CN114605552A - PRL2 antibody, and preparation method and application thereof - Google Patents

Abstract

The invention discloses a PRL2 antibody, a preparation method and application thereof, and relates to the technical field of biological detection, including obtaining serum by immunizing mice with PRL2 recombinant protein, crudely extracting immunoglobulin by saturated ammonium sulfate precipitation; extracting antibody by affinity chromatography PRL2 immunoglobulin G and the steps of coupling anti-PRL2 immunoglobulin G to fluorescein, the present invention specifically targets PRL2, and has important significance for early diagnosis and/or prognosis evaluation and treatment of inflammatory bone diseases.

Description

A kind of PRL2 antibody and its preparation method and application

technical field

The present invention relates to the technical field of biological detection, in particular to an antibody for detecting PRL2 and a detection method thereof.

Background technique

Inflammation is a double-edged sword, which can not only protect the human body from pathogen infection, but also is closely related to the occurrence and development of many diseases. Excessive inflammation of bone tissue is usually manifested in enhanced bone resorption capacity, which can be seen in inflammatory bone diseases such as rheumatoid arthritis, ankylosing spondylitis, periodontitis, and osteomyelitis. Excessive bone resorption can lead to impaired bone function, seriously affect the quality of life of patients, and be accompanied by a heavy socioeconomic burden. Therefore, new biomarkers for screening inflammatory bone diseases may provide new ideas for establishing early disease diagnosis models and prevention methods.

Osteoclasts are multinucleated cells formed by the fusion of monocytes/macrophages originating from common myeloid progenitor cells in the bone marrow. The main function in the body is to carry out bone resorption and maintain together with osteoblasts responsible for bone formation. Bone homeostasis. As a terminally differentiated cell, osteoclasts are prone to apoptosis after performing bone resorption. In order to maintain bone homeostasis, the osteoclast precursor cells in the human body-monocyte lineage cells (ie CD14+ cells) will continue to Microvessels that migrate to the joint eventually settle and further differentiate into osteoclasts. When osteoclasts accumulate in joints, that is, when bone resorption is stronger than bone formation, it can lead to a series of bone damage-related diseases. Therefore, finding important molecules that play a regulatory role in the process of osteoclast differentiation and bone resorption will have implications for the diagnosis and treatment of inflammatory bone diseases.

Phosphatase of Regenerating Liver 2 (PRL2) belongs to the protein tyrosine phosphatase family and is widely expressed in various tissues of the human body. The amino acid sequence of PRL2 is highly conserved and is completely identical between humans and mice. Previous studies on the PRL family were mostly related to tumors, and few studies were conducted in other diseases. It has been reported in the literature that the expression level of PRL2 is high in the process of osteoclast differentiation, suggesting that it may play a certain role in osteoclast differentiation. It has also been reported in the literature that in the state of inflammation, the expression level of PRL2 in myeloid cells (such as macrophages, neutrophils, etc.) will decrease rapidly. However, there is no relevant research report on the expression of PRL2 in osteoclast precursor cells and the role of PRL2 in the diagnosis of inflammatory bone disease.

For the diagnosis of inflammatory bone disease, in the early stage of the disease, clinical features are difficult to identify, and inflammatory markers such as ESR and C-reactive protein have no characteristic significance, and there is no clear biomarker yet. Therefore, with the support of the Shanghai Fifth Round of Public Health System Construction Three-Year Action Plan (GWV-10.1-XK13), the Shanghai Natural Science Foundation Project (19ZR1428500), and the National Natural Science Foundation General Project (81971486), we are committed It aims to find new diagnostic targets for inflammatory bone diseases and provide important technical basis for disease prevention and treatment.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the defects of the prior art, and to provide an antibody for detecting PRL2 and a preparation method and application thereof.

The present invention adopts the following technical solutions to realize:

One of the objects of the present invention is to provide a method for preparing a PRL2 antibody, characterized in that it comprises the following steps

Step 1. Immunize mice with PRL2 recombinant protein to obtain serum;

Step 2, crudely extract immunoglobulin by saturated ammonium sulfate precipitation method;

Step 3. Extract anti-PRL2 immunoglobulin G by affinity chromatography;

Step 4. Conjugate anti-PRL2 immunoglobulin G with fluorescein to obtain fluorescein-labeled anti-PRL2 immunoglobulin G.

Further, the step 1 specifically includes mixing 100 μg of PRL2 recombinant protein with an adjuvant to prepare a 100 μL mixed emulsion.

Further, in step 1, the mice were immunized three times with an interval of 2 weeks. The adjuvant used Freund's complete adjuvant in the first immunization, and Freund's incomplete adjuvant in the second and third immunizations. adjuvant.

Further, the mice described in step 1 are male C57BL/6 mice aged 6-8 weeks.

Further, the fluorescein/protein molar ratio of the fluorescein-labeled anti-PRL2 immunoglobulin G in step 4 is 2.5-6.5.

Another object of the present invention is to provide an antibody obtained by the above-mentioned preparation method.

The third object of the present invention is to provide the application of the antibody in the diagnosis of inflammatory bone disease

The fourth object of the present invention is to provide a detection reagent, the reagent component includes the antibody.

The fifth object of the present invention is to provide the application of the reagent in detecting the expression level of PRL2 in CD14+ cells of inflammatory bone disease.

Further, the described application specifically includes the following steps:

Step 1. Collect peripheral blood of subjects with intravenous anticoagulation;

Step 2. Use density gradient centrifugation to obtain peripheral blood mononuclear cells;

Step 3. Incubate the peripheral blood mononuclear cells with fluorescein-labeled anti-CD14 antibody and fluorescein-labeled anti-PRL2 immunoglobulin G in sequence;

Step 4. Use flow cytometry to detect the average fluorescence intensity of fluorescein FITC in CD14+ cells.

The beneficial effects of the present invention are:

Provided is an antibody for detecting the expression level of PRL2, and a method for detecting the expression level of PRL2 in human peripheral blood CD14+ cells. The experimental results of the present invention can illustrate that the expression level of PRL2 is related to inflammatory bone disease. It is closely related to the occurrence of inflammatory bone disease, which is manifested in that the expression level of PRL2 in CD14+ cells in peripheral blood of patients with inflammatory bone disease is significantly lower than that of normal individuals. Therefore, it can be used for early diagnosis and/or prognosis evaluation of inflammatory bone disease, and then used to guide inflammatory bone disease. disease treatment. The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings, so as to fully understand the purpose, characteristics and effects of the present invention.

Description of drawings

Fig. 1 is the immunoblotting method of the present invention to detect the protein level and statistical result of PRL2 in peripheral blood mononuclear cells of healthy controls (HC) and rheumatoid arthritis patients (RA);

Figure 2 shows the detection of peripheral blood mononuclear cells (PBMCs), B lymphocytes (CD19 cells), T lymphocytes (CD3 cells), monocytes in healthy controls (HC) and patients with rheumatoid arthritis (RA) by flow cytometry Protein levels of PRL2 in cells (CD14 cells).

Detailed ways

The following describes the embodiments of the present invention with reference to the accompanying drawings, so as to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the protection scope of the present invention is not limited to the embodiments mentioned herein.

In the embodiment of the present invention, the protein level of PRL2 in peripheral blood of patients with inflammatory bone disease can be detected by preparing an anti-PRL2 mouse polyclonal antibody, and then performing flow cytometry or immunoblotting.

Example 1. Preparation of anti-PRL2 mouse polyclonal antibody

1. PRL2 recombinant protein immunization of mice

Male C57BL/6 mice aged 6-8 weeks were selected and immunized according to the following steps, a total of 3 times, with an interval of 2 weeks each time:

For the first immunization, put 100 μg of PRL2 recombinant protein and an equal volume of Freund's complete adjuvant into the emulsifying needle tube, connect the emulsifying needle tube to the tee, push the needle tube repeatedly on ice for more than 1000 times, remove the needle tube, drop 1 drop to In water, the mixture is insoluble in water and the preparation is successful. The mixture was immunized subcutaneously on the back of mice at a dose of 100 μL per mouse;

During the second and third immunization, 100 μg of PRL2 recombinant protein was fully emulsified with an equal volume of incomplete Freund's adjuvant, and the injection method was the same as the first immunization;

Two weeks after the third immunization, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital, and blood was collected from the heart to obtain mouse serum.

2. Crude immunoglobulin extraction by saturated ammonium sulfate precipitation

The mouse serum was diluted with an equal volume of normal saline, added to a beaker with a magnetic stirring frame, and then slowly added dropwise 2 times the volume of saturated ammonium sulfate solution to the solution. At this time, the concentration of ammonium sulfate was about 66.7%. Place at 4°C and react for more than 3 hours;

Transfer the liquid in the beaker to a centrifuge tube, spin at 3000rpm for 20 minutes, and centrifuge at 4°C. Carefully discard the supernatant and keep the precipitate;

Dissolve the precipitate with a small volume of physiological saline until it is fully dissolved, add it to a beaker with a magnetic stirring frame, and slowly add 1/2 volume of saturated ammonium sulfate solution dropwise to the solution. At this time, the concentration of ammonium sulfate is about 33.3%. Place at 4°C and react for more than 3 hours;

Transfer the liquid in the beaker to a centrifuge tube, spin at 3000rpm for 20 minutes, and centrifuge at 4°C. Carefully discard the supernatant and keep the precipitate;

The precipitate was dissolved with 3 mL of TBS buffer, placed in a dialysis bag, and dialyzed at 4°C overnight.

(TBS buffer formulation: 25mM Tris-HCl, 137mM NaCl, pH 7.2～7.6)

3. Extraction of Immunoglobulin G by Affinity Chromatography

Agarose bead equilibration: aspirate 1 mL of Protein A/G PLUS-Agarose (purchased from Santa Cruz Biotechnology), add 10 mL of TBS buffer, spin at 1000 rpm, centrifuge at 4°C for 3 minutes, and carefully discard the supernatant;

The dialyzed crude immunoglobulin was added to the agarose beads, placed at 4°C, and rotated overnight;

Take out the agarose beads, spin at 1000rpm, centrifuge at 4°C for 3 minutes, and carefully discard the supernatant;

Add 10 mL of TBS buffer, resuspend the agarose beads, spin at 1000 rpm, centrifuge at 4 °C for 3 minutes, carefully discard the supernatant, repeat once, and wash off the non-specifically bound proteins;

Add 1mL of 0.1M Glycine-HCl (pH 2～3), resuspend the agarose beads, spin at 1000rpm, centrifuge at 4°C for 3 minutes, retain the supernatant, repeat once, and fully elute the immunoglobulin G (IgG);

The supernatant was placed in PBS and dialyzed overnight at 4°C.

4. Fluorescein-conjugated IgG

Preparation of cross-linking reaction solution: Weigh NaHCO ₃ 7.56g, Na ₂ CO ₃ 1.06g, NaCl 7.36g, ddH ₂ O to 1L respectively;

The IgG was placed in the cross-linking reaction solution and dialyzed at 4°C overnight;

Prepare 1mg/mL concentration of Fluorescein Isothiocyanate (FITC), dissolve in DMSO, slowly add 150μg FITC solution to each 1mg IgG, protect from light, and place at 4°C to react for more than 8h;

Slowly add 5M NH ₄ Cl to the solution at a ratio of 1:100 to make the final concentration 50 mM, protect from light, and place it at 4°C to stop the reaction for 2 h;

The FITC-IgG solution was placed in PBS and dialyzed at 4°C overnight to remove excess fluorescein;

Using a spectrophotometer, measure the absorbance of the solution at wavelengths of 280 nm and 495 nm, respectively, and substitute the following formula to calculate the fluorescein/protein molar ratio (F/P):

The formula for calculating the concentration of FITC-IgG conjugate is:

The F/P value can be between 2.5 and 6.5;

After the dialysis of FITC-IgG was completed, 0.1% NaN ₃ and 1% BSA were added, and the mixture was placed at 4° C. and stored in the dark.

Example 2. Detection of PRL2 levels in peripheral blood cells by flow cytometry and western blotting

1. Isolation of Human Peripheral Blood Mononuclear Cells

In this case, peripheral blood samples were collected from 17 healthy controls and 30 rheumatoid arthritis patients. Patients with rheumatoid arthritis were diagnosed according to the 1987 American Rheumatology Association revised classification criteria for rheumatoid arthritis. The clinical samples used in this study were informed to the patients and approved by the ethics committee.

Collect 5mL of peripheral venous anticoagulation from subjects and mix with equal volume of normal saline;

Add 15mL Lymphoprep ^TM separation reagent (purchased from STEMCELL Technologies) into the 50mL centrifuge tube, and then slowly add the peripheral blood diluted in the previous step to the upper layer of the separation reagent, do not damage the stratification;

Rotation speed 800g, 20 minutes, room temperature centrifugation, no brake;

After taking out the centrifuge tube, it is divided into four layers from top to bottom, which are: pale yellow plasma, white cloudy precipitate, colorless separation reagent, red blood cell and granulocyte layer. The white cloudy cloud is the required peripheral blood mononuclear cells. bloodmononuclear cells, PBMCs), carefully aspirate the PBMCs with a pipette and transfer to a new centrifuge tube;

Wash off the residual separation reagent, add 10 mL of normal saline, spin at 500 g, centrifuge at 4°C for 5 minutes, repeat once;

Discard the supernatant, resuspend the cells with 1 mL of FACS buffer (2% FBS, 0.1% NaN ₃ in PBS), and count the cells;

2. Flow Cytometry

5 μL of Fc receptor blocking antibody Human TruStainFcX ^™ (purchased from Biolegend) was added to 1×10 ⁶ PBMCs in a volume of 100 μL, and incubated at room temperature for 10 min;

Add 500 μL of FACS buffer, spin at 500 g for 5 minutes, and centrifuge at 4°C;

Discard the supernatant, resuspend the cells in 100 μL FACS buffer, and add 1 μL fluorescein-conjugated anti-human CD3 antibody (T lymphocyte marker), anti-human CD19 antibody (B lymphocyte marker), and anti-human CD14 antibody ( Monocyte marker) antibodies were purchased from Thermo Fisher Scientific, after mixing, protected from light, and incubated at 4°C for 30 min;

Add 500 μL of FACS buffer, spin at 500 g for 5 minutes, and centrifuge at 4°C;

Discard the supernatant, add 100 μL of Fixation/Permeabilization solution (purchased from BD Biosciences), protect from light, and incubate at 4°C for 10 min;

Add 500 μL washing buffer Perm/Wash ^™ Buffer (purchased from BD Biosciences), spin at 500 g for 5 minutes, and centrifuge at 4°C;

Discard the supernatant, resuspend the cells with 100 μL washing buffer, add 1 μL fluorescein-conjugated anti-PRL2 antibody, mix well, protect from light, and incubate at 4°C for 30 min;

Add 500 μL of washing buffer, spin at 500 g for 5 minutes, and centrifuge at 4 °C;

Discard the supernatant, resuspend the cells with 100 μL of wash buffer, collect data using a flow cytometer, and record the mean fluorescence intensity of the FITC channel in different cell subsets.

3. Immunoblotting

Take 1×10 ⁶ PBMCs, spin at 500g for 5 minutes, and centrifuge at 4°C;

Discard the supernatant, add 100 μL of cell lysis solution, mix well, lyse on ice for 20 minutes, and vortex every 5 minutes to achieve full lysis;

Centrifuge the cell lysate at 10,000g for 5 minutes at 4°C;

Transfer the supernatant to a new centrifuge tube, add 5× loading buffer, mix well, place in a metal bath at 100°C, and boil for 5 minutes;

Prepare SDS-PAGE gel: prepare separation gel and stacking gel of corresponding concentration according to the size of the protein to be analyzed;

Gel running: point the protein samples and protein markers prepared according to the above method into the gel holes, run the gel at a constant voltage, and control the running time according to the experimental requirements;

Transfer membrane: transfer the protein from the gel to the nitrocellulose membrane with a constant current, and the transfer process requires an ice bath;

Block, dissolve with 5% nonfat milk powder in TBS buffer, block at room temperature for 1h;

Incubation with primary antibody: Incubate the membrane with the antibody against the corresponding protein, and incubate at 4°C overnight;

Incubation of secondary antibody: recover the primary antibody and wash the membrane with 1×TBST, wash the membrane on a horizontal shaker for 3 times, 3 min each time, then add 5% nonfat milk powder containing the corresponding secondary antibody, and incubate at room temperature for 1 h;

Development: The membrane was washed with 1×TBST in the same manner as above, and after 3 times, the membrane was placed in a gel imager, and chemiluminescence solution was added dropwise for development.

4. Result analysis and statistics

Differences between the two samples were analyzed using a two-tailed Student's t-test, and the experimental results were analyzed using GraphPad Prism 9 software. *P<0.05, **P<0.01, ***P<0.001, NS means no statistical difference. When P<0.05, the difference of experimental results was statistically significant.

In the present invention, it is found that the PRL2 protein level of monocytes in the peripheral blood of patients with inflammatory bone disease is significantly lower than that of the healthy control group, while the protein levels of PRL2 in T and B lymphocytes do not change significantly. are precursor cells of osteoclasts. The invention can provide a new target for the diagnosis of inflammatory bone disease, and provide a technical basis for the treatment of inflammatory bone disease.

The preferred embodiments of the present invention have been described in detail above. It should be understood that many modifications and changes can be made according to the concept of the present invention by those skilled in the art without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the prior art according to the concept of the present invention shall fall within the protection scope determined by the claims.

Claims (10)

1. A preparation method of a PRL2 antibody is characterized by comprising the following steps

Step 1, immunizing a mouse by PRL2 recombinant protein to obtain serum;

step 2, crude extraction of immunoglobulin by saturated ammonium sulfate precipitation;

step 3, extracting anti-PRL 2 immunoglobulin G by affinity chromatography;

and 4, coupling the anti-PRL 2 immunoglobulin G with fluorescein to obtain fluorescein-labeled anti-PRL 2 immunoglobulin G.

2. The method according to claim 1, wherein the step 1 comprises mixing 100 μ g of PRL2 recombinant protein with an adjuvant to prepare 100 μ L of a mixed emulsion.

3. The method of claim 2, wherein step 1 comprises immunizing the mice 3 times, each 2 weeks apart, with Freund's complete adjuvant for the first immunization and Freund's incomplete adjuvant for the second and third immunizations.

4. The method of claim 1, wherein the mice in step 1 are male C57BL/6 mice that are 6-8 weeks old.

5. The method of claim 1, wherein the fluorescein-labeled anti-PRL 2 immunoglobulin G of step 4 has a fluorescein/protein molar ratio of 2.5 to 6.5.

6. An antibody obtained by the production method according to any one of claims 1 to 5.

7. The antibody according to claim 6 for use in diagnosis of inflammatory bone diseases.

8. A detection reagent, wherein the reagent component comprises an antibody of claim 6.

9. The use of the agent of claim 8 for detecting the expression level of CD14+ cells PRL2 in inflammatory bone diseases.

10. Use according to claim 7 or 9, in particular comprising the following steps:

step 1, collecting venous anticoagulation peripheral blood of a subject;

step 2, obtaining peripheral blood mononuclear cells by using a density gradient centrifugation method;

3, sequentially incubating the peripheral blood mononuclear cells with a fluorescein-labeled anti-CD 14 antibody and a fluorescein-labeled anti-PRL 2 immunoglobulin G;

and 4, detecting the average fluorescence intensity of fluorescein FITC in the CD14+ cells by using flow cytometry.

Images