A polypeptide for preparing one cut3 antibody and rabbit polyclonal antibody thereof, and application

Abstract

The application discloses a polypeptide for preparing ONECUT3 antibody and a rabbit polyclonal antibody of the polypeptide, and belongs to the technical field of biotechnology.The application provides a polypeptide for preparing ONECUT3 antibody, and provides a rabbit polyclonal purified antibody of the ONECUT3 antigen polypeptide and application thereof.The rabbit polyclonal purified antibody of the ONECUT3 antigen polypeptide obtained by the application has the specific recognition function of ONECUT3, almost no cross banding, and can reliably reflect the expression level of ONECUT3 protein at the cell level and the tissue level, and has the ability of detecting endogenous and exogenous expression of OUTCUT3 protein, and the detection ability is superior to the effect of a commercially available antibody.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a polypeptide for preparing ONECUT3 antibody and rabbit polyclonal antibody thereof and application. BACKGROUND

[0002] ONECUT3 and ONECUT1 (HNF6), ONECUT2 belong to the ONECUT family in the original homeobox transcription factor superfamily. Their common structure contains an N-terminal CUT domain and a C-terminal Homeobox domain. The 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. In liver cancer and pancreatic cancer cells, ONECUT1 can bind to the promoter regions of AFP, CyclinD1, Cdc25A, Cdk2 and E2F1 genes to participate in the regulation of cell proliferation and cell cycle, induce S phase cell proliferation, and activate 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 the proliferation and survival of tumor cells through androgen-dependent pathways. The above studies show that ONECUT1 and ONECUT2 in the ONECUT family abnormally express and participate in the pathogenesis of tumors. Whether ONECUT3 participates in the mechanism of occurrence and development of hematological tumors has not been reported.

[0003] The currently available ONECUT3 has the problems of low titer ratio and poor specificity, which is difficult to meet the research needs of ONECUT3 protein detection at the cellular and tissue levels. For example, in practical applications, the antibody of Novus (NBP1-91528) only has detection capability for exogenous overexpression of ONECUT3, and cannot detect endogenous expression of ONECUT3 protein in cells; the antibody of abcam (ab181450) has poor specificity due to the design of the antigen polypeptide sequence of the induced ONECUT3 protein polyclonal antibody, and has the problem of multiple bands during detection. SUMMARY

[0004] In view of the problems in the prior art, the purpose of the present application is to provide a technical scheme of a polypeptide for preparing ONECUT3 antibody and rabbit polyclonal antibody thereof and application.

[0005] The present application is specifically implemented by the following technical scheme:

[0006] The polypeptide for preparing the ONECUT3 antibody has a nucleotide sequence of CMNRWAEEPSTAPG, as shown in SEQ ID NO. 1.

[0007] The polypeptide is used for preparing the rabbit polyclonal antibody against the ONECUT3 antigen protein.

[0008] The method for preparing the rabbit polyclonal antibody against the ONECUT3 antigen protein by using the polypeptide comprises the following steps:

[0009] 1) coupling the antigen polypeptide of ONECUT3 with carrier proteins KLH and BSA respectively, and obtaining the coupling product after purification;

[0010] 2) immunizing rabbits with the coupling product, detecting the titer of the rabbit serum after multiple immunizations by using the ELISA method, strengthening the immunization once when the titer reaches the ideal value, and collecting the immune rabbit serum;

[0011] 3) using antigen affinity purification to obtain the polyclonal antibody.

[0012] The rabbit polyclonal antibody against the ONECUT3 antigen protein prepared by the above method is used for detecting endogenous and exogenous ONECUT3.

[0013] The rabbit polyclonal antibody against the ONECUT3 antigen polypeptide obtained by the method has the specific recognition function for ONECUT3, almost no cross-binding, and can reliably reflect the expression level of the ONECUT3 protein at the cell level and the tissue level, and has the ability to detect the endogenous and exogenous expression of the OUTCUT3 protein, and the detection ability is better than the effect obtained by the commercially available antibody. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is the amino acid sequence of Human ONECUT3.

[0015] Figure 2 It is the ONECUT protein structure analysis diagram.

[0016] Figure 3 It is the antibody peptide segment blocking experiment (Western-Blot diagram) in Example 2.

[0017] Figure 4 It is the knockdown verification antibody specificity experiment (Western-Blot diagram) in Example 3.

[0018] Figure 5 It is the endogenous ONECUT3 expression detection (Western-Blot diagram) of the bone marrow primary sample of the patient with myelodysplastic syndrome in Example 4.

[0019] Figure 6 Figure for the result of immunohistochemical detection of bone marrow tissue ONECUT3 expression in Example 5. DETAILED DESCRIPTION

[0020] The present application is further illustrated below in conjunction with examples.

[0021] Example 1: Design and synthesis of ONECUT3 polypeptide

[0022] 1. Design of antigen protein sequence of ONECUT3 antibody

[0023] The amino acid sequence of human ONECUT3 (UniProtKB-O60422) is shown as follows. Figure 1

[0024] The ONECUT3 protein structure analysis is shown as follows. Figure 2

[0025] According to the surface contactability, hydrophilicity and elasticity of each amino acid in the ONECUT3 protein, the antigenic determinant region of the ONECUT3 protein can be deduced, and therefore the designed peptide sequence is CMNRWAEEPSTAPG (human ONECUT3 469-482 aa).

[0026] 2. Artificially synthesized polypeptide

[0027] The artificially synthesized polypeptide adopts polypeptide solid-phase synthesis method, and the specific steps are as follows:

[0028] (1) Resin weighing: Wang resin is selected, for example, if 0.2 mmol is to be prepared, Wang resin 0.5 mmol / g is selected, that is, the amount of resin is 0.2 mmol divided by 0.5 mmol / g = 0.4 g swelling. Put the weighed resin into the reaction column, then swell with DCM for half an hour. The amount of DCM added is 2-3 times the height of the resin, that is, the resin is fully swelled. A small amount of air can be blown.

[0029] (2) Deprotection. (Cl-resin does not need to be protected in the first step, but after coupling with amino acids, it needs to be protected) The other two kinds of resin need to be deprotected (Fmoc), first dry DCM, then wash with DMF for 3 times to remove DCM, then deprotect with 20% hexahydropyridine, 5+10, that is, a total of two times, 5 minutes for the first time and 10 minutes for the second time, and wash with DMF in between.

[0030] (3) Wash after deprotection. Wash with DMF for 6 times.

[0031] ​​(4) Coupling. We now follow the 3-fold amount of amino acid, i.e. 0,2 mmol of resin, we throw the amount of amino acid 0.2 mmol * 3 = 0.6 mmol times its molecular weight is the mass to be weighed. In addition to the weighed activator DIC and DIEA, etc.

[0032] (5) Detection during coupling. Take resin (a little, about 15 or so, as long as the detection can be identified) with a dropper and a small test tube, respectively, drop 2 drops of A liquid (5 g of indantrione dissolved in 100 ml of ethanol), 2 drops of B liquid (80 g of phenol dissolved in 20 ml of ethanol), 2 drops of C liquid (2 ml of 0.001 M KCN added to 98 ml of heavy pyridine), put into heating for 3 minutes. Then take it out to see if the resin is transparent, if the solution color is deep, it can affect observation, the solution can be poured out, add a little DMF to wash, and then observe. If the resin is transparent, the next amino acid can be coupled. If the resin is not transparent, extend the reaction time, if it is still not transparent, re-coupling can be performed. Make sure that each step is completely reacted before coupling the next amino acid.

[0033] (6) Washing after coupling. After coupling is complete, wash with DMF 3 times.

[0034] (7) The above is the step of coupling one amino acid, repeat steps 3-6 until the last amino acid is coupled.

[0035] (8) After the last amino acid is coupled, deprotection, after deprotection, shrink, DMF wash 3 times, DCM 3 times, methanol 3 times, and dry the peptide resin.

[0036] (9) Weigh the completed dry peptide resin, put it into a 50 ml centrifuge tube, add the lysis solution (10 times ml per gram), such as: 1 g of peptide resin plus 10 ml of lysis solution. Add lysis solution D (TFA: TIS: Water = 95:2.5:2.5), react for 2 hours, shake every 10-15 minutes, so that it reacts fully, can be put into a shaker (25°C). After the reaction is complete, filter, pour the solution into ice ethyl ether to precipitate, then centrifuge, centrifuge 3-4 times.

[0037] (10) Put the centrifuged peptide into the fume hood and blow it for a while, then put it into a vacuum dryer to dry.

[0038] (11) Take a small amount to a sample tube, fill in the detection sheet and send it to the quality inspection department for mass spectrometry to determine whether the mass spectrum is correct, if correct, fill in the sheet and send the remaining peptide to purification.

[0039] 3. Polypeptide coupling

[0040] 1) Dissolve 20 mg of KLH in 2 mL of 5 mM EDTA aqueous solution.

[0041] 2) Weigh 8 mg Sulfo-SMCC completely dissolved in 50 μL DMSO, then add 150 μL 1 x PBS, mix well.

[0042] 3) Add Sulfo-SMCC solution dropwise to KLH, shake gently while adding (vigorous shaking will produce a precipitate), and place at room temperature for 1 h.

[0043] 4) Place the above activated KLH solution in a dialysis bag, clamp the dialysis clamp, dialyze in 2 L of 1 x PBS, and dialyze at 4°C in a refrigerator with magnetic stirring for 1 h.

[0044] 5) Change the new 1 x PBS and dialyze for 2 h, repeat once. Place the activated and dialyzed KLH in a 15 mL import centrifuge tube, and label the tube with the reagent name, time, and concentration, and store in a 4°C refrigerator.

[0045] 6) Weigh 4 mg of polypeptide (peptide sequence CMNRWAEEPSTAPG) and dissolve in 50 μL of DMSO, then add 200 μL of 1 x PBS, mix quickly, then immediately add KLH according to the ratio of polypeptide: KLH = 1 mg: 680 μg, and react overnight at 4°C in a refrigerator or for 2 h at room temperature.

[0046] 7) Place the above cross-linked KLH-peptide cross-linked complex in a dialysis bag, clamp the dialysis clamp, dialyze in 4 L of 1 x PBS, and dialyze overnight at 4°C in a refrigerator with magnetic stirring.

[0047] 8) Take the dialyzed KLH-peptide out of the clean 1.5 mL centrifuge tube, and divide it according to the immunization dose, and store in a -20°C refrigerator.

[0048] 4. Immunize rabbits to obtain antisera

[0049] 1) Animal selection: New Zealand white rabbits, weighing about 2.5 Kg, young and healthy. The fur should be shiny, and the animal should be active. Choose good animals and pre-culture for about 2 weeks. The purpose is to eliminate some unqualified animals to ensure smooth progress of the later experiment.

[0050] 2) Preparation before experiment: Label the rabbits.

[0051] 3) Antigen preparation:

[0052] 3.1) Take the antigen (coupling product in 3) out of the -20°C refrigerator and dissolve at room temperature to avoid repeated freezing and thawing.

[0053] 3.2) Draw the antigen (antigen completely mixed), the first immunization antigen concentration is 1 mg / ml, 0.5 ml per rabbit, and the second to fourth immunization antigen concentration is halved, with the same dose.

[0054] 3.3) Extract the adjuvant, the adjuvant and antigen are extracted at a volume ratio of 1:1. The first immunization uses complete adjuvant, and the second-fourth immunization uses incomplete adjuvant. When extracting, the adjuvant should be mixed evenly before being extracted into the syringe.

[0055] 3.4) Two syringes are connected by a needle tube and then emulsified completely. The emulsification standard is that the emulsified immunogen does not disperse when dropped into 37-degree water.

[0056] 4) Immunization: The rabbit is injected subcutaneously at multiple points, with each point being 0.2 ml.

[0057] Immunization time: The second immunization is performed 14 days after the first immunization, and the interval between the second and third immunizations is 7 days. The rabbit is sampled for serum detection 7 days after the third immunization, and the detection is qualified. After 7 days, the rabbit is immunized again, and the whole blood can be collected after 7 days of the last immunization.

[0058] 5. ELISA detection (indirect method)

[0059] 1) Coating: The known antigen is diluted to 1 μg / ml with coating buffer (Na2CO3 and NaHCO3 buffer), 50 μl is added to each polystyrene plate reaction well, and it is incubated at 4°C overnight. The next day, the solution in the well is discarded, and the well is washed with 1x TBST washing buffer at 180 μl per well.

[0060] 2) Blocking: 60 μl of 1% BSA (prepared with TBST) is added to each well for blocking, and it is incubated at 37°C for 1 hour. Then the blocking solution is discarded.

[0061] 3) Sample addition: A certain dilution of the sample to be tested (the sample to be tested is diluted according to a certain ratio) is added to the above-mentioned blocked reaction well, 50 μl. 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 the wells with 1x TBST washing buffer for 2 times at 180 μl per well.

[0062] 4) Add enzyme-labeled antibody: Freshly diluted secondary antibody-HRP (1:5K, diluted with 1% BSA) is added to the enzyme-labeled plate wells at 50 μl / well, and it is incubated at 37°C for 45 min. Then the blocking solution is discarded, and the wells are washed with 1x TBST washing buffer for 3 times at 180 μl per well.

[0063] 5) Color development by adding substrate solution: 100 μl of TMB substrate solution prepared on the spot is added to each reaction well, and it is reacted at 37°C for 5 min.

[0064] 6) Stop the reaction: Add 2M sulfuric acid 90 μl to each reaction well.

[0065] 7) Read the plate: Place the plate in a preheated microplate reader (450 nm) and read the plate, save the data, and analyze.

[0066] 6. Purify the antiserum with the antigen polypeptide to obtain purified antibody

[0067] 1) Wash the affinity column with 20 mL of pure water and 1 x PBS (pH 7.4) at a flow rate of 70 mL / h.

[0068] 2) Take 10 mL of serum to be purified in a 50 mL centrifuge tube, and filter it with a 0.45 μm pore size, 25 mm diameter microfiltration membrane.

[0069] 3) Load the filtered serum sample at a flow rate of 40 mL / h, and repeat once.

[0070] 4) Wash the column with 20 mL of 1 x PBS (pH 7.4) at a flow rate of 70 mL / h, and after 10 minutes, connect the protein detector. Adjust the instrument transmittance (T scale) to 100 during the washing process.

[0071] 5) Adjust the protein detector absorbance (1A scale) to 0, at which point open the computer desktop HD-A computer collector and set the full screen range to 5. Elute the antibody with glycine solution (pH 2.7, 0.2M) at a speed of 40 mL / h. At this time, press the green elution record button to start elution, and when the instrument reading starts to rise, start collecting the antibody.

[0072] 6) During the antibody collection process, adjust the pH of the antibody to around 7 with 1M sodium bicarbonate in a timely manner, and record the highest peak value of the elution peak.

[0073] 7) After the antibody collection is complete, adjust the pH to around 7, and record the volume of the eluted antibody. Then rinse the rubber tube connected to the collector with pure water.

[0074] 8) Wash the affinity column with 20 mL of 1 x PBS and pure water at a speed of 70 mL / h, then add 20% ethanol, seal, and store in a 4°C refrigerator.

[0075] 9) The purified antibody is sent for testing according to different requirements: ELISA, WB, ICC, etc.

[0076] Results

[0077] 1. Polypeptide information

[0078] Polypeptide Number Sequence Information HAPM1067-1 469-482a.a.CMNRWAEEPSTAPG

[0079] 2. Immunization cycle table

[0080]

[0081] 3. Detection results of serum ELISA

[0082]

[0083]

[0084] Conclusion: 5206 immune serum is qualified (based on ELISA judgment).

[0085] Definition: OD450 value of 1:256000 is P value (positive value), OD450 value of BSA hole is N value (negative value);

[0086] 5206 P / N = (0.494 + 0.367) / 2 ÷ (0.167 + 0.098) / 2 = 3.2 > 2.1, reaching the positive qualified standard.

[0087] Basic ELISA is qualified.

[0088] 5207 P / N = (0.420 + 0.428) / 2 ÷ (0.107 + 0.098) / 2 = 4.1 > 2.1, reaching the positive qualified standard.

[0089] Basic ELISA is qualified.

[0090] Example 2: Antibody peptide segment blocking experiment

[0091] (1) Dissolve 1 mg of peptide segment (HAPM1067-1) in enzyme-free sterilized water to a final concentration of 1 mg / ml;

[0092] (2) Take two 15 ml centrifuge tubes, one of which is configured with antibody and peptide segment premix solution, Rabbit Anti-ONECUT3 antibody (#5206, Conc. 0.5 mg / ml) and peptide segment are added to 5% skimmed milk, the molar ratio of antibody to peptide segment is 1:2, which is the blocked antibody; the other tube only adds Rabbit Anti-ONECUT3 antibody (#5206, Conc. 0.5 mg / ml) in 5% skimmed milk, i.e. the molar ratio of antibody to peptide segment is 1:0, which is the antibody control; 4°C overnight;

[0093] (3) Collect patient bone marrow mononuclear cells, add 50 μL SDS protein denaturation lysis solution according to 1x 10E6 cells, mix well by blowing, and lyse on ice for 10 min, Bioruptor ultrasonic (power High, ultrasonic 10 s, stop 10 s, 4-6 cycles);

[0094] (4) Equal amounts of samples are subjected to 10% SDS-PAGE gel electrophoresis;

[0095] (5) When bromophenol blue electrophoresis to the bottom, stop and PDVF membrane transfer membrane;

[0096] (6) 5% skim milk room temperature blocked 1h;

[0097] (7) After cutting the membrane, respectively, placed in the control antibody and blocked antibody, 4℃ refrigerator incubated overnight, 1x TBST membrane washing 3 times (10 min each time);

[0098] (8) Goat Anti-rabbit secondary antibody room temperature incubation 1h, 1x TBST membrane washing 3 times (10 min each time);

[0099] (9) After washing the membrane, add chemical developer (Pierce TM SuperSignal TM West Pico PLUS Chemiluminescent, Thermo), placed in the chemiluminescence developer (ChemiDoc MP imaging system, Bio-Rad) development.

[0100] (10) After elution with eluent (Restore PLUS Western Blot Stripping Buffer, Thermo) elution 15 min, 5% milk room temperature blocked 1h;

[0101] (11) Incubation Anti-Actin HRP (Huabio, ET1702-67) room temperature 1h, 1x TBST membrane washing 3 times (10 min each time); again add chemical developer (Pierce TM SuperSignal TM West Pico PLUS Chemiluminescent, Thermo), placed in the chemiluminescence developer (ChemiDoc MP imaging system, Bio-Rad) development.

[0102] Results: antibody control group (i.e. antibody to peptide = 1:0) can see ONECUT3 endogenous band (about 54kDa); antibody blocking group (i.e. antibody to peptide = 1:2), no signal in lane, suggesting that the ONECUT3 antibody can be blocked by the peptide (see Figure 3 ).

[0103] Example 3: knockdown verification antibody specificity experiment

[0104] (1) Construction of Tet-pLKO-hOC3 plasmid: Tet-pLKO-puro vector (purchased from the United States Addgene company) was double digested by Ecor I and Age I; the following base sequence was synthesized and inserted into the double enzyme cutting empty vector:

[0105] ShOC3 Forward: CCGGcagcatcccgcaggcaatc CTCGAGgattgcctgcgggatgctg TTTT (as set forth in SEQ ID NO. 2);

[0106] ShOC3 Reverse: AATTAAAAcagcatcccgcaggcaatc CTCGAGgattgcctgcgggatgctg (as set forth in SEQ ID NO. 3); After transformation, single colonies were picked for sequencing; the plasmid with correct sequence was extracted;

[0107] (2) Take 500 μl of serum-free Opti-MEM medium in two branches, add 40 μl of liposome transfection reagent PolyJet (Signagen) and 9 μg of plasmid respectively, mix the plasmid with the transfection reagent thoroughly, stand at room temperature for 10-15 min, then drop into the cell culture dish. The control group does not add doxycycline, and the knockdown group adds 1 μg / ml of doxycycline;

[0108] (3) Collect the control and knockdown cells after 72 h, add 50 μL SDS protein denaturation lysis solution according to 1x 10E6 cells, mix thoroughly, and lyse on ice for 10 min, and then perform ultrasonic treatment (power High, ultrasonic treatment for 10 s and stop for 10 s, 4-6 cycles) by using Bioruptor;

[0109] (4) Equal amounts of samples are subjected to 10% SDS-PAGE gel electrophoresis;

[0110] (5) Stop when the bromophenol blue electrophoresis reaches the bottom and perform PDVF membrane transfer;

[0111] (6) Block at room temperature for 1 h with 5% skimmed milk;

[0112] (7) Incubate the primary antibody Rabbit Anti-ONECUT3 antibody (#5206, Conc. 1 mg / ml) overnight in a 4°C refrigerator, and wash the membrane with 1x TBST for 3 times (10 min each time);

[0113] (8) Incubate the secondary antibody Goat Anti-rabbit at room temperature for 1 h, and wash the membrane with 1x TBST for 3 times (10 min each time);

[0114] (9) After washing the membrane, add chemical developing solution (Pierce TM SuperSignal TMWest Pico PLUS Chemiluminescent, Thermo), and developed in a chemiluminescent imager (ChemiDoc MP Imaging System, Bio-Rad).

[0115] (10) After eluted with elution buffer (Restore PLUS Western Blot Stripping Buffer, Thermo) for 15 min, 5% milk was used to block at room temperature for 1 h;

[0116] (11) Incubated with Anti-Actin HRP (Huabio, ET1702-67) at room temperature for 1 h, washed the membrane with 1x TBST for 3 times (10 min each time), and then added the chemiluminescent solution and developed in a chemiluminescent imager (ChemiDoc MP Imaging System, Bio-Rad).

[0117] Results: The endogenous band of ONECUT3 (about 54 kDa) was observed in the control group (i.e. -DOX group); no signal was observed in the knockdown group (i.e. +DOX group), indicating that the antibody specifically recognized human ONECUT3 protein (see Figure 4 ). Example 4: Detection of endogenous ONECUT3 expression in bone marrow primary samples of patients with myelodysplastic syndrome (WB)

[0118] (1) Construction of pcDNA3.1-hOC3 plasmid: The human ONECUT3 CDS sequence was synthesized in pcDNA3.1-empty (completed by Fenghui Biological), and the plasmid was sequenced and identified;

[0119] (2) Take a 10 cm culture dish as an example, when the 293T cells grow to 70% density, take 500 μl of serum-free Opti-MEM medium in two branches, add 40 μl of lipofectamine transfection reagent PolyJet (Signagen) and 9 μg of plasmid respectively, mix the plasmid with the transfection reagent, stand at room temperature for 10-15 min, and then drop into the cell culture dish;

[0120] (3) Collect the control and knockdown cells after 72 h, add 100 μL SDS protein denaturation lysis buffer according to 1x 10E6 cells, mix well by blowing, and lyse on ice for 10 min, and then use Bioruptor ultrasonic (power High, ultrasonic for 10 s and stop for 10 s, 4-6 cycles) as a positive control;

[0121] (4) Take 2 samples of bone marrow samples from patients with myelodysplastic syndrome with complex karyotype (P1 and P2), bone marrow samples from patients with myelodysplastic syndrome with normal karyotype (P3 and P4), and 5 ml of each bone marrow sample and healthy donor bone marrow samples (D1 and D2) in a 15 ml centrifuge tube;

[0122] (5) Collect single nuclear cells by relative density gradient centrifugation with Ficoll human lymphocyte separation medium, count the cells, and mix 1x 10E6 cells with 50 μL SDS protein denaturation lysis solution, blow and mix, lyse on ice for 10 min, and perform Bioruptor ultrasonication (power High, ultrasonic for 10 s and stop for 10 s, 4-6 cycles); as a detection sample;

[0123] (6) 3 μL of the positive sample (1x loading volume) and 15 μL of the sample to be detected are subjected to 10% SDS-PAGE gel electrophoresis;

[0124] (7) Stop when the bromophenol blue electrophoresis reaches the bottom and perform PDVF membrane transfer;

[0125] (8) Block with 5% skim milk at room temperature for 1 h;

[0126] (9) Rabbit Anti-ONECUT3 antibody (#5206, Conc. 1 mg / ml) primary antibody is incubated overnight in a 4°C refrigerator, and the membrane is washed with 1x TBST for 3 times (10 min each time);

[0127] (10) Goat Anti-rabbit secondary antibody is incubated at room temperature for 1 h, and the membrane is washed with 1x TBST for 3 times (10 min each time);

[0128] (11) After washing the membrane, chemical developing solution (Pierce TM SuperSignal TM West Pico PLUS Chemiluminescent, Thermo) is added, and the membrane is developed in a chemiluminescence developer (ChemiDoc MP imaging system, Bio-Rad).

[0129] (12) After elution with eluent (Restore PLUS Western Blot Stripping Buffer, Thermo) for 15 min, block with 5% milk at room temperature for 1 h;

[0130] (13) Incubate Anti-Actin HRP (Huabio, ET1702-67) at room temperature for 1 h, wash the membrane with 1x TBST for 3 times (10 min each time), add the chemiluminescent reagent again, and develop in the chemiluminescent imager (ChemiDoc MP Imaging System, Bio-Rad).

[0131] Results: Using ONECUT3 antibody as the primary antibody, endogenous and exogenous ONECUT3 protein expression can be detected, and the expression of ONECUT3 in bone marrow cells of patients with myelodysplastic syndrome with complex karyotype group is relatively higher than that in normal karyotype group, and the expression of ONECUT3 in bone marrow cells of healthy donors is low (see Figure 5 ).

[0132] Example 5: Immunohistochemical detection of ONECUT3 expression in bone marrow tissue

[0133] (1) Paraffin section deparaffinization to water: put the section into xylene I 8 min-xylene II 8 min-xylene III 8 min-anhydrous ethanol I 5 min-anhydrous ethanol II 5 min-85% alcohol 5 min-75% alcohol 5 min, and wash with tap water for 2 min.

[0134] (2) Antigen repair: put the tissue section into the repair box filled with citric acid antigen repair buffer (PH 6.0) in the microwave oven for antigen repair, medium heat for 8 min to boiling, stop heating for 8 min, and then turn to low heat for 7 min. During this process, prevent the buffer from evaporating too much, and do not dry the slice. After natural cooling, put the slide into PBS (PH 7.4) and shake for 3 times, 5 min each time.

[0135] (3) Circle drawing: use a group-specific group drawing pen to draw a small circle with a spacing of 3-4 mm from the tissue periphery, and then add enough PBS to ensure that the subsequent sequentially added blocking serum, primary antibody, secondary antibody, and color developing agent can completely cover the tissue without flowing along the slide.

[0136] (4) Block endogenous peroxidase: add endogenous peroxidase in the kit to the section, 50-100 ul per section, incubate at room temperature for 25 min in the dark, and put the slide into PBS (PH 7.4) and shake for 3 times, 5 min each time.

[0137] (5) Serum blocking: add 3% BSA evenly to the tissue in the group circle, and block at room temperature for 30 min.

[0138] (6) Add primary antibody: gently shake off the blocking solution, and add Rabbit Anti-ONECUT3 antibody (1:100) prepared in PBS to the section, and incubate the section flat in the wet box at 4° overnight.

[0139] (7) Add secondary antibody: Place the slide in PBS (pH 7.4) and shake on a decolorizing shaker for 3 times, 5 min each time. After slightly shaking dry the section, add the universal secondary antibody (HRP labeled) for the hypersensitive rabbit mouse to cover the tissue in the circle, incubate at room temperature for 50 min.

[0140] (8) Restain the cell nucleus: Stain with hematoxylin for 2-3 min, wash with tap water, differentiate in 1% hydrochloric acid alcohol differentiation solution for a few seconds, wash with tap water, return to blue in ammonia water for 15-30 s, and wash with running water.

[0141] (9) Dehydrate and mount the section: sequentially place the section in 75% alcohol for 5 min, 85% alcohol for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, xylene I for 5 min, take out the section from xylene, slightly dry, and mount with neutral balsam.

[0142] (10) Microscope examination, image collection and analysis.

[0143] 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 is 10x under the microscope, and the right is 40x under the microscope. (See Figure 6 ).

[0144] In summary, the rabbit polyclonal purified antibody of the anti-ONECUT3 antigen polypeptide obtained by the application has specific recognition function for ONECUT3, almost no banding, and can reliably reflect the expression level of ONECUT3 protein at the cell level and tissue level, and has the ability to detect endogenous and exogenous expression of OUTCUT3 protein. SEQUENCE LISTING <110> The First Affiliated Hospital of Medical College of Zhejiang University <120> A polypeptide for preparing ONECUT3 antibody and rabbit polyclonal antibody thereof, and application <160> 3 <170> SIPOSequenceListing 1.0 <210> 1 <211> 14 <212> PRT <213> Artificial Sequence <400> 1 Cys Met Asn Arg Trp Ala Glu Glu Pro Ser Thr Ala Pro Gly 1 5 10 <210> 2 <211> 52 <212> DNA <213> Artificial Sequence <400> 2 ccggcagcat cccgcaggca atcctcgagg attgcctgcg ggatgctgtt tt 52 <210> 3 <211> 52 <212> DNA <213> Artificial Sequence <400> 3 aattaaaaca gcatcccgca ggcaatcctc gaggattgcc tgcgggatgc tg 52

Claims

1. A polypeptide for the preparation of an ONECUT3 antibody, characterized in that, The amino acid sequence of the polypeptide is as shown in SEQ ID NO.

1.

2. Use of the polypeptide of claim 1 in the preparation of rabbit polyclonal antibodies against the ONECUT3 antigen protein.

3. A method of preparing a rabbit polyclonal antibody against an ONECUT3 antigen protein using the polypeptide of claim 1, characterized by, The method comprises the following steps: 1) coupling the polypeptide for preparing the ONECUT3 antibody with a carrier protein KLH, and obtaining a coupling product after purification; 2) immunizing rabbits with the coupling product, collecting the rabbit serum after multiple immunizations, detecting the titer by ELISA, and collecting the immune rabbit serum after one-time booster immunization when the titer reaches an ideal value; 3) using antigen affinity purification to obtain the polyclonal antibodies.

Images