Preparation method and application of chicken dkk1 protein polyclonal antibody

Abstract

The application discloses a preparation method of chicken DKK1 protein polyclonal antibody, and relates to the technical field of poultry genetic engineering, which comprises the following steps: (1) constructing a chicken DKK1 gene recombinant expression plasmid by using a nucleotide shown in SEQ ID No. 1 and a prokaryotic expression vector; (2) transforming host cells with the chicken DKK1 gene recombinant expression plasmid, then screening and inducing expression to obtain a His-DKK1 fusion protein and purify the same; and (3) immunizing animals with the purified His-DKK1 fusion protein, and then obtaining chicken DKK1 protein polyclonal antibody through serum separation and purification. The application further provides application of the chicken DKK1 protein polyclonal antibody in a chicken DKK1 protein detection reagent or kit. The application can specifically recognize DKK1 protein in chicken tissues at different development stages, and provides an important tool for functional research of DKK1 on chickens.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of poultry genetic engineering technology, and particularly relates to a preparation method and application of chicken DKK1 protein polyclonal antibody. BACKGROUND

[0002] Dickkopf-related protein 1 (DKK1) is a typical inhibitor of Wnt / β-catenin signaling pathway, and belongs to the DKK family (the rest includes DKK2, DKK3 and DKK4). DKK1 is a secreted glycoprotein, mainly secreted by osteoblasts and bone cells, and has an important regulatory effect on cell life activities, thus playing an important role in animal growth and development.

[0003] At present, the commercial DKK1 antibody is mainly a mouse or human antibody, and these DKK1 antibodies cannot specifically recognize DKK1 protein in chicken tissues, which greatly limits the research and application of DKK1 in chickens. Therefore, it is of great significance to develop a kind of DKK1 protein that can specifically recognize DKK1 protein in chicken tissues. SUMMARY

[0004] The present application discloses a preparation method and application of chicken DKK1 protein polyclonal antibody, which can specifically recognize DKK1 protein in chicken tissues.

[0005] The technical scheme adopted by the present application is as follows:

[0006] One of the purposes of the present application is to provide a nucleotide for constructing a chicken DKK1 gene recombinant expression plasmid, and the sequence of the nucleotide is shown in SEQ ID No. 1.

[0007] The second purpose of the present application is to provide a preparation method of chicken DKK1 protein polyclonal antibody, which comprises the following steps:

[0008] (1) The above nucleotide is cloned into a prokaryotic expression vector with a His tag by using PCR amplification and recombinase ligation technology, and a chicken DKK1 gene recombinant expression plasmid is constructed;

[0009] (2) The chicken DKK1 gene recombinant expression plasmid is transformed into host cells, and then screened and induced to express to obtain a His-DKK1 fusion protein and purify the same; the His-DKK1 fusion protein contains amino acids shown in SEQ ID No. 2;

[0010] (3) The purified His-DKK1 fusion protein is used to immunize animals, and then chicken DKK1 protein polyclonal antibody is obtained by serum separation and purification.

[0011] As a kind of preference, the prokaryotic expression vector is pET-28a(+).

[0012] Specifically, the step (2) comprises the following steps:

[0013] (201) the chicken DKK1 gene recombinant expression plasmid is introduced into host cell, and the transformation product is obtained;

[0014] (202) the transformation product is coated on the TB plate containing kanamycin, and the positive clone is screened;

[0015] (203) IPTG induction method is used to induce host cell to express His-DKK1 fusion protein;

[0016] (204) the NI 2+ affinity chromatography column is used to separate and purify His-DKK1 fusion protein.

[0017] As a kind of preference, the host cell is BL21 (DE3) competent cell.

[0018] As a kind of preference, the NI 2+ affinity chromatography column is Ni-IDA agarose gel.

[0019] Specifically, the step (3) comprises the following steps:

[0020] (301) the purified His-DKK1 fusion protein is mixed with equal amount of complete Freund's adjuvant, and then the animal is immunized for the first time;

[0021] (302) the purified His-DKK1 fusion protein is mixed with equal amount of incomplete Freund's adjuvant, and then the animal is immunized for 2-3 times, and the interval time between each immunization is 2 to 3 weeks;

[0022] (303) blood is collected to prepare antiserum;

[0023] (304) the NI 2+ affinity chromatography column is used to separate and purify the antiserum, and the chicken DKK1 protein polyclonal antibody is obtained.

[0024] Further, the immunized animal is rabbit, mouse or rat.

[0025] The third object of the present application is to provide the polyclonal antibody in chicken DKK1 protein detection reagent or kit.

[0026] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0027] (1) This invention utilizes gene recombination technology to prepare a polyclonal antibody against chicken DKK1 protein by designing suitable target nucleotides. Experiments show that the polyclonal antibody against chicken DKK1 protein prepared in this invention exhibits high specificity, capable of specifically recognizing DKK1 protein in chicken tissues at different developmental stages, thus making it suitable for use in chicken DKK1 protein detection reagents or kits. Therefore, this invention provides an important tool for functional studies of DKK1 in chickens and is highly suitable for large-scale application.

[0028] (2) The present invention uses pET-28a(+) as a prokaryotic expression vector, which not only has a good expression effect of His-DKK1 fusion protein, but also has soluble expression, which is very beneficial for subsequent purification.

[0029] (3) This invention uses NI 2+ Affinity chromatography columns can specifically bind to proteins with His tags but not to proteins without His tags, thereby achieving efficient selective purification and effectively maintaining the stability of the higher-order structure of biological macromolecules.

[0030] (4) The raw materials used in gene recombination of this invention are inexpensive, easy to obtain, and have a high degree of antibody separation and purification, good animal immunization effect, and appropriate antibody titer, which effectively ensures the quality of commercialized chicken DKK1 protein polyclonal antibody. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the enzyme digestion identification results of the recombinant expression plasmid pET-28a(+)-DKK1 in an embodiment of the present invention, wherein 1 is the recombinant expression plasmid before enzyme digestion, 2 is the recombinant expression plasmid after enzyme digestion, and M is the DNA marker.

[0032] Figure 2 This is a schematic diagram of the His-DKK1 fusion protein expression identification results in an embodiment of the present invention, where M is the protein marker, 1 is the induced host cell BL21 (pET-28a(+)-empty vector), 2 is the uninduced host cell BL21 (pET-28a(+)-DKK1), 3 is the induced host cell BL21 (pET-28a(+)-DKK1), 4 is the supernatant after lysis of the induced host cell BL21 (pET-28a(+)-DKK1), and 5 is the precipitate after lysis of the induced host cell BL21 (pET-28a(+)-DKK1).

[0033] Figure 3Figure showing the results of purification of the His-DKK1 fusion protein of the present application, wherein M represents a protein marker, 1 represents the supernatant after disruption of the induced host cells BL21 (pET-28a(+)-DKK1), 2 represents the flow-through, and 3-4 represent the purified His-DKK1 fusion protein collected after elution.

[0034] Figure 4 Figure showing the results of Western Blot identification of the purified His-DKK1 fusion protein of the present application, wherein M represents a protein marker, 1 represents the purified His-DKK1 fusion protein, and the arrow indicates the target protein band.

[0035] Figure 5 Figure showing the results of identification of the purity of the chicken DKK1 protein polyclonal antibody of the present application, wherein M represents a protein marker, 1 represents 0.5 mg / mL BSA, and 2 represents the purified chicken DKK1 protein polyclonal antibody.

[0036] Figure 6 Figure showing the results of mass spectrometry detection of the purified chicken DKK1 protein polyclonal antibody of the present application.

[0037] Figure 7 Figure showing the results of detection of DKK1 protein in chicken skin tissue using the chicken DKK1 protein polyclonal antibody of the present application. DETAILED DESCRIPTION

[0038] In order to make the objectives, technical solutions and advantages of the present application clearer, the present application is further described in detail below with reference to the embodiments.

[0039] EMBODIMENT

[0040] The present embodiment provides a chicken DKK1 protein polyclonal antibody for facilitating the research and application of DKK1 in chickens. The preparation of the chicken DKK1 protein polyclonal antibody described above mainly comprises three processes, i.e., cloning of the chicken DKK1 gene, expression and purification of the chicken DKK1 recombinant protein, and preparation of the chicken DKK1 protein polyclonal antibody, which are described in detail below.

[0041] I. Cloning of the chicken DKK1 gene

[0042] The recombinant expression plasmid of the chicken DKK1 gene was constructed by PCR amplification and recombinase ligation technology, wherein the nucleotides shown in SEQ ID No. 1 were cloned into the vector pET-28a(+), and the recombinant expression plasmid was named as pET-28a(+)-DKK1.

[0043] SEQ ID No. 1:

[0044] CATATGGGTGGTGGTCTGAGCAGCAATGCCATTAAGGTGGGTGCAGCCGCAAGCGCAGCCCCGGCAGCAGCAGCACCTGCACCTTTTGATGGCAGCAATAAGGCACCGCCGGCAGCAACCCGCCAGCCTTTTCCGTGTAGTGAAGATGAAGATTGTGGTCCGGATGAATTTTGCGGTGGCGCAGCCCGCGGCGGTGGTCCTCCTTTATGCCTGGCATGTCGCCGCCGCCGCAAACGTTGTCTGCGCGATGCAATGTGCTGTCCGGGCACCGCATGTAGCAATGGCCTGTGCACCCCGCCGGAACCGCCGCCTGGTGCACCTGAACTGGATGAACCGGGTCCGGAAGCACTGCCGCGCCGTACACCGGCTCCGGCATGGCCTCCTGATGCCAAAGGTGAAGAAGGTGACTTTTGTCTGCGTAGCAGCGATTGCGCAGCAGGCCTGTGCTGCGCACGTCATTTTTGGAGCAAAATTTGCAAACCGGTTCTGCGTGAAGGCCAGGTGTGTACCCGCCATCGCCGTAAAGGTGCACATGGTCTGGAAATTTTTCAGCGCTGCCCGTGTGCAGAAGGTATGGCCTGCCGCCCGCAGCGTGATCGTGGCGGTCCTGATGCAAGCCGCCTGCATACCTGCCAGCGTCATTAACTCGAG, 651 bp.

[0045] PCR amplification reaction system was 10 μmol·L -1 1 μL of each of the upstream and downstream primers, 100 ng·μL -1 Gene synthesis product (recombinant expression plasmid pET-28a(+)-DKK1) 0.3 μL, 5 U / μL Pfu DNA polymerase 0.5 μL, 10×Taq Pfubuffer 5 μL, add ddH2O to 50 μL, reaction program was 95℃, 3 min; 95℃, 25 s, 62℃, 20 s, 72℃, 40 s, 25 cycles; 72℃, 1 min. The PCR product was analyzed by gel electrophoresis, and the product was recovered for use.

[0046] The recombination enzyme ligation reaction system is: 4 μL of PCR product recovered by gel purification, 3.5 μL of linearized pET-28a(+) vector, and 2.5 μL of recombination DNA ligase. The recombination enzyme DNA ligase ligation reaction procedure is: 50°C water bath for 25 min, and then placed at room temperature for 2-3 min to reduce the temperature, and then transformed.

[0047] II. Expression and purification of chicken DKK1 recombinant protein

[0048] 1. 1 μL of pET-28a(+)-DKK1 recombinant expression plasmid was added to 100 μL of BL21(DE3) competent cells (host cells), and placed on ice for 20 min, then heated at 42°C for 90 s, quickly placed on ice for 5 min, added with 600 μL of TB medium, and then incubated at 37°C and 220 r / min for 1 h, and then coated on a TB plate containing 50 μg / mL kanamycin, and the positive clones were selected for enzyme digestion identification, and the results are shown in Figure 1 The obtained host cells were named as: BL21(pET-28a(+)-DKK1).

[0049] 2. The IPTG induction method was used to induce the host cells to express His-DKK1 fusion protein (recombinant protein antigen), and the specific steps are as follows:

[0050] The host cells BL21(pET-28a(+)-DKK1) were inoculated in a test tube containing 3 ml of TB culture solution containing 50 μg / mL kanamycin, and incubated at 37°C and 220 r / min overnight; the next day, inoculated in 30 ml of TB culture solution containing 50 μg / mL kanamycin at a ratio of 1:100, and incubated at 37°C and 220 r / min until the OD600 of the bacterial cells was 0.6-0.8.

[0051] Then, 1 ml of the culture was taken out, centrifuged at 10000 r / min and room temperature for 2 min, the supernatant was discarded, and the bacterial cell pellet was resuspended with 100 μL of 1x SDS loading buffer. Then, IPTG was added to the remaining culture to a final concentration of 0.2 mM, and the culture was incubated at 37°C and 220 r / min for 4 h to induce the expression of the fusion protein.

[0052] Then, 1 ml of the culture was taken out, centrifuged at 10000 r / min and room temperature for 2 min, the supernatant was discarded, and the bacterial cell pellet was resuspended with 100 μL of 1x SDS loading buffer. Then, the remaining culture was centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the pellet was resuspended with PBS.

[0053] After the resuspension solution was broken by ultrasonic, the supernatant and the precipitate were taken and resuspended in loading buffer, and then 12% SDS-PAGE detection analysis was performed, and the bands were shown by Coomassie brilliant blue staining, as shown in Figure 2 The target protein mainly exists in the supernatant.

[0054] 3, the purified His-DKK1 fusion protein was separated and purified by using a Ni 2+ affinity chromatography column, specifically as follows:

[0055] First, the supernatant solution was loaded into a Ni-IDA Binding-Buffer pre-equilibrated Ni-IDA-Sepharose CL-6B affinity chromatography column at a flow rate of 0.5 ml / min using a low-pressure chromatography system; and the column was washed with Ni-IDA Binding-Buffer at a flow rate of 0.5 ml / min until the OD280 value of the effluent reached the baseline.

[0056] Then, the column was washed with Ni-IDA Washing-Buffer (20 mM Tris-HCl, 20 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 ml / min until the OD280 value of the effluent reached the baseline, and then the target protein was eluted with Ni-IDA Elution-Buffer (20 mM Tris-HCl, 250 mM imidazole, 0.15 M NaCl, pH 8.0) at a flow rate of 1 ml / min, and the effluent was collected.

[0057] The collected effluent was added to a dialysis bag and dialyzed overnight using 20 mM Tris-HCl, 0.15 M NaCl, pH 8.0. Finally, 12% SDS-PAGE analysis was performed, and the results are shown in Figure 3 It can be seen that the purified His-DKK1 fusion protein has high purity. Western Blot analysis was performed on the purified His-DKK1 fusion protein using His antibody as the primary antibody and HRP-labeled goat anti-mouse IgG polyclonal antibody (purchased from Nanjing Zhongding Biotechnology Co., Ltd.) as the secondary antibody, and the results are shown in Figure 4 As can be seen from the figure, a target band appears at the expected size.

[0058] The His-DKK1 fusion protein prepared in this example contains the amino acid shown in SEQ ID No. 2.

[0059] SEQ ID No. 2:

[0060] MGGGLSSNAI KVGAAASAAPAAAAPAPFDGSNKAPPAATRQPFPCSEDEDCGPDEFCGGAARGGGPPLCLACRR RRKRCLRDAMCCPGTACSNGLCTPPEPPPGAPELDEPGPEALPRRTPAPAWPPDAKGEEGDFCLRSSDCAAGLCCARH FWSKICKPVLREGQVCTRHRRKGAHGLEI FQRCPCAEGMACRPQRDRGGPDASRLHTCQRH.

[0061] III. Preparation of chicken DKKl protein polyclonal antibody

[0062] 1. Immunization of animals

[0063] After the concentration of the purified His-DKKl fusion protein was determined by BCA concentration determination kit, two New Zealand white rabbits (2-2.5 kg) were immunized subcutaneously with 400 μg each time. The first immunization was with an equal amount of complete Freund's adjuvant, and the subsequent immunizations were with an equal amount of incomplete Freund's adjuvant, 2-3 times, with an interval of 2-3 weeks between each immunization.

[0064] 2. Preparation of antiserum

[0065] After immunization, blood was collected for testing, and the titer of the antiserum against DKKl protein was determined by indirect ELISA. When the titer was greater than 1:50,000, final blood collection was performed to prepare the antiserum.

[0066] 3. Antibody purification

[0067] The His-DKKl fusion protein was coupled with agarose medium to prepare an antigen affinity purification chromatography column. The prepared antiserum was mixed with an equal amount of PBS and slowly loaded. After the antibody was bound, it was eluted with glycine elution buffer to obtain the chicken DKKl protein polyclonal antibody.

[0068] The purity, concentration, and titer determination of the chicken DKKl protein polyclonal antibody are described below.

[0069] The chicken DKKl protein polyclonal antibody was dialyzed in PBS at 4°C overnight, and the purity, concentration, and titer determination were performed every other day.

[0070] 1. The concentration of the obtained DKKl antibody was determined by BCA concentration determination kit, and the result was 0.8 mg / mL. The purity of the purified antibody was observed by SDS-PAGE electrophoresis and Coomassie blue staining, and the results are shown in Figure 5As shown in the figure, the purity of the purified DKK1 antibody is more than 85%. According to the analysis of the identification results of SDS-PAGE and WB, the protein molecular weight is larger than the theoretical molecular weight due to the protein structure. Further LC-MS identification is carried out, and the identification result is shown in Figure 6 Figure 2, the matching degree is more than 62%, and the subsequent experiment is continued.

[0071] 2. Purification of DKK1 antibody indirect ELISA titer detection steps:

[0072] The purified His-DKK1 protein antigen is diluted to 5 μg / mL with PBS coating solution (pH = 7.4), 100 μL per well, and refrigerated at 4°C overnight. The next day, discard the coating solution, wash the plate with PBST 3 times for 5 min each time, then add 200 μL blocking solution to each well, and incubate at 37°C for 1 h.

[0073] Take out the enzyme-labeled plate, discard the inner liquid, and wash the plate with PBST once. Dilute the purified antibody 1:500, 3-fold gradient dilution, 100 μL per well, set the negative control hole of other serum antibodies, and incubate at 37°C for 1 h. Take out the enzyme-labeled plate, discard the inner liquid, wash the plate 3 times, and add 100 μL of diluted HRP-labeled goat anti-rabbit (1:50000 dilution) enzyme-labeled secondary antibody to each well, and incubate at 37°C for 1 h. Take out the enzyme-labeled plate, discard the inner liquid, wash the plate with PBST 4 times for 5 min each time, add 100 μL of TMB color developing solution to each well, and determine the color developing time according to the color depth. Generally, 37°C, 15 min; add 100 μL of 1M HCL solution to each well to terminate the reaction, and immediately read the OD value at 450 nm on the enzyme-labeled instrument. The dilution corresponding to the hole with an OD value greater than 2.1 times the OD value of the set negative control is defined as the titer of the sample.

[0074] The titer detection results are shown in Table 1. As shown in Table 1, the titer of the purified DKK1 antibody is greater than 512000, and the titer is suitable.

[0075] Table 1. Purified DKK1 antibody indirect ELISA detection results

[0076]

[0077] The following describes the detection of the expression of chicken DKK1 protein in chicken tissues by the above chicken DKK1 protein polyclonal antibody.

[0078] The expression of DKK1 protein in chicken skin tissue is detected by Western Blot technology, and the specific operation is as follows:

[0079] (1) Collect Jiangsu Lihua Huashan chicken embryo skin samples at 8, 9, and 11 days of incubation, and freeze them in liquid nitrogen and then transfer them to a -80°C refrigerator for storage.

[0080] (2) Take 300 mg of each preserved skin tissue sample, add 0.8 ml of RIPA lysis buffer (purchased from Cel Signaling Technology) and a protein inhibitor mixture with a final concentration of 1 mmol / L, and manually homogenize 20 times with a glass homogenizer. Transfer the homogenate to a 1.5 ml centrifuge tube and centrifuge at 12000 r / min and 4℃ for 5 min. Take the supernatant and determine the protein concentration using a BCA concentration kit.

[0081] (3) After preparing 4% stacking gel and 10% separating gel, load 30 μg of protein into each well; keep the stacking gel at a constant voltage of 90 V and the separating gel at a constant voltage of 200 V. When the bromophenol blue reaches the bottom of the gel, electrotransfer it onto the NC membrane (300 mA constant current, 1.5 h); after electrotransfer, place the NC membrane in 5% skim milk blocking solution and block it at 37 °C for 1 h; then use the blocking solution to dilute the purified chicken DKK1 protein polyclonal antibody to 1:1000 and the mouse anti-β-actin internal control primary antibody polyclonal antibody (ERWAN) to 1:3000, and incubate at 37 °C for 1.5 h or at 4 °C overnight.

[0082] (4) Remove the membrane and wash it three times with TBST for 5 min each time; dilute the HRP-labeled goat anti-mouse / rabbit IgG secondary antibody to 1:5000 with blocking buffer and incubate at 37℃ for 1.5 h; remove the membrane and wash it four times with TBST for 5 min each time; perform ECL chemiluminescence staining and fixation; scan the film and analyze the gray value of the target band using the UVP gel image processing system Labworks 4.6 software. The results are as follows: Figure 7 As shown in Table 2.

[0083] Depend on Figure 7 As can be seen, the rabbit anti-chicken DKK1 protein antibody prepared in this embodiment can specifically detect DKK1 protein at the expected size of 28-40 kDa. Table 2 shows that DKK1 protein expression can be detected in chicken embryo skin tissue at different developmental stages.

[0084] Table 2. Results of DKK1 protein expression in chicken embryo skin tissue detected by Western blotting.

[0085] Embryonic age DKK1 band gray value β-actin band gray value Gray ratio value 8 25998 108448 0.2397 8 27166 119557 0.2272 9 23194 119203 0.1945 9 23126 110931 0.2084 9 20107 118110 0.1702 11 7543 118844 0.063 11 26463 119463 0.2215

[0086] In summary, this invention designs a polyclonal antibody against chicken DKK1 protein, which can specifically recognize DKK1 protein in chicken tissues at different developmental stages, providing a very good guarantee for the research and application of DKK1 in chickens.

[0087] The above embodiments only express the specific implementation of the present application, which is described in more detail and in more detail, but cannot be understood as a limitation on the protection scope of the present application. It should be noted that for ordinary skilled in the art, without departing from the technical scheme of the present application, a number of modifications and improvements can be made, which are within the scope of protection of the present application.

Claims

1. A method for preparing a polyclonal antibody against chicken DKK1 protein, characterized in that, The method comprises the following steps: (1) cloning the nucleotide sequence shown in SEQ ID No. 1 into a prokaryotic expression vector with a His tag by PCR amplification and recombinase ligation technology to construct a chicken DKK1 gene recombinant expression plasmid; (2) transforming host cells with the chicken DKK1 gene recombinant expression plasmid, then screening and inducing expression to obtain a His-DKK1 fusion protein and purify the same; the His-DKK1 fusion protein contains an amino acid sequence shown in SEQ ID No. 2; (3) immunizing animals with the purified His-DKK1 fusion protein, and then obtaining a chicken DKK1 protein polyclonal antibody through serum separation and purification.

2. The method for preparing chicken DKK1 protein polyclonal antibody according to claim 1, characterized in that, The prokaryotic expression vector is pET-28a(+).

3. The method for preparing chicken DKK1 protein polyclonal antibody according to claim 1 or 2, characterized in that, The step (2) comprises the following steps: (201) introducing the chicken DKK1 gene recombinant expression plasmid into host cells to obtain a transformation product; (202) coating the transformation product on a TB plate containing kanamycin to screen positive clones; (203) inducing the host cells to express the His-DKK1 fusion protein by IPTG induction method; (204)NI 2+ His-DKK1 fusion protein was isolated and purified using affinity chromatography column.

4. The method of claim 3, wherein the chicken DKKl protein polyclonal antibody is prepared by the steps of: (a) immunizing a chicken with a chicken DKKl protein; (b) collecting serum from the chicken; and (c) purifying the chicken DKKl protein polyclonal antibody from the serum. The host cells are BL21(DE3) competent cells.

5. The method of claim 4, wherein the chicken DKKl protein polyclonal antibody is prepared by the steps of: (a) immunizing a chicken with a chicken DKKl protein; (b) collecting serum from the chicken; and (c) purifying the chicken DKKl protein polyclonal antibody from the serum. The NI 2 + The affinity chromatography column was Ni-IDA agarose gel.

6. The method for preparing chicken DKK1 protein polyclonal antibody according to claim 1 or 2 or 4 or 5, characterized in that, The step (3) comprises the following steps: (301) immunizing animals with the purified His-DKK1 fusion protein mixed with an equal amount of complete Freund's adjuvant for the first time; (302) immunizing animals with the purified His-DKK1 fusion protein mixed with an equal amount of incomplete Freund's adjuvant for 2-3 times, and the interval between each immunization is 2-3 weeks; (303) collecting blood to prepare antisera; (304)NI 2+ The chicken DKK1 protein polyclonal antibody was obtained by separating and purifying the antiserum by affinity chromatography column.

7. The method for preparing a polyclonal antibody against chicken DKK1 protein according to claim 6, characterized in that, The immunized animals are rabbits, mice or rats.

8. Use of the polyclonal antibody in any one of claims 1-7 in the preparation of a chicken DKK1 protein detection reagent or kit.

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