26 1.08 0.61 0.35

[0070] When the titer test result is OD 稀释 / OD 空白 When the value is greater than 2.1, it is positive. From the results in Table 1, it can be seen that the titer of the MMP12-2 monoclonal antibody has reached more than 1:1024000, indicating that the monoclonal antibody has good activity and can be used in subsequent experiments.

[0071] Test Example 4

[0072] Use of the kit:

[0073] The kit containing the MMP12-2 monoclonal antibody prepared in Example 3 was used to determine the content of MMP12 protein in human blood. A total of 15 healthy volunteers were selected, and their serum and whole blood were taken as samples, and 3 biological replicates were performed for each sample. 50 μL of sample and 50 μL of MMP12-2 antibody coated with carboxyl magnetic beads were added to a 96-well plate, shaken and mixed, reacted at 37°C for 15 minutes, magnetically separated after the reaction, and washed 3 times with 150 μL of washing solution; 50 μL of acridinium ester-labeled anti-mouse IgG antibody was added, shaken and mixed, reacted at 37°C for 10 minutes, magnetically separated after the reaction, and washed 3 times with 150 μL of washing solution. 50 μL of luminescent substrate solution A and B were added, shaken and mixed, and the chemiluminescence intensity was immediately detected using a fluorescence meter.

[0074] The calibration material was tested three times in the same way, and the standard curve was drawn with the concentration value as the horizontal axis and the average luminescence value as the vertical axis ( Figure 4 ). The content of MMP12 protein in the sample to be tested was calculated according to the standard curve, and the results are shown in Table 2.

[0075] Table 2

[0076]

[0077] The serum and whole blood of volunteers No. 4, No. 9 and No. 15 were taken as samples, and 10 biological replicates were performed for each sample to test the stability of the kit. The results are shown in Table 3.

[0078] Table 3

[0079]

[0080]

[0081] Table 2 and Table 3 show that the content of MMP12 protein in healthy human serum and whole blood ranges from 200 to 400 pg / mL, which is consistent with the normal value range of human MMP12 protein content. This shows that the kit of the present invention can quantitatively detect the content of MMP12 protein in human serum and whole blood specimens, and has good stability, which can meet the needs of basic research and clinical diagnosis.

[0082] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A monoclonal antibody against MMP12 protein, characterized in that: The monoclonal antibody comprises a heavy chain variable region as shown in SEQ ID NO.7 and a light chain variable region as shown in SEQ ID NO.

8.

2. Use of the anti-MMP12 protein monoclonal antibody according to claim 1 in the preparation of a kit for specifically detecting MMP12 protein.

3. The use according to claim 2, characterized in that The kit for specifically detecting MMP12 protein comprises a magnetic particle component, an acridinium ester labeling component, an acridinium ester luminescent substrate liquid, and an MMP12 protein calibrator.

4. The use according to claim 3, characterized in that The preparation method of the magnetic particle component is as follows: (1) Activate the carboxyl groups on the surface of carboxyl magnetic beads using EDC Solution and NHS Solution; (2) Mix the anti-MMP12 monoclonal antibody described in claim 1 with the carboxyl magnetic beads activated in step (1), and couple at 25°C for 2 hours; (3) Use Blocking Solution to block the sites on the surface of carboxyl magnetic beads that are not bound to antibodies; (4) The magnetic beads prepared in step (3) are dispersed and washed by ultrasonication, and diluted with magnetic bead storage solution for later use.

5. The use according to claim 4, characterized in that The composition of the magnetic bead storage solution described in step (4) is: 6% Tris-HCl buffer, 0.2% Tween-20, 0.85% NaCl, 2% BSA, 0.2% Proclin-300, and the rest is ultrapure water.

6. The use according to claim 3, characterized in that The preparation method of the acridinium ester labeling component is: The goat anti-mouse IgG antibody and the acridinium ester working solution were mixed and reacted at room temperature in the dark for 1 hour; the labeling termination buffer was added and mixed at room temperature; and the acridinium ester-labeled goat anti-mouse IgG antibody was purified by a desalting column to obtain the acridinium ester-labeled goat anti-mouse IgG antibody.

7. The use according to claim 6, characterized in that The labeling termination buffer is composed of a mixed aqueous solution of lysine and NaHCO3, wherein the concentration of the lysine is 0.55 mol / L and the concentration of the NaHCO3 is 0.2 mol / L.

8. The use according to claim 3, characterized in that The acridinium ester luminescent substrate solution is 0.05% H2O2 and 100mmol / L NaOH solution.

9. The use according to claim 2, characterized in that The sample detected by the kit is whole blood or serum.

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