Preparation method and application of Muscovy duck MAPK1 protein polyclonal antibody

Abstract

The invention discloses a preparation method and application of a Muscovy duck MAPK1 protein polyclonal antibody, and belongs to the technical field of biology. According to the invention, DNAStar software, AI-assisted protein three-dimensional structure prediction and other bioinformatics methods are adopted to accurately determine the 4th-364th amino acids of MAPK1 as immunogens, and protein expression, purification and antibody preparation methods are optimized to obtain the preparation method: expressing an MAPK1 recombinant protein with an amino acid sequence of SEQ ID NO.2, and immunizing a rabbit with the recombinant protein to obtain the Muscovy duck MAPK1 protein polyclonal antibody. The titer of the obtained polyclonal antibody can reach 1: 102400, and the polyclonal antibody has the advantages of high recognition efficiency and strong specificity. The MAPK1 polyclonal antibody prepared in the invention has high targeting recognition capability at the tissue level, can be used for immunofluorescence visualization research of Muscovy duck ovarian tissue protein expression, and provides key technical support for subsequent function exploration of MAPK1 in follicular development and reproduction regulation.

Description

Technical Field

[0001] The present invention relates to a method for preparing a Muscovy duck polyclonal antibody, in particular to a method for preparing a Muscovy duck MAPK1 protein polyclonal antibody, and belongs to the field of biotechnology. Background Art

[0002] The Muscovy duck (Cairinamoschata), a key lean waterfowl species in my country, boasts rapid growth, strong disease resistance, and excellent meat quality, and is widely distributed in southern my country. However, its strong nesting habit, low fecundity, low egg production, and long rearing cycle severely restrict its industrial development and economic benefits. Therefore, effectively improving the reproductive efficiency of Muscovy ducks has become a hot topic in current research. Ovarian development is a key physiological factor determining the reproductive capacity of Muscovy ducks. Granulosa cells, as key functional cells within the follicle, play a central role in regulating sex hormone synthesis and follicular development. Their functional status is directly related to normal follicular growth and ovulation. Therefore, systematically analyzing the molecular regulatory mechanisms of granulosa cells has important theoretical and practical significance for deepening the understanding of the regulatory network of ovarian function in Muscovy ducks, improving their reproductive performance, and conducting molecular breeding.

[0003] The mitogen-activated protein kinase (MAPK) signaling pathway is a classic intracellular signal transduction system, widely involved in various life processes such as cell proliferation, differentiation, apoptosis, and stress response. MAPK1 (also known as ERK2), a key member of the MAPK family, is a key effector molecule in the extracellular signal-regulated kinase (ERK) pathway. Studies have shown that MAPK1 can be activated by follicle-stimulating hormone (FSH) in mammalian ovarian granulosa cells and participates in multiple reproductive processes such as steroid hormone synthesis, follicle development, and ovulation regulation. However, systematic studies on the expression characteristics of MAPK1 in waterfowl ovarian tissue, its localization and function in granulosa cells, and its biological role in economically important waterfowl such as Muscovy ducks remain unclear. Currently, there are no commercially available antibodies targeting the Muscovy duck MAPK1 protein. Due to differences in protein sequences between species, existing antibodies targeting mammalian ERK1 / 2 often suffer from low recognition efficiency and insufficient specificity in poultry. Furthermore, traditional antigen design methods are often limited to simple physicochemical parameter predictions, such as isoelectric point and hydrophilicity, making it difficult to fully and accurately reflect the spatial structure and immunogenicity of protein antigenic determinants. This also limits the development of antibodies against the Muscovy duck MAPK1 protein. Therefore, developing an antibody tool that can specifically recognize the Muscovy duck MAPK1 protein will not only help establish a molecular detection platform for ovarian development in poultry, but also provide key technical support for in-depth analysis of the mechanism of action of MAPK1 in the regulation of waterfowl reproduction. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for preparing a polyclonal antibody against Muscovy duck MAPK1 protein with high recognition efficiency and strong specificity.

[0005] Technical solution: The present invention provides a method for preparing a polyclonal antibody against Muscovy duck MAPK1 protein, comprising the following steps: step 1, expressing a recombinant MAPK1 protein having an amino acid sequence of SEQ ID NO.2; step 2, immunizing a rabbit with the recombinant protein to obtain a polyclonal antibody against Muscovy duck MAPK1 protein.

[0006] The present invention uses bioinformatics methods including DNAStar software and AI-assisted protein three-dimensional structure prediction (AlphaFold3) to systematically analyze the physicochemical properties and structural characteristics of the Muscovy duck MAPK1 protein, breaking through the conventional "empirical interception" method, accurately analyzing the hydrophilicity and antigenicity of MAPK1, and accurately evaluating the spatial conformation and stability of the antigen fragment. Ultimately, the amino acids 4 to 364 of MAPK1 were identified as immunogens, achieving the accuracy and scientificity of antigen fragment selection, and significantly improving the expression efficiency and immunogenicity of the antigen.

[0007] SEQ ID NO. 1 in the sequence listing is the nucleic acid sequence encoding the recombinant MAPK1 protein; positions 157-1239 at the 5' end are derived from the nucleic acid sequence of the MAPK1 protein (GenBank: KF482375.2), and SEQ ID NO. 1 was obtained after codon optimization based on E. coli preferences. SEQ ID NO. 2 is the amino acid sequence of the recombinant MAPK1 protein, wherein positions 53-413 at the N-terminus are derived from the amino acid sequence of the MAPK1 protein (GenBank: AII25436.1).

[0008] Rabbits are selected as immune hosts in the present invention because rabbits have a wider range of immunogen recognition than mice and can recognize more complex antigen epitopes.

[0009] The titer, specificity and adaptability of the polyclonal antibody against the Muscovy duck MAPK1 protein were systematically evaluated by ELISA, Western blot, immunohistochemistry and immunofluorescence, and it was proved that the titer of the polyclonal antibody against the Muscovy duck MAPK1 protein prepared by the present invention could reach 1:102400, and Western blot could specifically recognize a 48 kDa band, showing the advantages of high recognition efficiency and strong specificity.

[0010] Preferably, in the first step, HB-PET autoinduction medium is used to express the MAPK1 recombinant protein.

[0011] The use of HB-PET self-induction medium with glycerol as the main energy source instead of the traditional IPTG induction method not only significantly increased the expression level of the target protein, but also obtained a higher proportion of soluble protein, avoiding the problem of inclusion body formation in traditional methods.

[0012] Preferably, in the first step, magnetic beads are used to purify the MAPK1 recombinant protein.

[0013] Choosing magnetic bead purification technology (HisSep Ni-NTAMagBeads) instead of traditional nickel column purification can effectively improve purification efficiency and protein purity and reduce operational difficulty.

[0014] HB-PET autoinduction culture and magnetic bead purification enhance protein expression efficiency, purity, and structural integrity. Increased protein purity and quality directly enhance immune stimulation, ensuring the production of high-titer antibodies.

[0015] Preferably, after the MAPK1 recombinant protein is eluted with an eluent containing imidazole, the imidazole is removed by dialysis.

[0016] Dialysis technology can gently and thoroughly remove high-concentration imidazole residues in the eluate, greatly reducing the irritation and immune stress response of the immunogen to the animal, improving the animal's tolerance to the antigen, thereby enhancing the intensity of the animal's immune response, ensuring the stable production of high-titer antibodies, and safeguarding animal welfare.

[0017] Preferably, in the second step, polyclonal antibodies against Muscovy duck MAPK1 protein are prepared using serum pools.

[0018] The serum pool strategy uses sera from three rabbits with similar immune titers to form a serum pool. This can solve the common batch difference problem in polyclonal antibody preparation and make the antibody titer stable between batches.

[0019] Preferably, in the second step, an adjuvant is used together with the recombinant protein to immunize rabbits.

[0020] Preferably, in the second step, multiple immune stimulations are used to prepare polyclonal antibodies against Muscovy duck MAPK1 protein.

[0021] Preferably, the multiple times is three times.

[0022] Preferably, the three immune stimulations are performed on day 0, day 21 and day 42 respectively.

[0023] Highly effective adjuvants and scientifically designed immunization procedures can make the rabbit's immune memory response and antibody production more efficient and lasting.

[0024] The present invention also provides application of the Muscovy duck MAPK1 protein polyclonal antibody in visualization research of Muscovy duck ovarian tissue protein expression.

[0025] Analysis of the localization characteristics of MAPK1 in combination with the granulosa cell marker FSHR revealed that MAPK1 protein is primarily expressed in granulosa cells and theca of follicles, with subcellular localization primarily in the cytoplasm and extending into the perinuclear region. Therefore, this antibody is suitable for visualizing protein expression in Muscovy duck ovarian tissue, providing a key technical tool and theoretical foundation for subsequent exploration of the function of MAPK1 in follicular development and reproductive regulation.

[0026] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: 1. The polyclonal antibody prepared by the method for preparing the Muscovy duck MAPK1 protein has a titer of up to 1:102400, exhibiting high recognition efficiency and strong specificity. This helps establish a molecular detection platform for ovarian development in poultry and further elucidates the mechanism of action of MAPK1 in waterfowl reproductive regulation. 2. The MAPK1 polyclonal antibody prepared by the present invention has high target recognition ability at the tissue level and can be used for immunofluorescence visualization of protein expression in Muscovy duck ovarian tissue. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Bioinformatics analysis diagram of MAPK1 protein (A is the signal peptide structure prediction. B is the transmembrane structure prediction. C is the protein three-dimensional structure model prediction (the green area is the immunogenic sequence). D is the protein antigenicity and hydrophilicity prediction (the green box is the immunogenic segment));

[0028] Figure 2 Diagram of MAPK1 gene amplification and recombinant expression vector identification (A is PCR amplification of the Muscovy duck MAPK1 gene. B is enzyme digestion identification of the pET30a-MAPK1 recombinant vector. M1 is DL2000 DNA Marker. M2 is DL15000 DNA Marker. 1 is the PCR amplification product of the MAPK1 gene. 2 is the EcoR I single enzyme digestion identification of the pET30a-MAPK1 recombinant expression vector. 3 is the Xho I single enzyme digestion identification of the pET30a-MAPK1 recombinant expression vector. 4 is the EcoR I / Xho I double enzyme digestion identification of the pET30a-MAPK1 recombinant expression vector. 5 is the pET30a-MAPK1 recombinant expression vector);

[0029] Figure 3 The expression, purification and identification diagram of MAPK1 recombinant protein (M is the protein molecular weight standard. 1 is the whole bacteria of broken bacteria. 2 is the precipitate of broken bacteria. 3 and 4 are the supernatants of broken bacteria. 5 is the purified protein. 6 is the dialyzed protein);

[0030] Figure 4 Figure 1 shows the immunization procedure, titer, and specificity detection of MAPK1 protein polyclonal antibodies (A is a schematic diagram of the immunization procedure. B is the ELISA test for the titer of MAPK1 protein polyclonal antibodies. C is the Western blot verification of the specificity of MAPK1 protein polyclonal antibodies);

[0031] Figure 5Figure 4 shows the identification of FSHR expression in granulosa cells of Muscovy duck follicles (AF is immunofluorescence (IFA) detection of specific marker FSHR expression in granulosa cells. GH is Western blot detection of FSHR and GAPDH expression in granulosa cells);

[0032] Figure 6 The expression and localization diagram of MAPK1 protein in granulosa cells of Muscovy duck follicles (AF are immunofluorescence detection of MAPK1 expression and localization in granulosa cells. GH are Western blot detection of MAPK1 protein and internal reference protein GAPDH expression in granulosa cells);

[0033] Figure 7 Immunohistochemical expression and localization of MAPK1 protein in Muscovy duck follicle tissue (A is immunohistochemical staining of MAPK1 in granulosa cells and theca regions of follicles, with brown signals localized in the cytoplasm; B is FSHR as a positive control for granulosa cell markers, with black arrows indicating positive staining areas in the granulosa cell layer. The upper row was taken with a 20× objective lens, and the lower row was taken with an 80× objective lens);

[0034] Figure 8 This is the immunofluorescence double staining localization map of MAPK1 and FSHR in the follicular tissue of Muscovy duck (AD is Merge, MAPK1 (red), FSHR (yellow) and DAPI-stained nucleus (blue). The white arrows indicate the spatially consistent expression areas of MAPK1 and FSHR in the granulosa cell layer. The upper row is the objective lens of 20×, and the lower row is the objective lens of 80×). DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0036] The immunized hosts were 4- to 5-week-old SPF-grade male New Zealand white rabbits, provided by Pizhou Dongfang Breeding Co., Ltd. Muscovy ducks were provided by Taizhou Fengda Agriculture and Animal Husbandry Technology Co., Ltd. The pET30a(+) plasmid was maintained by the Jiangsu Provincial Key Laboratory of High-Tech Research in Veterinary Biopharmaceuticals. All other experimental materials were commercially available.

[0037] Example 1

[0038] This embodiment provides a method for preparing a polyclonal antibody against Muscovy duck MAPK1 protein, comprising the following steps: step 1, expressing a recombinant MAPK1 protein having an amino acid sequence of SEQ ID NO. 2; and step 2, immunizing a rabbit with the recombinant protein to obtain a polyclonal antibody against Muscovy duck MAPK1 protein.

[0039] The MAPK1 recombinant protein described in step 1 was obtained through the following experiments: 1.1 bioinformatics analysis; 1.2 gene cloning and prokaryotic recombinant expression vector construction; 1.3 recombinant protein expression, purification and identification.

[0040] 1.1 Bioinformatics analysis

[0041] To evaluate the potential of MAPK1 protein as an immunogen, we used various bioinformatics tools to predict its physicochemical properties. Specifically, online tools were used to predict the isoelectric point, signal peptide, transmembrane structure, three-dimensional protein structure, and subcellular localization of the Muscovy duck MAPK1 protein sequence (GenBank: AII25436.1) (see Table 1). DNAStar software was used to analyze its hydrophilicity and antigenicity.

[0042] Table 1 Online tools for bioinformatics analysis

[0043] Online Tools use Website IPCprotein Theoretical isoelectric point analysis http: / / isoelectric.org / index.html SignalP-5.0 Signal peptide prediction https: / / services.healthtech.dtu.dk / services / SignalP-5.0 / TMHMM-2.0 Transmembrane structure prediction https: / / services.healthtech.dtu.dk / services / TMHMM-2.0 / AlphaFold3 3D structure modeling https: / / alphafoldserver.com / PSORTIIPrediction Subcellular localization prediction https: / / psort.hgc.jp / form2.html

[0044] The results showed that the MAPK1 protein consists of 368 amino acids, with a molecular weight of approximately 42 kDa and a theoretical isoelectric point of 6.05. The signal peptide prediction results showed that the possibility of its presence of a signal peptide was low (0.62%) ( Figure 1 A); transmembrane structure analysis confirmed that it is a non-transmembrane protein ( Figure 1 B); Spatial structural simulations showed that the protein has a typical tertiary folding conformation and is composed of multiple functional domains ( Figure 1 C). DNAStar software analysis showed that MAPK1 has good antigenicity and hydrophilicity ( Figure 1 D). Subcellular localization prediction results showed that the protein was mainly localized in the cytoplasm (60.9%) and the nucleus (21.7%). Through the above combination strategy, the 4th to 364th amino acids ( Figure 1 C, 1D green area) as the immunogen, ensuring the high antigenicity, stability and good spatial conformation of the immunogen, achieving the accuracy and scientificity of antigen fragment selection, and significantly improving the expression efficiency and immunogenicity of the antigen.

[0045] 1.2 Cloning of Muscovy duck MAPK1 gene and construction of prokaryotic recombinant expression vector

[0046] Specific PCR amplification primers were designed based on the MAPK1 gene sequence in the NCBI database (GenBank: KF482375.2):

[0047] F:5′-ATGGCGGCGGTGTCGG-3′;

[0048] R: 5′-TTAAGATGGATATCCTGGCTGGAATCTAGC-3′.

[0049] The total RNA of the ovarian tissue of Muscovy duck was extracted by Trizol and reverse transcribed into cDNA. The MAPK1 gene was amplified by PCR, and Suzhou Jinweizhi Biotechnology Co., Ltd. was commissioned to optimize the codons and synthesize the nucleotide sequence (SEQ ID NO.1). The resulting recombinant protein contains 421 amino acids (SEQ ID NO.2), with a predicted molecular weight of 48kDa and a theoretical isoelectric point of 5.91. The synthesized nucleotide sequence was cloned into the prokaryotic expression vector pET30a using the homologous recombination method. After the bacterial solution PCR and plasmid single and double enzyme digestion were confirmed to be correct, it was sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The correctly sequenced recombinant vector was named pET30-MAPK1.

[0050] The MAPK1 gene was amplified by PCR using the cDNA of the ovarian tissue of the black-feathered Muscovy duck as a template. The product size was approximately 1143 bp, which was in line with the expectation ( Figure 2 A). The amplified product was cloned into the pET30a expression vector by homologous recombination. Enzyme digestion verification showed that single enzyme digestion produced a 6473 bp band, and double enzyme digestion products were 5390 bp and 1083 bp, respectively, which were consistent with expectations ( Figure 2 B) Sequencing results showed that the inserted sequence had a 99.8% similarity to the MAPK1 gene sequence in the NCBI database. In summary, the pET30a-MAPK1 recombinant prokaryotic expression vector was successfully constructed.

[0051] 1.3 Expression, purification and identification of recombinant MAPK1 protein

[0052] The recombinant plasmid pET30-MAPK1 was transformed into BL21 competent cells and cultured in HB-PET autoinduction medium until the logarithmic growth phase. The cells were then inducible at 20°C for 20 hours. After ultrasonic disruption, the whole cells, pellet, and supernatant were collected. The expression of recombinant MAPK1 protein in each fraction was assayed by SDS-PAGE electrophoresis (Coomassie Brilliant Blue staining) and Western blot (identified using an HRP-conjugated Anti-His Tag antibody). Following the instructions for HisSep Ni-NTA Agarose Resin, the supernatant was bound to magnetic beads at 4°C overnight. The beads were washed three times with a wash buffer containing 50 mM imidazole. The target protein was eluted with an eluent containing 200 mM imidazole. The eluent was dialyzed to remove imidazole, filtered through a 0.22 μm filter, and the protein concentration was determined using the BCA assay. The protein was then stored at -70°C until further use.

[0053] The results are as follows Figure 3As shown, a specific band with a molecular weight of approximately 48 kDa was detected in the total bacterial suspension (lane 1) and the soluble supernatant (lanes 3 and 4). The signal was weaker in the precipitate (lane 2), suggesting that the protein is primarily expressed in a soluble form. High-purity protein was obtained after purification using Ni-NTA magnetic beads (lane 5). After dialysis (lane 6), a clear band was observed with no degradation, indicating a stable protein structure. Western blot analysis further confirmed the expression of the His-tagged protein, consistent with the SDS-PAGE results. These results demonstrate the successful preparation of the MAPK1 immunogen, namely, the recombinant MAPK1 protein.

[0054] The polyclonal antibody against the Muscovy duck MAPK1 protein in step 2 was obtained and evaluated by the following experiments: 2.1 Preparation of polyclonal antibody against MAPK1 protein; 2.2 Determination of antibody titer by indirect ELISA.

[0055] 2.1 Preparation of MAPK1 protein polyclonal antibodies

[0056] An immunization group and a saline control group were established, with 3 New Zealand white rabbits in each group. The dialyzed MAPK1 recombinant protein was adjusted to 500 μg / mL and mixed with QuickAntibody-Rabbit8W adjuvant at a 1:1 ratio. The New Zealand white rabbits were initially immunized with 200 μL / rabbit via intramuscular injection. Booster immunizations were performed using the same method on days 21 and 42 after the initial immunization. On day 56, terminal serum was collected by cardiac puncture and titer assayed (see immunization procedure for details). Figure 4 A). Serum from the normal saline group served as a negative control.

[0057] 2.2 Indirect ELISA determination of antibody titer

[0058] Recombinant MAPK1 protein was adjusted to 2 μg / mL, with 0.1 mL / well coated overnight at 4°C. Blocking was performed with 3% skim milk at 37°C for 2 h. Test serum and negative control serum were serially diluted 2-fold, with 100 μL / well incubated at 37°C for 1 h. After washing five times with PBST, 100 μL of HRP-conjugated goat anti-rabbit IgG (1:5000 dilution) was added to each well. The cells were incubated at 37°C for 1 h, washed five times with PBST, and developed with TMB at room temperature for 15 min. After color development, the D450 nm value was measured. A D450 nm value of 2.1 times higher than that of the negative control serum was considered positive. Three independent biological replicates (n = 3) were performed for each sample. GraphPad Prism 9.5 was used to plot the mean ± standard deviation.

[0059] The ELISA results showed that the titer of the polyclonal antibody against Muscovy duck MAPK1 protein prepared in this example could reach 1:102400, and it had a strong immune response ability ( Figure 4B). Western blot was performed using MAPK1 protein as the coating antigen and rabbit serum as the primary antibody. A specific band of approximately 48 kDa was detected, and no nonspecific binding was observed ( Figure 4 C), verified the high specificity of the antibody.

[0060] Example 2

[0061] In this example, based on the polyclonal antibody against Muscovy duck MAPK1 protein prepared in Example 1, the expression and localization characteristics of MAPK1 protein in Muscovy duck ovarian granulosa cells and tissues were explored by Western blot, immunohistochemistry (IHC), and immunofluorescence (IF) methods.

[0062] The following experiments were carried out:

[0063] (1) Isolation and identification of granulosa cells from the ovary of Muscovy duck

[0064] The isolation and culture methods of granulosa cells from the ovary of Muscovy ducks were based on the previous research of our research group (Zhang Lei, Guo Zhihui, Yi Anwang, et al. In vitro isolation, culture and identification of granulosa cells from preovulatory follicles of Gaoyou ducks [J]. Jiangsu Agricultural Sciences, 2022, 50(24):136-141. DOI:10.15889 / j.issn.1002-1302.2022.24.020.). Among them, a portion of cells were fixed with 4% paraformaldehyde and the expression of granulosa cell-specific marker FSHR was detected by indirect immunofluorescence assay; the other portion of cells were lysed by RIPA and total protein was extracted, and the expression of FSHR protein was detected by Western blot.

[0065] (2) Indirect immunofluorescence assay (IFA)

[0066] Granulosa cells were fixed with 4% paraformaldehyde and blocked overnight with PBS containing 10% fetal bovine serum at 4°C. A 1:500 dilution of FSHR protein polyclonal antibody (or MAPK1 protein polyclonal antibody) was added and incubated for 2 h at 37°C. A 1:1000 dilution of FITC-conjugated goat anti-rabbit IgG was used as a secondary antibody for 1 h at 37°C. After DAPI staining, the cells were observed under a fluorescence microscope.

[0067] (3) Western blot identification

[0068] Protein samples were subjected to SDS-PAGE electrophoresis at 100 V and then transferred to a membrane at 400 mA for 30 min. The transferred NC membrane was blocked with 5% skim milk overnight at room temperature. The membrane was incubated with FSHR protein polyclonal antibody (1:2000 dilution) (or MAPK1 polyclonal antibody) and mouse anti-GAPDH antibody (1:1000 dilution) as primary antibodies for 1 h at room temperature. HRP-conjugated goat anti-rabbit IgG and goat anti-mouse IgG were used as secondary antibodies (1:5000 dilution) for 1 h at room temperature. The membrane was visualized using an ECL luminescence imaging system.

[0069] (4) Tissue section preparation

[0070] Fresh Muscovy duck follicle tissue was obtained and fixed with 4% paraformaldehyde for 24 hours. The tissue was then dehydrated with gradient ethanol (70%, 80%, 90%, 95%, 100%, 20 minutes each), transparentized with xylene (3 times, 10 minutes each), and embedded in paraffin. After embedding, 4 μm thick paraffin sections were prepared and attached to polylysine-treated slides for later use. The sections were dried at 65°C for 1 hour before use. Before staining, the tissue was dewaxed with xylene (3 times, 5 minutes each), hydrated with gradient ethanol (100%, 100%, 95%, 70%, 5 minutes each), and washed with distilled water (2 times, 5 minutes each) for subsequent immunohistochemistry or immunofluorescence staining.

[0071] (5)IHC

[0072] Rehydrated tissue sections were incubated in 3% H₂O₂ methanol solution at room temperature for 10 minutes to block endogenous peroxidases and then washed three times with PBS. Microwave antigen retrieval was performed for 10 minutes using 0.01 mol / L sodium citrate buffer (pH 6.0). After cooling to room temperature, sections were blocked with 10% goat serum for 30 minutes. Rabbit anti-MAPK1 polyclonal antibody (1:200 dilution) was then added, and sections were incubated overnight at 4°C. Sections were washed three times with PBS, and HRP-conjugated goat anti-rabbit IgG secondary antibody (1:500 dilution) was added and incubated at 37°C for 30 minutes. The sections were developed with DAB, counterstained with hematoxylin, dehydrated, and cleared before mounting with neutral gum. Antibody against FSHR, a granulosa cell marker, was used as a positive control staining to verify localization accuracy.

[0073] (6)IF

[0074] Rehydrated tissue sections were microwave-fixed in 0.01 mol / L sodium citrate buffer for 10 minutes. After cooling, sections were incubated in 0.5% Triton X-100 permeabilization buffer for 15 minutes and washed three times with PBS. After blocking with 10% goat serum for 30 minutes, rabbit anti-MAPK1 polyclonal antibody (1:200 dilution) and mouse anti-FSHR antibody (1:200 dilution) were added dropwise, respectively, and incubated overnight at 4°C in the dark. After washing with PBS, HRP-conjugated secondary antibody working solution was added, incubated at room temperature for 30 minutes, and TAS fluorescent dye was added dropwise, incubated at room temperature for 30 minutes, and washed with PBS. Nuclei were stained with DAPI, and sections were washed with PBS, air-dried, and mounted.

[0075] The above test results are as follows:

[0076] 1. Identification of Muscovy duck ovarian granulosa cells

[0077] Immunofluorescence results showed that the cells showed FSHR positive fluorescence under 10× and 20× fields of view, mainly located in the cell membrane and cytoplasm ( Figure 5 A~F). Western blot results showed that the FSHR-specific band of about 78kDa was detected in the cell lysate, and the internal reference GAPDH band was located at 36kDa ( Figure 5 G~H). The results showed that the obtained cells had the molecular marker expression characteristics of typical granulosa cells.

[0078] 2. Expression and localization analysis of MAPK1 protein in granulosa cells

[0079] To evaluate the applicability of MAPK1 polyclonal antibodies at the cellular level, immunofluorescence and Western blot were used to detect the expression of MAPK1 protein in granulosa cells. The results showed that the MAPK1 fluorescence signal was significant and mainly distributed in the cytoplasm and perinuclear area. DAPI staining showed that the cell nucleus was clear ( Figure 6 A~F). Western blot detected a MAPK1 protein-specific band of approximately 42kDa, which was consistent with the GAPDH signal, indicating that the sample quality was reliable ( Figure 6 G-H). The results indicate that MAPK1 polyclonal antibodies can be used to study the expression and localization of MAPK1 protein in granulosa cells.

[0080] 3. Immunohistochemical expression and localization analysis of MAPK1 protein in Muscovy duck follicle tissue

[0081] To evaluate the applicability of MAPK1 polyclonal antibodies at the tissue level, immunohistochemistry was used to detect the expression and localization of MAPK1 protein in the ovarian tissue of Muscovy ducks. The results showed that MAPK1 was positively stained brown in the granulosa cell layer and theca region of the follicles, mainly distributed in the cytoplasm ( Figure 7A). FSHR is a positive control for granulosa cell markers, and its localization is basically consistent with that of MAPK1 ( Figure 7 B).

[0082] 4. Immunofluorescence expression and localization analysis of MAPK1 protein in Muscovy duck follicle tissue

[0083] To further clarify the cellular localization characteristics of MAPK1 in ovarian tissue, double immunofluorescence staining was used to detect the spatial distribution of MAPK1 and granulosa cell marker FSHR. The results showed that MAPK1 (red) and FSHR (yellow) signals were mainly concentrated in the granulosa cell layer of the follicle and had good spatial consistency in the cytoplasm ( Figure 8 A–D). DAPI nuclear staining further confirms the cell layer structure. This result suggests that MAPK1 is primarily localized in FSHR-positive granulosa cells, demonstrating that the prepared MAPK1 polyclonal antibody has high target recognition ability at the tissue level and can be used for immunofluorescence visualization studies.

[0084] In summary, the above evaluation preliminarily clarified the expression and localization characteristics of MAPK1 protein in the granulosa cells and tissues of the Muscovy duck ovary, indicating that this antibody is suitable for the visualization study of protein expression in the Muscovy duck ovary tissue, and provides a key technical tool and theoretical basis for in-depth revelation of the role of MAPK1 in the ovarian development and reproductive regulation of Muscovy duck.

Claims

1. A method for preparing a polyclonal antibody against Muscovy duck MAPK1 protein, characterized in that: The following steps are involved: Step 1: expressing the MAPK1 recombinant protein with the amino acid sequence of SEQ ID NO.2; Step 2: immunizing rabbits with the recombinant protein to obtain a polyclonal antibody against the Muscovy duck MAPK1 protein.

2. The preparation method according to claim 1, characterized in that In the first step, HB-PET autoinduction medium was used to express recombinant MAPK1 protein.

3. The preparation method according to claim 1, characterized in that In the first step, recombinant MAPK1 protein was purified using magnetic beads.

4. The preparation method according to claim 3, characterized in that After eluting the recombinant MAPK1 protein with an eluent containing imidazole, the imidazole was removed by dialysis.

5. The preparation method according to claim 1, characterized in that In the second step, polyclonal antibodies against Muscovy duck MAPK1 protein were prepared from serum pools.

6. The preparation method according to claim 1, characterized in that In the second step, rabbits are immunized with the recombinant protein together with an adjuvant.

7. The preparation method according to claim 1, characterized in that In the second step, multiple immune stimulations were used to prepare polyclonal antibodies against Muscovy duck MAPK1 protein.

8. The preparation method according to claim 7, characterized in that The multiple times is three times.

9. The preparation method according to claim 8, characterized in that The three immune stimulations were conducted on day 0, day 21 and day 42 respectively.

10. Use of the polyclonal antibody against Muscovy duck MAPK1 protein according to claim 1 in a visualization study of protein expression in Muscovy duck ovarian tissue.

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