WO2023108897A1

WO2023108897A1 - Polypeptide for preparing onecut3 antibody, and rabbit polyclonal antibody and application thereof

Info

Publication number: WO2023108897A1
Application number: PCT/CN2022/078194
Authority: WO
Inventors: 佟红艳; 罗颖婉; 朱双虹; 郎伟
Original assignee: 浙江大学医学院附属第一医院
Priority date: 2021-12-16
Filing date: 2022-02-28
Publication date: 2023-06-22
Also published as: CN114395024A

Abstract

A polypeptide for preparing an ONECUT3 antibody, and a rabbit polyclonal antibody and an application thereof, relating to the technical field of biology. On one hand, a polypeptide for preparing an ONECUT3 antibody is provided; on the other hand, an anti-ONECUT3 antigen polypeptide rabbit polyclonal purified antibody and an application thereof are provided. The obtained anti-ONECUT3 antigen polypeptide rabbit polyclonal purified antibody has a specific recognition function for ONECUT3, almost has no contaminant band, reliably reflects the expression level of cell-level and tissue-level ONECUT3 proteins, has the capability of detecting endogenously and exogenously expressed ONECUT3 proteins, and has the detection capability superior to the effect achieved by commercial antibodies available on markets.

Description

A kind of polypeptide for preparing ONECUT3 antibody and its rabbit polyclonal antibody, application

technical field

The invention belongs to the field of biotechnology, and specifically relates to a polypeptide for preparing ONECUT3 antibody, a rabbit polyclonal antibody and applications thereof.

Background technique

ONECUT3, ONECUT1 (HNF6), ONECUT2 belong to the ONECUT family in the original homeobox (homeobox) transcription factor superfamily. Their common structure contains N-terminal CUT domain and C-terminal Homeobox domain. These three transcription factors play an important role in physiological processes such as embryonic development, tissue formation and differentiation. ONECUT1 dynamically regulates the expression of genes related to cell adhesion and migration through the TGF-beta signaling pathway in embryonic development, and its abnormal expression plays an important role in the progression and metastasis of some malignant tumors. For example, in liver cancer and pancreatic cancer cells, ONECUT1 can Combined with the promoter regions of genes such as AFP, CyclinD1, Cdc25A, Cdk2 and E2F1, it participates in the regulation of cell proliferation and cell cycle, induces the increase of cells in S phase, and activates the TGF-beta signaling pathway. Abnormally high expression of ONECUT1 can also promote the proliferation and metastasis of colorectal cancer cells. In metastatic castration-resistant prostate cancer (mCRPC), ONECUT2 promotes tumor cell proliferation and survival through an androgen-dependent pathway. The above studies have shown that the abnormal expression of ONECUT1 and ONECUT2 in the ONECUT family is involved in the pathogenesis of tumors. Whether ONECUT3 is involved in the occurrence and development of hematological tumors has not been reported yet.

At present, ONECUT3 on the market has the problems of low potency and poor specificity, and it is difficult to meet the scientific research needs of ONECUT3 protein detection at the cell level and tissue level. For example, in practical applications, the antibody of Novus (NBP1-91528) only has the ability to detect the exogenous overexpression of ONECUT3, but cannot detect the endogenously expressed ONECUT3 protein; The reason for the design of the antigen polypeptide sequence of the antibody is that the specificity is not good, and there are problems of multiple bands in the detection.

Contents of the invention

Aiming at the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for preparing the polypeptide of ONECUT3 antibody and its rabbit polyclonal antibody and its application.

The present invention is specifically realized through the following technical solutions:

The polypeptide for preparing the ONECUT3 antibody has a nucleotide sequence of CMNRWAEEPSTAPG, as shown in SEQ ID NO.1.

The application of the polypeptide in the preparation of rabbit polyclonal antibody against ONECUT3 antigen protein.

The method for preparing a rabbit polyclonal antibody against ONECUT3 antigen protein by using said polypeptide comprises the following steps:

1) Coupling the antigenic polypeptide of ONECUT3 with carrier proteins KLH and BSA respectively, and obtaining the coupling product after purification;

2) Take the coupling product to immunize rabbits, take the rabbit serum after multiple immunizations, and use the ELISA method to detect the titer. After the titer reaches the ideal value, boost the immunization once, and collect the immunized rabbit serum;

3) Using antigen affinity purification to obtain polyclonal antibodies.

Application of the rabbit polyclonal antibody against ONECUT3 antigen protein prepared above in detecting endogenous and exogenous ONECUT3.

The rabbit polyclonal purified antibody of the anti-ONECUT3 antigen polypeptide obtained in the present invention has a specific recognition function for ONECUT3, almost no miscellaneous bands, and reliably reflects the expression level of ONECUT3 protein at the cell level and tissue level, and has the ability to detect endogenous And the ability to exogenously express OUTCUT3 protein, the detection ability is better than the effect obtained by the commercial antibody on sale.

Description of drawings

Figure 1 is the amino acid sequence of Human ONECUT3;

Figure 2 is a structural analysis diagram of ONECUT protein;

Fig. 3 is the antibody peptide blocking experiment (Western-Blot figure) in embodiment 2;

Fig. 4 is the knockdown verification antibody specificity experiment (Western-Blot figure) in embodiment 3;

Fig. 5 is the endogenous ONECUT3 expression detection (Western-Blot figure) of the bone marrow primary sample of the myelodysplastic syndrome patient in embodiment 4;

FIG. 6 is a graph showing the results of immunohistochemical detection of bone marrow tissue ONECUT3 expression in Example 5. FIG.

Detailed ways

The present invention will be further described below in conjunction with the examples.

Embodiment 1: Design and synthesis of ONECUT3 polypeptide

1. Design of the antigenic protein sequence of the ONECUT3 antibody

The amino acid sequence of Human ONECUT3 (UniProtKB-O60422) is shown in Figure 1.

The structural analysis of ONECUT3 protein is shown in Figure 2.

According to the surface accessibility, hydrophilicity and elasticity of each amino acid in ONECUT3 protein, the epitope region of ONECUT3 protein can be deduced, so the designed peptide sequence is CMNRWAEEPSTAPG (human ONECUT3 469-482aa).

2. Synthetic peptides

Artificially synthesized peptides adopt the peptide solid-phase synthesis method, and the specific steps are as follows:

(1) Weighing of resin: choose Wang resin, such as: to make 0.2mmol, choose resin Wei 0.5mmol/g, that is, the amount of weighing resin is 0.2mmol divided by 0.5mmol/g=0.4g swelling. Put the weighed resin into the reaction column, and then swell it with DCM for half an hour. The amount of DCM added is 2 to 3 times the height of the resin, that is to fully swell the resin completely. Can be aerated slightly.

(2) To protect. (Cl-resin should not be protected in the first step, but it must be deprotected after coupling with amino acids) The other two resins must be deprotected (Fmoc), first drain the DCM, and then wash with DMF 3 times to remove the DCM , and then deprotected with 20% hexahydropyridine, 5+10 twice in total, the first time was 5 minutes, the second time was 10 minutes, and the middle was washed once with DMF.

(3) Wash after deprotection. Wash 6 times with DMF.

(4) Coupling. We are now throwing amino acids in 3 times the amount, that is, 0,2mmol of resin, the amount of amino acids we put in is 0.2mmol*3=0.6mmol multiplied by its molecular weight is the mass to be weighed. In addition, add and weigh the activator DIC and DIEA etc.

(5) Detection during coupling. Use a dropper to take resin (a little, about 15 particles, as long as it can be identified by detection) and a small test tube, add 2 drops of A solution (5g ninhydrin dissolved in 100ml ethanol), 2 drops of B solution (80g Dissolve phenol in 20ml of ethanol), 2 drops of solution C (2ml of 0.001M KCN added to 98ml of redistilled pyridine), and heat for 3 minutes. Then take it out to see if the resin is transparent. If the color of the solution is too deep to affect the observation, you can pour out the solution, add a little DMF to wash it, and then observe. If the resin is detected to be transparent, the next amino acid can be coupled. If the resin is opaque, prolong the reaction time. If it is still not transparent, re-casting can be carried out. Be sure to complete the reaction at each step before coupling the next amino acid.

(6) Washing after coupling. After coupling, DMF was washed 3 times.

(7) The above is the step of coupling an amino acid, repeat steps 3-6 until the coupling of the last amino acid is completed.

(8) After the last amino acid is coupled, deprotect it, shrink after deprotection, wash 3 times with DMF, 3 times with DCM, 3 times with methanol, and drain the peptide resin.

(9) Weigh the completed dry peptide resin, put it into a 50ml centrifuge tube, and add lysate (10 times ml per gram), such as: 1g peptide resin plus 10ml lysate. Add lysate solution D (TFA:TIS:Water=95:2.5:2.5), react for 2 hours, shake every 10-15 minutes, let it fully react, and put it in a shaker (25°C). After the reaction is completed, filter with suction, pour the solution into glacial ether for precipitation, and then centrifuge for 3-4 times.

(10) Put the centrifuged peptide in a fume hood to blow for a while, and then put it in a vacuum desiccator to dry it.

(11) Take a small amount into the sample tube, fill in the test form and send it to the quality inspection department for mass spectrometry to determine whether the mass spectrum is correct. If it is correct, fill in the form for the remaining peptide and send it for purification.

3. Peptide Conjugation

1) Dissolve 20mg KLH in 2mL, 5mM EDTA aqueous solution.

2) Weigh 8mg Sulfo-SMCC and dissolve it completely in 50μL DMSO, then add 150μL 1×PBS and mix well.

3) Add the Sulfo-SMCC solution dropwise to KLH, shake gently while adding (violent shaking will produce precipitation), and place at room temperature for 1h.

4) Put the above-mentioned activated KLH solution into a dialysis bag, clamp the dialysis clamp, and dialyze in 2L of 1×PBS for 1 hour under magnetic stirring in a refrigerator at 4°C.

5) Replace with new 1×PBS and dialyze for 2 hours, repeat once. Put the KLH activated and dialyzed into a 15mL imported centrifuge tube, and mark the reagent name, time and concentration on the tube, and store it in a refrigerator at 4°C.

6) Weigh 4 mg of polypeptide (the peptide sequence is CMNRWAEEPSTAPG), dissolve it in 50 μL of DMSO, then add 200 μL of 1×PBS, mix quickly, then immediately add KLH according to the ratio of polypeptide: KLH=1 mg: 680 μg, and keep at 4°C Refrigerate overnight or react for 2 hours at room temperature.

7) Put the above-mentioned cross-linked KLH-peptide cross-linked complex into a dialysis bag, clamp the dialysis clamp, and dialyze in 4 L of 1×PBS overnight under magnetic stirring in a refrigerator at 4°C.

8) Take out the dialyzed KLH-peptide into a clean 1.5mL centrifuge tube, aliquot according to the immune dose, and store in a -20°C refrigerator.

4. Immunize rabbits and obtain antiserum

1) Animal selection: the rabbits are New Zealand white rabbits, young and middle-aged with a weight of about 2.5Kg. Animals were selected as healthy animals with shiny coat color and free movement. Choose a good animal and pre-raise it for about 2 weeks. The purpose is to eliminate some unqualified animals so that the later experiments can be carried out smoothly.

2) Preparation before the experiment: the rabbits were marked.

3) Antigen preparation:

3.1) Take the antigen (the coupling product in 3) out of the -20°C refrigerator and dissolve at room temperature, avoiding repeated freezing and thawing.

3.2) Extract the antigen (the antigen is completely mixed), the antigen concentration is 1mg/ml for the first immunization, 0.5ml/rabbit, the antigen concentration is halved for the second immunization-fourth immunization, and the dose remains unchanged.

3.3) Extract the adjuvant, the adjuvant and the antigen are extracted in a volume ratio of 1:1. Complete adjuvant was used for the first immunization, and incomplete adjuvant was used for the second to fourth immunization. When drawing, the adjuvant should be fully mixed and then drawn into the syringe.

3.4) The two syringes are fully emulsified after being docked with a syringe connecting tube. The emulsification standard is: the emulsified immunogen is dripped into 37°C water and does not disperse, which is qualified.

4) Immunization: Rabbits were injected subcutaneously at multiple points, 0.2ml per point.

Immunization time: The second immunization is carried out on the 14th day after the first immunization, and the interval between the second immunization and the third immunization is 7 days. On the 7th day after the third immunization, a small sample of serum was taken from the middle ear artery of the rabbit, and the test was qualified. After 7 days, the immunization was added, and the whole blood could be collected 7 days after the immunization.

5. ELISA detection (indirect method)

1) Coating plate: Dilute the known antigen to 1 μg/ml with coating buffer (coating buffer: Na ₂ CO ₃ and NaHCO ₃ buffer), add 50 μl to each reaction well of polystyrene plate, 4°C Overnight, the next day, discard the solution in the well, and wash once with 1x TBST washing buffer at 180 μl per well.

2) Blocking: add 60 μl of 1% BSA (prepared in TBST) to each well for blocking, and incubate at 37° C. for 1 hour. Then discard the blocking solution.

3) Sample addition: add a certain dilution of the sample to be tested (dilute the sample to be tested according to a certain ratio), 50 μl to the above-mentioned blocked reaction well. At the same time, set up positive control wells (positive serum) and negative control wells (BSA). Incubate at 37°C for 1 hour, then discard the blocking solution and wash twice with 1x TBST washing buffer at 180 μl per well.

4) Add enzyme-labeled antibody: add freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) to the wells of the enzyme-labeled plate at 50 μl/well, incubate at 37°C for 45min, then discard the blocking solution. Wash 3 times with 1x TBST wash buffer at 180 μl per well.

5) Color development by adding substrate solution: add 100 μl of temporarily prepared TMB substrate solution to each reaction well, and react at 37° C. for 5 minutes.

6) Termination of the reaction: 90 μl of 2M sulfuric acid was added to each reaction well.

7) Plate reading: place the microplate plate in a preheated microplate reader (450nm) for reading, save the data, and analyze.

6. Purify the antiserum by using the antigen polypeptide to obtain the purified antibody

1) Fully wash the affinity chromatography column with 20mL pure water and 1×PBS (pH7.4) at a flow rate of 70mL/h.

2) Take 10 mL of the serum to be purified in a 50 mL centrifuge tube, and filter it with a microporous membrane with a pore size of 0.45 μm and a diameter of 25 mm.

3) Load the suction-filtered serum sample at a flow rate of 40 mL/h, and repeat once.

4) Wash the column with 20mL 1×PBS (pH7.4) at a flow rate of 70mL/h. After 10min, connect the protein detector, and adjust the light transmittance (T gear) of the instrument to 100 during the cleaning process.

5) Adjust the absorbance of the protein detector (1A file) to 0. At this time, turn on the HD-A computer collector on the computer desktop, and adjust the full screen range to 5, and use glycine solution (pH 2.7, 0.2M) to The antibody is eluted at a speed of 40mL/h. At this time, the green elution record button is pressed to start elution, and the antibody is collected when the reading of the instrument starts to rise.

6) During the antibody collection process, adjust the pH value of the antibody to about 7 in time with 1M sodium bicarbonate, and record the highest peak of the elution peak.

7) After the antibody is collected, adjust the pH value to about 7, and record the volume of the eluted antibody, and then rinse the rubber tube connected to the collector with pure water.

8) Wash the affinity chromatography column with 20mL 1×PBS and pure water at a rate of 70mL/h in sequence, then add 20% ethanol, seal, and store in a 4°C refrigerator.

9) The purified antibody is sent for inspection according to different requirements: ELISA, WB, ICC, etc.

result

1. Peptide information

多肽编号Peptide number	序列信息sequence information
HAPM1067-1HAPM1067-1	469-482a.a.CMNRWAEEPSTAPG469-482a.a.CMNRWAEEPSTAPG

2. Periodic table of immunity

3. Serum ELISA test results

Conclusion: 5206 immune serum is qualified (based on ELISA judgment).

Definition: the OD450 value of 1:256000 is P value (positive value), and the OD450 value of BSA hole is N value (negative value);

5206P/N＝(0.494+0.367)/2÷(0.167+0.098)/2＝3.2>2.1 reached the positive standard.

Basic ELISA qualified.

5207P/N＝(0.420+0.428)/2÷(0.107+0.098)/2＝4.1>2.1 reached the positive standard.

Basic ELISA qualified.

Example 2: Antibody Peptide Blocking Experiment

(1) Dissolve 1 mg of peptide (HAPM1067-1) in enzyme-free sterilized water, with a final concentration of 1 mg/ml;

(2) Take two 15ml centrifuge tubes, one of which is equipped with antibody and peptide premix, Rabbit Anti-ONECUT3 antibody (#5206, Conc.0.5mg/ml) and peptide are added to 5% skimmed milk, antibody and peptide The peptide molar ratio is 1:2, which is a blocked antibody; the other tube is only added Rabbit Anti-ONECUT3 antibody (#5206, Conc.0.5mg/ml) in 5% skimmed milk, that is, the molar ratio of antibody to peptide is 1:0, as the antibody control; premix overnight at 4°C;

(3) Collect the patient's bone marrow mononuclear cells, add 50 μL SDS protein denaturing lysate to 1x 10E6 cells, pipette and mix, place on ice for 10 minutes, Bioruptor ultrasound (high power, ultrasound 10s off 10s, 4-6 cycles) ;

(4) Electrophoresis of an equal amount of samples on a 10% SDS-PAGE gel;

(5) When bromophenol blue electrophoresis reaches the bottom, stop and perform PDVF membrane transfer;

(6) Block with 5% skimmed milk at room temperature for 1 hour;

(7) After cutting the membrane, place it in the control antibody and blocking antibody, incubate overnight in the refrigerator at 4°C, wash the membrane 3 times with 1×TBST (10 min each time);

(8) Goat Anti-rabbit secondary antibody was incubated at room temperature for 1 hour, and the membrane was washed 3 times with 1×TBST (10 minutes each time);

(9) Add a chemical developer (Pierce ^TM SuperSignal ^TM West Pico PLUS Chemiluminescent, Thermo) after washing the membrane, and place it in a chemiluminescent developer (ChemiDoc MP imaging system, Bio-Rad) for development.

(10) After elution with eluent (Restore PLUS Western Blot Stripping Buffer, Thermo) for 15 minutes, block with 5% milk at room temperature for 1 hour;

(11) Incubate Anti-Actin HRP (Huabio, ET1702-67) at room temperature for 1 h, wash the membrane 3 times with 1 × TBST (10 min each time); add chemical developer (Pierce ^TM SuperSignal ^TM West Pico PLUS Chemiluminescent, Thermo) again, set It was developed in a chemiluminescent imager (ChemiDoc MP imaging system, Bio-Rad).

Results: The endogenous band of ONECUT3 (about 54kDa) can be seen in the antibody control group (i.e. antibody ratio to peptide = 1:0); in the antibody blocking group (i.e. antibody ratio to peptide = 1:2), there is no signal in the lane, suggesting that ONECUT3 Antibodies can be blocked by this peptide (see Figure 3).

Example 3: Knockdown verification antibody specificity experiment

(1) Construction of the Tet-pLKO-hOC3 plasmid: The Tet-pLKO-puro vector (purchased from Addgene, USA) was double-digested with Ecor I and Age I; the following base sequence was synthesized and inserted into a double-digested blank:

ShOC3Forward: CCGGcagcatcccgcaggcaatcCTCGAGgattgcctgcgggatgctgTTTT (as shown in SEQ ID NO.2);

ShOC3Reverse: AATTAAAAcagcatcccgcaggcaatcCTCGAGgattgcctgcgggatgctg (as shown in SEQ ID NO.3); after transformation, single clone sequencing is picked; the correct plasmid is extracted for sequencing;

(2) Taking a 10cm culture dish as an example, transfect Hela cells when they grow to 70% density, take two 500μl serum-free Opti-MEM medium, add 40μl liposome transfection reagent PolyJet (Signagen) and plasmid 9 μg, add the plasmid to the transfection reagent and mix well, let it stand at room temperature for 10-15 minutes, then drop it into the cell culture dish, add 1 μg/ml doxycycline to the control group, and add 1 μg/ml doxycycline to the knockdown group;

(3) Collect control and knockdown cells after 72 hours, add 50 μL SDS protein denaturing lysate to 1x 10E6 cells, mix well by pipetting, place on ice for 10 minutes, Bioruptor ultrasonic (power High, ultrasonic 10s off 10s, 4-6 cycle);

(4) Electrophoresis of an equal amount of samples on a 10% SDS-PAGE gel;

(6) Block with 5% skimmed milk at room temperature for 1 hour;

(7) Rabbit Anti-ONECUT3 antibody (#5206, Conc.1mg/ml) was incubated overnight in a refrigerator at 4°C, and the membrane was washed 3 times with 1×TBST (10 min each time);

(11) Incubate Anti-Actin HRP (Huabio, ET1702-67) at room temperature for 1 hour, wash the membrane 3 times with 1 × TBST (10 minutes each time), add chemical developer again, and place it in a chemiluminescence developer (ChemiDoc MP imaging system, Developed in Bio-Rad).

Results: The endogenous band of ONECUT3 (about 54kDa) can be seen in the control group (i.e. -DOX group); there is no signal in the lane of the knockdown group (i.e. +DOX group), suggesting that the antibody specifically recognizes human ONECUT3 protein (see Figure 4) .

Example 4: Detection of endogenous ONECUT3 expression in primary bone marrow samples from patients with myelodysplastic syndrome (WB)

(1) Construction of pcDNA3.1-hOC3 plasmid: the humanONECUT3 CDS sequence was synthesized in pcDNA3.1-empty (completed by Fenghui Bio), and the plasmid was sequenced and identified;

(2) Taking a 10cm culture dish as an example, transfect 293T cells when they grow to 70% density, take two 500μl serum-free Opti-MEM medium, add liposome transfection reagent PolyJet (Signagen) 40μl and plasmid 9 μg, add the plasmid to the transfection reagent and mix well, let it stand at room temperature for 10-15 minutes, and then drop it into the cell culture dish;

(3) Collect control and knockdown cells after 72 hours, add 100 μL SDS protein denaturing lysate to 1x 10E6 cells, mix well by pipetting, place on ice for 10 minutes, Bioruptor ultrasonic (power High, ultrasonic 10s off 10s, 4-6 cycle); as a positive control;

(4) Take 2 bone marrow samples from patients with myelodysplastic syndrome with complex karyotype (P1 and P2), and samples from patients with myelodysplastic syndrome with normal karyotype (P3 and P4). Each bone marrow, healthy Donor bone marrow samples (D1 and D2), take 5ml of each bone marrow sample in a 15ml centrifuge tube;

(5) Collect mononuclear cells by relative density gradient centrifugation with Ficoll Human Lymphocyte Separation Medium, perform cell counting, add 50 μL SDS protein denaturing lysate for 1x 10E6 cells, pipette and mix, place on ice for 10 min, and Bioruptor ultrasonic ( High power, ultrasonic 10s stop 10s, 4-6 cycles); as a test sample;

(6) Electrophoresis on 10% SDS-PAGE gel of 3 μL of positive ginseng sample (1x loading to make up the volume) and 15 μL of the sample to be tested;

(7) When bromophenol blue electrophoresis reaches the bottom, stop and perform PDVF membrane transfer;

(8) Block with 5% skimmed milk at room temperature for 1 hour;

(9) The primary antibody of Rabbit Anti-ONECUT3 antibody (#5206, Conc.1mg/ml) was incubated overnight in a refrigerator at 4°C, and the membrane was washed 3 times with 1×TBST (10min each time);

(10) Incubate Goat Anti-rabbit secondary antibody at room temperature for 1 hour, wash the membrane with 1×TBST 3 times (10 minutes each time);

(11) Add a chemical developer (Pierce ^TM SuperSignal ^TM West Pico PLUS Chemiluminescent, Thermo) after washing the membrane, and place it in a chemiluminescent developer (ChemiDoc MP imaging system, Bio-Rad) for development.

(12) After elution with eluent (Restore PLUS Western Blot Stripping Buffer, Thermo) for 15 minutes, block with 5% milk at room temperature for 1 hour;

(13) Incubate Anti-Actin HRP (Huabio, ET1702-67) at room temperature for 1 hour, wash the membrane 3 times with 1 × TBST (10 minutes each time), add chemical developer again, and place it in a chemiluminescence developer (ChemiDoc MP imaging system, Developed in Bio-Rad).

Results: Using ONECUT3 antibody as the primary antibody, the expression of endogenous and exogenous ONECUT3 protein can be detected, and the expression of ONECUT3 in bone marrow cells of patients with myelodysplastic syndrome accompanied by complex karyotype was higher than that of normal karyotype group, and healthy donors The expression of ONECUT3 in the bone marrow cells of the group was low (see Figure 5).

Example 5: Immunohistochemical detection of ONECUT3 expression in bone marrow tissue

(1) Paraffin sections were dewaxed to water: put the sections in xylene Ⅰ 8 min-xylene Ⅱ 8 min-xylene Ⅲ 8 min-absolute ethanol Ⅰ 5 min-absolute ethanol Ⅱ 5 min-85% alcohol 5 min-75% alcohol 5 min, washed in tap water for 2 min .

(2) Antigen retrieval: place the tissue slices in a repair box filled with citric acid antigen retrieval buffer (PH 6.0) and carry out antigen retrieval in a microwave oven, from medium heat to boiling for 8 minutes and then to medium-low heat for 7 minutes. Excessive evaporation of the buffer should be prevented and slides should not be dried. After natural cooling, slides were placed in PBS (pH7.4) and washed three times on a decolorizing shaker, 5 min each time.

(3) Draw a circle: Use a special histochemical pen to draw a small circle with a distance of 3-4 mm from the tissue along the outer contour of the tissue, and then add enough PBS to ensure that the subsequent addition of blocking serum, primary antibody, and secondary Resist, and chromogen completely cover the tissue without running off the slide.

(4) Block endogenous peroxidase: add endogenous peroxidase in the kit to the slices, incubate 50-100ul per slice at room temperature in the dark for 25min, and place slides in PBS (PH7.4) Shake and wash 3 times on a decolorizing shaker, 5 min each time.

(5) Serum blocking: 3% BSA was added dropwise in the histochemical circle to evenly cover the tissue, and blocked at room temperature for 30 min.

(6) Add primary antibody: Shake off the blocking solution gently, add Rabbit Anti-ONECUT3 antibody (1:100) prepared in PBS at a certain ratio dropwise on the slice, and lay the slice flat in a wet box at 4° to incubate overnight.

(7) Add secondary antibody: place slides in PBS (PH7.4) and shake and wash 3 times on a decolorizing shaker, 5 min each time. After the slices were shaken dry, the tissues were covered with hypersensitive rabbit-mouse universal secondary antibody (HRP-labeled) dropwise in the circle, and incubated at room temperature for 50 minutes.

(8) Counterstaining cell nuclei: stain with hematoxylin for about 2-3 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol differentiation solution for a few seconds, rinse with tap water, turn blue solution with ammonia water for 15-30 seconds, rinse with running water.

(9) Dehydration and sealing: Put the slices in 75% alcohol for 5 min-85% alcohol for 5 min--absolute ethanol I 5 min-absolute ethanol II 5 min-xylene I 5 min for dehydration and transparency, and take the slices out of xylene to dry slightly , neutral gum mount.

(10) Microscopic examination, image acquisition and analysis.

Results: Using ONECUT3 antibody as the primary antibody, the expression of endogenous ONECUT3 protein in bone marrow tissue can be detected, mainly in the nucleus; the left side is under 10x microscope, and the right picture is under 40x microscope. (See Figure 6).

In summary, the rabbit polyclonal purified antibody against ONECUT3 antigen polypeptide obtained in the present invention has a specific recognition function for ONECUT3, almost no miscellaneous bands, and reliably reflects the expression level of ONECUT3 protein at the cell level and tissue level. The ability to express endogenous and exogenous OUTCUT3 protein was tested.

Claims

A polypeptide for preparing ONECUT3 antibody, characterized in that the nucleotide sequence of the polypeptide is as shown in SEQ ID NO.1.
The use of the polypeptide according to claim 1 in the preparation of rabbit polyclonal antibody against ONECUT3 antigen protein.
Utilize the polypeptide described in claim 1 to prepare the method for the rabbit polyclonal antibody of anti-ONECUT3 antigenic protein, it is characterized in that comprising the following steps:

1) Coupling the antigenic polypeptide of ONECUT3 with carrier proteins KLH and BSA respectively, and obtaining the coupling product after purification;

2) Take the coupling product to immunize rabbits, take the rabbit serum after multiple immunizations, and use the ELISA method to detect the titer. After the titer reaches the ideal value, boost the immunization once, and collect the immunized rabbit serum;

3) Using antigen affinity purification to obtain polyclonal antibodies.
Application of the rabbit polyclonal antibody against ONECUT3 antigen protein prepared according to claim 3 in detecting endogenous and exogenous ONECUT3.