Preparation method and application of chicken DKK1 protein polyclonal antibody

Abstract

The invention discloses a preparation method of a chicken DKK1 protein polyclonal antibody, and relates to the technical field of poultry genetic engineering, and the method comprises the following steps: (1) constructing a chicken DKK1 gene recombinant expression plasmid by using nucleotide as shown in SEQ ID No.1 and a prokaryotic expression vector; (2) transforming the chicken DKK1 gene recombinant expression plasmid into a host cell, then performing screening and induced expression to obtain a His-DKK1 fusion protein, and purifying the His-DKK1 fusion protein; and (3) immunizing an animal with the purified His-DKK1 fusion protein, and performing serum separation and purification to obtain the chicken DKK1 protein polyclonal antibody. The invention also provides an application of the chicken DKK1 protein polyclonal antibody in a chicken DKK1 protein detection reagent or kit. The DKK1 protein in chicken tissues in different development stages can be specifically recognized, and an important tool is provided for functional research of DKK1 on chickens.

Description

Technical Field

[0001] The invention relates to the technical field of poultry genetic engineering, and in particular to a preparation method and application of a chicken DKK1 protein polyclonal antibody. Background Art

[0002] Dickkopf-related protein 1 (DKK1) is a typical inhibitor of the Wnt / β-catenin signaling pathway and belongs to the DKK family (the others include DKK2, DKK3 and DKK4). As a secretory glycoprotein, DKK1 is mainly secreted by osteoblasts and osteocytes, and has an important regulatory effect on cell life activities, so it plays an important role in animal growth and development.

[0003] At present, commercialized DKK1 antibodies are mainly antibodies against mice or humans. These DKK1 antibodies cannot specifically recognize DKK1 proteins in chicken tissues, which greatly limits the research and application of DKK1 in chickens. Therefore, it is of great significance to develop a DKK1 protein that can specifically recognize DKK1 proteins in chicken tissues. Summary of the invention

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

[0005] The technical solution adopted by the present invention is as follows:

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

[0007] The second object of the present invention is to provide a method for preparing a chicken DKK1 protein polyclonal antibody, comprising the following steps:

[0008] (1) using PCR amplification and recombinase ligation technology, cloning the above nucleotides into a prokaryotic expression vector with a His tag to construct a chicken DKK1 gene recombinant expression plasmid;

[0009] (2) transforming the chicken DKK1 gene recombinant expression plasmid into a host cell, then screening and inducing expression to obtain a His-DKK1 fusion protein and purifying it; the His-DKK1 fusion protein contains the amino acids shown in SEQ ID No. 2;

[0010] (3) Immunizing animals with the purified His-DKK1 fusion protein, and then obtaining chicken DKK1 protein polyclonal antibodies through serum separation and purification.

[0011] Preferably, 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 was introduced into the host cell to obtain the transformation product;

[0014] (202) The transformation products were spread on TB plates containing kanamycin to screen for positive clones;

[0015] (203) The IPTG induction method was used to induce host cells to express the His-DKK1 fusion protein;

[0016] (204) Using NI 2+ The His-DKK1 fusion protein was separated and purified by affinity chromatography.

[0017] Preferably, the host cell is a BL21 (DE3) competent cell.

[0018] As a preferred embodiment, the NI 2+ The affinity chromatography column was Ni-IDA agarose gel.

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

[0020] (301) The purified His-DKK1 fusion protein was mixed with an equal amount of complete Freund's adjuvant and used to immunize animals for the first time;

[0021] (302) The purified His-DKK1 fusion protein was mixed with an equal amount of incomplete Freund's adjuvant for immunization 2-3 times, with an interval of 2 to 3 weeks between each immunization;

[0022] (303) blood collection for preparation of antiserum;

[0023] (304) Using NI 2+ The antiserum was separated and purified by affinity chromatography to obtain the chicken DKK1 protein polyclonal antibody.

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

[0025] The third object of the present invention is to provide the use of the above-mentioned polyclonal antibody in a chicken DKK1 protein detection reagent or kit.

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

[0027] (1) The present invention prepares a chicken DKK1 protein polyclonal antibody by designing a suitable target nucleotide and utilizing gene recombination technology. Experiments show that the chicken DKK1 protein polyclonal antibody prepared by the present invention has strong specificity and can specifically identify DKK1 protein in chicken tissues at different developmental stages, thereby being applied to chicken DKK1 protein detection reagents or kits. Therefore, the present invention provides an important tool for functional research of DKK1 in chickens, and is very suitable for large-scale promotion and application.

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

[0029] (3) The present invention adopts 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 the gene recombination of the present invention are low-cost, simple and easy to obtain, and the antibody separation and purification degree is high, the animal immunization effect is good, and the antibody titer is appropriate, which effectively guarantees the quality of the commercialization of chicken DKK1 protein polyclonal antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the result of enzyme digestion identification of the recombinant expression plasmid pET-28a(+)-DKK1 in the 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 a DNA marker.

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

[0033] Figure 3Schematic diagram of the purification results of the His-DKK1 fusion protein in the embodiment of the present invention, wherein M is a protein marker, 1 is the supernatant after the induced host cell BL21 (pET-28a (+) -DKK1) is disrupted, 2 is the effluent, and 3 to 4 are the purified His-DKK1 fusion proteins collected after elution.

[0034] Figure 4 Schematic diagram of Western Blot identification results of the purified His-DKK1 fusion protein in the embodiment of the present invention, wherein M is a protein marker, 1 is a purified His-DKK1 fusion protein, and the band indicated by the arrow is the target protein band.

[0035] Figure 5 The figure is a schematic diagram of the purity identification result of the chicken DKK1 protein polyclonal antibody in the embodiment of the present invention, wherein M is a protein marker, 1 is 0.5 mg / mL BSA, and 2 is a purified chicken DKK1 protein polyclonal antibody.

[0036] Figure 6 This is a schematic diagram of the mass spectrometry detection results of the polyclonal antibody against chicken DKK1 protein purified in the examples of the present invention.

[0037] Figure 7 This is a schematic diagram of the results of detecting DKK1 protein in chicken skin tissue with a chicken DKK1 protein polyclonal antibody in an embodiment of the present invention. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments.

[0039] Example

[0040] This embodiment provides a chicken DKK1 protein polyclonal antibody for promoting the research and application of DKK1 in chickens. The preparation of the chicken DKK1 protein polyclonal antibody mainly includes three processes: cloning of the chicken DKK1 gene, expression and purification of the chicken DKK1 recombinant protein, and preparation of the chicken DKK1 protein polyclonal antibody. The following is a detailed introduction to the three processes.

[0041] 1. Cloning of Chicken DKK1 Gene

[0042] The chicken DKK1 gene recombinant expression plasmid was constructed, specifically: the nucleotide shown in SEQ ID No. 1 was cloned into the vector pET-28a(+) by PCR amplification and recombinase ligation technology to construct a recombinant expression plasmid, and it was named: pET-28a(+)-DKK1.

[0043] SEQ ID No.1:

[0044] , 651bp.

[0045] The PCR amplification reaction system was 10 μmol·L -1 1 μL each of upstream and downstream primers, 100 ng·μL -1 0.3 μL of gene synthesis product (recombinant expression plasmid pET-28a(+)-DKK1), 0.5 μL of 5U / μL Pfu DNA polymerase, 5 μL of 10×Taq Pfubuffer, add ddH2O to 50 μL, the reaction program is 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 recombinase ligation reaction system is: 4 μL of PCR product recovered by gel purification, 3.5 μL of pET-28a(+) linearized vector, and 2.5 μL of recombinant DNA ligase. The recombinase DNA ligation reaction procedure is: 50°C water bath for 25 minutes, place at room temperature for 2-3 minutes to lower the temperature, and then perform transformation.

[0047] 2. Expression and purification of chicken DKK1 recombinant protein

[0048] 1. Pipette 1 μL of pET-28a(+)-DKK1 recombinant expression plasmid and add it to 100 μL of BL21(DE3) competent cells (host cells). Place it on ice for 20 minutes, heat shock it at 42°C for 90 seconds, quickly place it on ice for 5 minutes, add 600 μL of TB medium, shake it at 37°C and 220 r / min for 1 hour, then spread it on a TB plate containing 50 μg / mL kanamycin, select positive clones for enzyme digestion identification, and the results are shown in the table. Figure 1 The obtained host cells were named: 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), as follows:

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

[0051] Next, take out 1 ml of the culture, centrifuge at 10000 r / min and room temperature for 2 min, discard the supernatant, and resuspend the bacterial pellet with 100 μl 1× SDS loading buffer. Then, add IPTG to the remaining culture to a final concentration of 0.2 mM, and shake at 37°C and 220 r / min for 4 h to induce the expression of the fusion protein.

[0052] Then, take out 1 ml of the culture, centrifuge it at 10000 r / min and room temperature for 2 min, discard the supernatant, resuspend the bacterial pellet with 100 μl 1× SDS loading buffer, centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the pellet with PBS.

[0053] After the resuspension was ultrasonically disrupted, the supernatant and precipitate were respectively taken and resuspended in the loading buffer, and then analyzed by 12% SDS-PAGE and Coomassie brilliant blue staining. Figure 2 As shown, the target protein was mainly present in the supernatant.

[0054] 3. Use NI 2+ The His-DKK1 fusion protein is separated and purified by affinity chromatography, specifically:

[0055] First, using a low-pressure chromatography system, the supernatant solution was loaded onto a Ni-IDA-Sepharose CL-6B affinity chromatography column pre-equilibrated with Ni-IDA Binding-Buffer at a flow rate of 0.5 ml / min; the column was rinsed 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] Next, wash with Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml / min until the OD280 value of the effluent reaches the baseline, and then elute the target protein with Ni-IDA Elimination-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml / min, and collect the effluent.

[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. The results are shown in Figure 2. Figure 3 As shown, it can be seen that the purified His-DKK1 fusion protein has high purity. 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, the purified His-DKK1 fusion protein was analyzed by Western Blot. The results are shown in Figure 4 ,As can be seen from the figure, the target band appears at the expected size.

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

[0059] SEQ ID No.2:

[0060] MGGGLSSNAI KVGAAASAAPAAAAPAPFDGSNKAPPAATRQPFPCSEDEDCGPDEFCGGAARGGGPPLCLACRR RRKRCLRDAMCCPGTACSNGLCTPPEPPPGAPELDEPGPEALPRRTPAWPPDAKGEEGDFCLRSSDCAAGLCCARH FWSKICKPVLREGQVCTRHRRKGAHGLEI FQRCPCAEGMACRPQRDRGGPDASRLHTCQRH.

[0061] 3. Preparation of chicken DKK1 protein polyclonal antibody

[0062] 1. Animal immunization

[0063] After the concentration of the purified His-DKK1 fusion protein was measured using a BCA concentration determination kit, two New Zealand white rabbits (2-2.5 kg) were immunized subcutaneously with 400 μg / time. The first immunization was mixed with an equal amount of complete Freund's adjuvant, and then it was mixed with an equal amount of incomplete Freund's adjuvant for 2-3 times, with an interval of 2 to 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 DKK1 protein was determined by the indirect ELISA method. When the titer was greater than 1:50,000, a final blood collection was performed to prepare the antiserum.

[0066] 3. Antibody Purification

[0067] The His-DKK1 fusion protein was coupled to an agarose medium to prepare an antigen affinity purification chromatography column, the prepared antiserum was mixed with an equal amount of PBS and then slowly loaded, and after the antibody was bound, it was eluted with a glycine elution buffer to obtain a chicken DKK1 protein polyclonal antibody.

[0068] The following describes the purity, concentration and titer determination of the above-mentioned chicken DKK1 protein polyclonal antibody.

[0069] The chicken DKK1 protein polyclonal antibody was dialyzed in PBS at 4°C overnight, and the purity, concentration and titer were determined the next day.

[0070] 1. The concentration of the obtained DKK1 antibody was measured using a 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 Brilliant Blue staining. The results are shown in Figure 5, it can be seen from the figure that the purity of DKK1 antibody after purification is above 85%. According to the analysis of SDS-PAGE and WB identification results, the molecular weight of the protein is larger than the theoretical molecular weight due to the protein structure. Further LC-MS identification, the identification results are as follows Figure 6 As shown, the matching degree is more than 62%, and the subsequent experiments are continued.

[0071] 2. Indirect ELISA titer detection steps for purified DKK1 antibody:

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

[0073] Take out the ELISA plate, discard the internal solution, wash the plate once with PBST; after the purified antibody is diluted 1:500, dilute it 3 times in a gradient manner, 100 μL per well, set up other serum antibody negative control wells, and keep the temperature at 37°C for 1 hour; take out the ELISA plate, discard the internal solution, wash the plate 3 times, add 100 μL of diluted HRP-labeled goat anti-rabbit (1:50000 dilution) enzyme-labeled secondary antibody to each well, keep the temperature at 37°C for 1 hour, then take out the ELISA plate, discard the internal solution, wash the plate 4 times with PBST, each time for 5 minutes; add 100 μL of TMB color development solution to each well, and determine the color development time according to the depth of the color, generally 37°C, 15 minutes; add 100 μL of 1M HCL solution to each well to terminate the reaction, and immediately read the reading at 450nm on the ELISA reader, and the dilution corresponding to the well with an OD value greater than 2.1 times the set negative control OD value is defined as the titer of the sample.

[0074] The titer test results are shown in Table 1. It can be seen from Table 1 that the titer of the purified DKK1 antibody is greater than 512,000, and the titer is appropriate.

[0075] Table 1 Indirect ELISA test results of purified DKK1 antibody

[0076]

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

[0078] Western Blot technology was used to detect the expression of DKK1 protein in chicken skin tissue. The specific operation was as follows:

[0079] (1) Skin samples of Jiangsu Lihua Huashan chicken embryos were collected on the 8th, 9th and 11th days of incubation, quickly frozen in liquid nitrogen and then stored in a -80°C refrigerator.

[0080] (2) Take 300 mg of each sample of preserved skin tissue, add 0.8 ml of RIPA lysis buffer (purchased from CellSigning Technology) and a protease inhibitor mixture with a final concentration of 1 mmol / L, manually homogenize 20 times up and down with a glass homogenizer, transfer the homogenate to a 1.5 ml centrifuge tube, centrifuge at 12000 r / min and 4°C 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% separation gel, 30 μg of protein was loaded into each well; the stacking gel was kept at a constant voltage of 90 V and the separation gel was kept at a constant voltage of 200 V. When bromophenol blue ran to the bottom of the gel, it was electrotransferred to the NC membrane (300 mA constant current, 1.5 h); after the electrotransfer, the NC membrane was placed in 5% skim milk blocking solution and blocked at 37°C for 1 h; then the purified chicken DKK1 protein polyclonal antibody was diluted to 1:1000 with the blocking solution, and the mouse anti-β-actin internal reference primary antibody polyclonal antibody (ERWAN) was diluted to 1:3000, and incubated at 37°C for 1.5 h or at 4°C overnight.

[0082] (4) The membrane was removed and then washed with TBST for 3 times, 5 min each time; the HRP-labeled goat anti-mouse / rabbit IgG secondary antibody was diluted to 1:5000 with blocking solution and incubated at 37°C for 1.5 h; the membrane was removed and washed with TBST for 4 times, 5 min each time; ECL chemiluminescence was used for color development and fixation; the film was scanned and the gray value of the target band was analyzed using the UVP gel image processing system Labworks4.6 software. The results are shown in the figure. Figure 7 , as shown in Table 2.

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

[0084] Table 2 Western Blot detection of DKK1 protein expression in chicken embryo skin tissue

[0085] Embryonic age DKK1 band gray value Gray value of β-actin band Grayscale ratio 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, the present invention designs a chicken DKK1 protein polyclonal antibody that can specifically recognize the 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-mentioned embodiments only express the specific implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the protection scope of the present invention. It should be pointed out that for ordinary technicians in this field, several variations and improvements can be made without departing from the technical solution concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing a chicken DKK1 protein polyclonal antibody, characterized in that: The following steps are involved: (1) Using PCR amplification and recombinase ligation technology, the nucleotide sequence shown in SEQ ID No. 1 was cloned into a prokaryotic expression vector with a His tag to construct a chicken DKK1 gene recombinant expression plasmid; (2) transforming the chicken DKK1 gene recombinant expression plasmid into a host cell, then screening and inducing expression to obtain a His-DKK1 fusion protein and purifying it; the His-DKK1 fusion protein contains the amino acids shown in SEQ ID No. 2; (3) Immunizing animals with the purified His-DKK1 fusion protein, and then obtaining chicken DKK1 protein polyclonal antibodies through serum separation and purification.

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

3. The method for preparing a chicken DKK1 protein polyclonal antibody according to claim 1 or 2, characterized in that: The step (2) comprises the following steps: (201) The chicken DKK1 gene recombinant expression plasmid was introduced into the host cell to obtain the transformation product; (202) The transformation products were spread on TB plates containing kanamycin to screen for positive clones; (203) The IPTG induction method was used to induce host cells to express the His-DKK1 fusion protein; (204) Using NI 2+ The His-DKK1 fusion protein was separated and purified by affinity chromatography.

4. The method for preparing a chicken DKK1 protein polyclonal antibody according to claim 3, characterized in that: The host cell is a BL21 (DE3) competent cell.

5. The method for preparing a chicken DKK1 protein polyclonal antibody according to claim 4, characterized in that: The NI 2 + The affinity chromatography column was Ni-IDA agarose gel.

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

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

8. Use of the polyclonal antibody according to any one of claims 1 to 7 in a reagent or kit for detecting chicken DKK1 protein.

Images