IRF8 polyclonal antibody as well as preparation method and application thereof

Abstract

The invention discloses an IRF8 polyclonal antibody as well as a preparation method and application thereof, and belongs to the technical field of biology. A prokaryotic expression vector of IRF8 is successfully constructed through methods of RT-PCR, cloning, double enzyme digestion and the like, high-concentration and high-purity target protein is obtained by adopting a nickel column affinity purification technology after induced expression, a New Zealand white rabbit is used as a polyclonal antibody preparation object, recombinant protein and a Freund's adjuvant are mixed, pachymaran is added for co-emulsification treatment, and the recombinant protein is obtained. The immune effect is improved; the determination result shows that when the antigen coating concentration is 1.25 mu g / mL, the titer reaches 1: 2048000, and the titer of the polyclonal antibody added with the pachymaran group is obviously higher than that of a normal control group. The IRF8 polyclonal antibody has excellent specificity and sensitivity to poultry animals, provides a reliable tool for expression detection, positioning analysis and function exploration of IRF8 protein in poultry related disease research, and has important application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an IRF8 polyclonal antibody, its preparation method, and its application. Background Technology

[0002] Broilers ascites syndrome (BAS), also known as pulmonary hypertension syndrome (PHS) or heart failure syndrome, is a common non-infectious nutritional and metabolic disease in modern broiler production. It is characterized by massive intraperitoneal fluid accumulation, right ventricular dilation and hypertrophy, and heart failure. This disease is most prevalent in fast-growing broilers aged 3-5 weeks, especially in cold seasons, at high altitudes, or in poorly ventilated chicken houses. The root cause is that cardiopulmonary function development lags behind muscle growth, leading to pulmonary hypertension under conditions of hypoxia, high-energy diets, or environmental stress, ultimately resulting in right heart failure and systemic circulatory congestion, thus forming ascites. Currently, there is no specific vaccine for broiler ascites syndrome; prevention and control mainly rely on comprehensive management measures.

[0003] Poria cocos is a dried sclerotium used both as food and medicine. Belonging to the genus Poria in the family Polyporaceae, it is a sclerotium fungus that parasitizes pine roots and has been recorded in ancient texts such as *Fifty-Two Prescriptions* and *Shennong's Classic of Materia Medica*. Poria cocos contains various components, including polysaccharides, triterpenes, steroids, and small molecules. Its main active component, poria cocos polysaccharide, accounts for 70%-90% of the sclerotium's dry weight, with β-1,3-glucan as the main chain and β-1,6-glucan as the branch chain. Modern pharmacological studies have shown that poria cocos has immunomodulatory, antioxidant, antitumor, antibacterial, and hepatoprotective effects. In immunomodulation, poria cocos polysaccharide can act as a "biological response modifier," promoting macrophage polarization towards the M1 (pro-inflammatory) or M2 (repair) phenotype, enhancing complement system activity, and increasing the secretion of cytokines such as IL-1, TNF-α, and IFN-γ, thereby coordinating innate and acquired immunity to combat tumors, infections, and chronic inflammation.

[0004] Interferon regulatory factor 8 (IRF8), formerly known as interferon consensus sequence binding protein (ICSBP), is an important member of the interferon regulatory factor (IRF) family. The IRF8 gene is widely expressed in the immune system, playing a crucial role, especially in myeloid cells (such as dendritic cells, macrophages, and B cells). IRF8 expression is also high in immune-related tissues such as the spleen, lymph nodes, thymus, lungs, and kidneys, which are important sites for immune cell development, differentiation, and function.

[0005] Transcriptomic analysis of broiler pulmonary artery tissue revealed that IRF8 expression was significantly upregulated during broiler ascites syndrome (BAS), suggesting a potential regulatory role in the development and progression of BAS. However, chicken-derived specific antibodies against this protein are currently lacking, and its mechanism of action in poultry diseases has been rarely reported in domestic and international research. Summary of the Invention

[0006] In response to the content mentioned in the background art, this invention prepares a high-titer, high-specificity rabbit anti-chicken IRF8 polyclonal antibody by prokaryotic expression of exogenous genes and the addition of Poria cocos polysaccharide for animal immunization. This not only fills the gap in chicken-derived IRF8 antibodies, but also provides important experimental tools and theoretical references for in-depth exploration of the pathogenesis of nutritional metabolic diseases such as ascites syndrome in broilers.

[0007] This invention provides a method for preparing an IRF8 polyclonal antibody, comprising the following steps: Step 1: Insert the IRF8 target gene into the T3 cloning vector, transform the ligation mixture into DH5α competent cells, screen for positive strains, and extract the recombinant cloning plasmid. Step 2: Double digestion of the obtained recombinant cloning plasmid and PET empty vector to insert the target gene into the PET empty vector, transformation of the ligation product into competent E. coli cells, screening for positive strains, plasmid extraction to obtain recombinant expression plasmid, induction of expression to collect recombinant protein and purification. Step 3: Use the purified recombinant protein and Poria cocos polysaccharide as an antigen mixture to immunize rabbits, and separate the serum to obtain rabbit anti-chicken IRF8 polyclonal antibody.

[0008] Furthermore, the amount of Poria cocos polysaccharide added in step three is 80-120 times that of the recombinant protein.

[0009] Furthermore, in step three, the recombinant protein at 0.05 mg / mL, with the addition of 5 mg / mL of Poria cocos polysaccharide, is mixed with Freund's complete adjuvant in an equal mass ratio to form an antigen mixture.

[0010] Furthermore, the IRF8 target gene has the nucleotide sequence shown in SEQ ID NO.1; the recombinant protein has the amino acid sequence shown in SEQ ID NO.2.

[0011] Further, the method for obtaining the target gene in step one is as follows: RNA is extracted from pulmonary artery tissue of broiler chickens, and cDNA is obtained by reverse transcription. The obtained cDNA is used as a template to amplify the IRF8 target gene by PCR. The primers used to amplify the IRF8 target gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0012] Furthermore, the PCR amplification program was as follows: 95℃ for 3 min, 95℃ for 15 s, 68℃ for 15 s, 72℃ for 1 min, repeated for 35 cycles; and finally 72℃ for 5 min.

[0013] Further, the specific operation of transforming the ligation mixture into DH5α competent cells in step one is as follows: mix the ligation mixture with 100 μL of DH5α competent cells and incubate on ice for 30 min; heat shock in a 42℃ water bath for 45 s, then quickly transfer to an ice bath and incubate for 2 min; add 700 μL of antibiotic-free sterile liquid culture medium to a centrifuge tube, mix well, and then revive at 37℃ and 200 rpm for 60 min; then spread evenly on a liquid culture medium solid culture plate, and invert the plate in a 37℃ incubator for overnight culture.

[0014] Furthermore, the double enzyme digestion described in step two uses... BamH I and Hind III endonuclease; the specific operation for transforming the ligation product into competent E. coli cells is as follows: place the ligation product at 4℃ for 12-16 h, and then transform it into competent E. coli cells for expansion culture. The specific operation is the same as that for transforming into DH5α competent cells.

[0015] The present invention also provides an IRF8 polyclonal antibody obtained by the above preparation method.

[0016] The present invention also provides the application of the above-mentioned IRF8 polyclonal antibody in the preparation of a drug that specifically recognizes broiler ascites syndrome or IRF8 protein.

[0017] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention successfully constructed a prokaryotic expression vector for IRF8 using RT-PCR, cloning, and double enzyme digestion. High-concentration, high-purity target protein was obtained through nickel column affinity purification, providing a crucial antigenic basis for the preparation of high-titer antiserum. Compared to direct protein extraction from tissues, the prokaryotic expression combined with affinity purification method is simpler and less costly, suitable for large-scale preparation of polyclonal antibodies, and possesses good economic benefits and application prospects.

[0018] 2. This invention uses New Zealand white rabbits as the preparation material for polyclonal antibodies. IRF8 recombinant protein is mixed with Freund's adjuvant, and Poria cocos polysaccharide is added for emulsification to improve the immune effect. The test results show that when the antigen coating concentration is 1.25 μg / mL, the titer reaches 1:2048000. The titer of the polyclonal antibody prepared with the addition of Poria cocos polysaccharide is significantly higher than that of the normal control group, which proves that Poria cocos polysaccharide can improve the antibody titer by regulating the body's immune function. It also shows that the prepared antiserum has good sensitivity.

[0019] 3. The rabbit anti-chicken IRF8 protein polyclonal antibody prepared by this invention has excellent specificity and sensitivity to poultry. Its recognition efficacy is closely related to the high conservation of the poultry IRF8 gene, providing a reliable tool for the expression detection and functional study of IRF8 protein in poultry-related diseases. Attached Figure Description

[0020] Figure 1 For IRF8 protein, predict the transmembrane region (A), signal peptide (B), hydrophilicity / hydrophobicity (C), and secondary structure / antigenic epitope (D).

[0021] Figure 2 Electrophoresis results for IRF8 cloning and expression vectors. A: IRF8 gene polymerase chain reaction amplification product (714bp); lanes 1-3: IRF8 gene PCR amplification product, lane 4: negative control. B: Colony PCR results of cloned recombinant plasmids; lanes 1-9: IRF8 gene fragment inserted into pEASY®-T3-IRF8 recombinant plasmid, lane 10: negative control. C: Double enzyme digestion identification of recombinant plasmids PET32a-IRF8 and pET-32a empty vector; lane 1: recombinant plasmid PET32a-IRF8, lane 2: PET-32a empty vector. D: Colony PCR results of expression vector PET32a-IRF8; lanes 1-3: target fragment inserted into PET32a-IRF8 expression recombinant plasmid, lane 4: negative control. Lane M: DNA marker (DL2000).

[0022] Figure 3 Comparison results of IRF8 recombinant plasmid structure sequence determination.

[0023] Figure 4 Prokaryotic expression and purification of recombinant IRF8 protein. A: Successfully expressed recombinant IRF8 protein; Lane 1: Blank control (without IPTG), Lane 2: Total protein expressed in prokaryotes. B: Identification of IRF8 protein expression form; Lane 1: Blank control (without IPTG), Lane 2: Protein supernatant after fragmentation, Lane 3: Protein precipitate after fragmentation. C: Solubility assessment of recombinant IRF8 inclusion body protein; Lane 1: Supernatant after dissolution of IRF8 inclusion body protein. D: Dialysis and concentration of recombinant protein. Lane 1: Concentrated recombinant IRF8 protein (45 kDa). The size of standard protein markers in the electrophoresis images ranges from 20 to 150 kDa.

[0024] Figure 5 The results show the titer of the IRF8 antiserum. The X-axis represents the dilution ratio of rabbit anti-chicken IRF8 positive (with added Poria cocos polysaccharide) and negative sera; the Y-axis represents the absorbance of the antiserum at an excitation wavelength of 450 nm.

[0025] Figure 6 Comparison of IRF8 antiserum titer results from different immunization groups.

[0026] Figure 7 This is the result of Western blot analysis of IRF8 protein expressed in prokaryotes. Lanes 1-3: 3 replicates.

[0027] Figure 8 The effect of BAS on IRF8 protein expression levels. A: Lane 1: Normal group, Lane 2: Ascites group. B: In the statistical analysis of IRF8 relative to β-Tubulin protein expression levels, the results are expressed as mean ± SE (n = 3).

[0028] Figure 9 The expression changes of IRF8 protein in multiple tissues of broiler chickens are shown. A: Immunohistochemical staining results of IRF8 protein in the pulmonary artery, lung, heart, kidney, and liver tissues of normal and ascites broiler chickens (400×). B: Relative positive area of ​​IRF8 protein in the normal and ascites groups of broiler chickens. Statistical data are expressed as mean ± standard error (n = 3).

[0029] Figure 10 Immunohistochemical staining results of IRF8 protein in the pulmonary artery, lung, heart, kidney and liver tissues of different animals (400×). Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below in conjunction with embodiments. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0032] Example 1: Prokaryotic expression of chicken IRF8 Main test materials Table 1 Main Reagents

[0033] Table 2 Main Instruments and Equipment

[0034] Preparation of main test reagents (1) Preparation of bacterial culture reagent LB liquid medium (200 mL system): 2 g sodium chloride, 2 g tryptone, 1 g yeast extract, add 190 mL double-distilled water, shake until fully dissolved, adjust the pH of the solution to 7.4, and then bring the volume to 200 mL with double-distilled water. Autoclave before use: 121℃, 30 min.

[0035] LB medium solid (100 mL system): Add 1.5 g agarose powder to the culture medium and stir evenly.

[0036] IPTG working solution: Weigh 2.0 g of IPTG solid and pour it into a Shu Niu bottle containing 18 mL of double-distilled water. Shake well until completely dissolved, add double-distilled water to 20 mL, filter for sterilization, and finally store in a -20℃ refrigerator.

[0037] Ampicillin (100 mg / mL): Weigh 10 g Amp and mix with 90 mL of double-distilled water. After dissolving, bring the volume to 100 mL. Filter the solution using a 0.22 μm pore size filter to remove bacteria. Finally, store the solution in a -20°C freezer.

[0038] (2) Preparation of reagents for agarose gel electrophoresis Preparation of 100×TAE solution: Pour 0.01 mol EDTA, 11.4 mL glacial acetic acid, and 48.4 g Tris into a flask containing 95 mL double-distilled water, shake thoroughly until completely dissolved, add double-distilled water to bring the total to 100 mL, and place at room temperature.

[0039] Preparation of 1×TAE solution: Take 10 mL of 100×TAE solution into a container, pour in 990 mL of double-distilled water to make 1 L, and place at room temperature.

[0040] 1% nucleic acid gel: 0.2 g C 24 H 38 O 19 20 mL of TAE nucleic acid electrophoresis solution (1×) was heated for 1 min to fully melt the solution. Then, 4 μL of nucleic acid fuel (EB) was added, and the mixture was thoroughly mixed before being poured into a 10-well gel plate and cooled for 20 min.

[0041] (3) SDS-polyacrylamide gel electrophoresis reagent formulation SDS-PAGE electrophoresis buffer solution (1 L): Tris 3 g, Gly 18.4 g, SDS 1 g, add ddH2O to make up to 1 L.

[0042] 10% SDS stock solution (100 mL): Mix 10 g of SDS with 100 mL of double-distilled water thoroughly.

[0043] Protein gel staining solution: Take 0.625 g of Coomassie Brilliant Blue solid powder, 100 mL of methanol, and 25 mL of glacial acetic acid and pour them into a glass bottle containing 125 mL of distilled water. Shake to dissolve completely.

[0044] Protein glue decolorizing solution: Pour 100 mL of methanol and 25 mL of glacial acetic acid into a glass bottle containing 125 mL of distilled water, and shake to mix thoroughly.

[0045] (4) Protein inclusion body dissolving reagent formulation Bacterial lysate: 500 mM sodium chloride, 0.05 mol / L sodium dihydrogen phosphate (pH 8.0).

[0046] Inclusion body washing solution: 50 mM sodium chloride, 50 mM Tris base, 1% Triton-X-100, 1 mM disodium ethylenediaminetetraacetate (EDTA disodium) (pH 8.0).

[0047] Inclusion body dissolution solution: 0.1 mol / L sodium dihydrogen phosphate, 10 mM Tris base, 8 mol urea (pH 8.0).

[0048] Purification column equilibration solution: 0.1 mol / L sodium dihydrogen phosphate, 10 mM Tris base, 8 mol urea (pH 8.0).

[0049] Washing solution: 10 mM Tris base, 0.2 mol / L sodium dihydrogen phosphate, 6 mol urea, 0.02 mol / L imidazole (pH 8.0).

[0050] Eluent: 10 mM Tris base, 0.1 mol / L sodium dihydrogen phosphate, 8 mol urea, (0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, 0.5 mol / L) imidazole (pH 8.0).

[0051] (5) Preparation of protein dialysis refolding reagent Dialysis refolding solution A: 0.05 M Tris-HCl, with urea added to achieve final concentrations of 1 M, 2 M, 4 M, and 6 M (pH adjusted to 8.0).

[0052] Dialysis refolding solution B: 0.05 M Tris-HCl, 2 mM EDTA, 0.1% sodium lauroyl sarcosinate, 2 mM dithiothreitol, 0.5 mol / L urea (pH adjusted to 8.0).

[0053] Dialysis refolding solution C: 0.05 M Tris-HCl, 0.05 M sodium chloride, 0.2 mol / L urea (pH adjusted to 8.0).

[0054] 1. Bioinformatics analysis of IRF8 in broiler chickens

[0055] 2. RNA extraction A suitable amount of pulmonary artery tissue was cut into a 1.5 mL EP tube, and 1 L of TRIzol lysis buffer was added. After low-temperature magnetic grinding, the tube was allowed to stand for 5 min. The tube was then centrifuged at 12000×g for 5 min at 4°C. 800 μL of the supernatant was pipetted into another EP tube. 200 μL of chloroform was added to the lysis buffer, and the mixture was gently mixed by inverting until white. The mixture was allowed to stand for 5 min. The tube was then centrifuged at 12000×g for 15 min at 4°C. The resulting supernatant was transferred to a new EP tube. Approximately 600 μL of isopropanol was added, and the mixture was allowed to stand for 10 min. The tube was then centrifuged at 12000×g for 10 min at 4°C until a white precipitate appeared. The supernatant was discarded and the tube was slowly poured off. 1 mL of 75% ethanol solution was slowly added along the tube wall, taking care not to touch the precipitate. The tube was inverted and centrifuged at 7500×g for 5 min at 4°C. This ethanol washing was repeated twice. After extraction, dry the sample for 5-10 minutes with the lid off. Add DEPC water to dissolve the sample in 10 μL. After extraction, take 0.5 μL of the sample, dilute it 10-fold, and test the RNA OD. A260 / A280 The ratio and concentration.

[0056] 3. Reverse transcription of RNA The reaction was performed according to the instructions of the one-step cDNA synthesis kit, and the reaction system is shown in the table below.

[0057] Table 3 cDNA synthesis system

[0058] Program settings: 42℃ / 5min, 85℃ / 5s, 4℃ / ∞.

[0059] 4. Primer design and synthesis IRF8 gene-specific primers were designed by the company using Primer Premier software according to experimental requirements, and then synthesized by Qingke Biotechnology Co., Ltd. The primer synthesis and purification method was PAGE.

[0060] Table 4 Gene Primer Sequences

[0061] 5. Target gene amplification Table 5 PCR reaction system

[0062] The amplification program for the IRF8 gene was as follows: 95℃ for 3 min, 95℃ for 15 s, 68℃ for 15 s, 72℃ for 1 min, cycle number 35; 72℃ for 5 min.

[0063] The obtained PCR products were added to 1% agarose gels at a rate of 5 μL / well, and the electrophoresis results were identified using the Gel DocXR+ system.

[0064] 6. Purification of PCR products The PCR products were recovered using the Quick Gel Extraction Kit manufactured by Beijing TransGen Biotech Co., Ltd., and the operation was strictly performed in accordance with the manufacturer's instructions.

[0065] 7. Ligation and transformation of cloning vectors According to the Peasy-T3 Cloning Vector instructions, the purified target gene amplification product was inserted into the T3 cloning vector to construct a recombinant plasmid. The cloning ligation system is shown in the table below.

[0066] Table 6 TA Cloning System

[0067] Gently mix the ligation mixture with 100 μL of DH5α competent cells and incubate on ice for 30 min. Heat shock at 42°C for 45 s, then quickly transfer to ice and incubate for 2 min. Add 700 μL of antibiotic-free sterile liquid medium (LB) to a centrifuge tube, mix well, and incubate at 37°C and 200 rpm for 60 min. Then, evenly spread the mixture onto LB solid culture plates (IRF8 recombinant vector antibiotic: Amp; concentration: 100 μg / mL), and incubate inverted at 37°C overnight.

[0068] 8. Screening and identification of strains Using a sterile, enzyme-free 10 μL pipette tip, a single clone from an LB agar plate was picked and transferred to 5 mL of LB liquid medium containing the corresponding antibiotic. The culture was incubated at 220 rpm and 37°C for 8–10 h. The amplified bacterial culture was then subjected to PCR to determine whether the target gene had been successfully inserted into the cloning vector. The specific system is shown in the table below.

[0069] Table 7. PCR identification of bacterial culture

[0070] The PCR amplification procedure and gel electrophoresis for the IRF8 gene were the same as described above. Positive bacterial cultures identified by PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for first-generation nucleic acid sequencing of the gene.

[0071] 9. Double digestion of IRF8 cloning vector and PET-32a(+) Following the instructions of the plasmid extraction kit, plasmids were extracted from the sequenced bacterial culture. The extracted clonal recombinant plasmids and expression vectors were then processed. BamH I and Hind The digestion system using endonuclease III is shown in the table below.

[0072] Table 8. Double Enzyme Digestion System

[0073] After complete digestion of the vector by incubating in a 37°C water bath for 1 hour, the digested product was purified using a commercially available purification kit from Beijing Solarbio Biotechnology Co., Ltd.

[0074] 10. Ligation and transformation of genes with their corresponding expression vectors In this experiment, the target gene fragment was inserted into the corresponding PET series empty vector to construct a recombinant expression plasmid. The specific experimental system is shown in the table below.

[0075] Table 9. Construction system of recombinant plasmid for target gene

[0076] The ligation product was placed in a 4°C refrigerator for 12-16 h and then transformed into competent Escherichia coli cells (BL21(DE3)-IRF8) for expansion culture. The specific experimental procedures were the same as the transformation steps described above.

[0077] 11. Screening and identification of positive bacterial colonies Identify positive colonies according to the aforementioned steps, and extract plasmids from bacterial solutions whose nucleic acid sequences have been correctly detected, following the instructions of the plasmid extraction kit.

[0078] 12. Identification of recombinant expression plasmids by double enzyme digestion In this experiment, the obtained PET32a-IRF8 recombinant expression plasmid was digested with BamHI and HindIII restriction endonucleases for detection. The specific procedures are shown in the table below.

[0079] Table 10 Double enzyme digestion verification system

[0080] The enzyme was digested at 37℃ for 1 h, and gel electrophoresis was used to verify whether the target gene was successfully inserted into the expression vector.

[0081] 13. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) Prepare separating gels of appropriate concentrations based on the molecular weight of the protein samples to be tested (the proportion of acrylamide added varies). The concentration of the stacking gel is generally 5%. The specific components of this gel are shown in the table below. For a 10-well SDS-PAGE gel, load 10 μL of the sample and 1.5 μL of protein marker. After loading, run the gel at 80 V for 20-30 min, then increase the voltage to 120 V until the marker is completely dispersed. After electrophoresis, immerse the gel in an appropriate amount of Coomassie Brilliant Blue staining solution for 4-6 h, then destain for approximately 12 h. This allows you to observe whether the target protein has been successfully induced.

[0082] Table 11 SDS-PAGE Gel Formulation

[0083] 14. High-level expression of recombinant proteins (1) The bacterial strain that was positive by PCR and correctly sequenced was transferred to 500 mL of freshly prepared LB medium, supplemented with 500 μL of Amp (100 mg / mL), and cultured at 220 rpm and 37℃ until OD. 600 It is 0.6-0.8.

[0084] (2) Add IPTG to LB (0.2 mM), 220 rpm, 37℃, and keep for 6 h.

[0085] 15. Identification of recombinant protein expression forms 200 mL of LB medium (400 μL of Amp + 5 mL of positive bacterial culture) was used for expansion culture, followed by the addition of 400 μL of IPTG. Expression was induced at 22℃ and 160 rpm for 10 h. All bacterial culture was centrifuged in a 50 mL EP tube to form a precipitate. 30 mL of PBS was added and mixed thoroughly, followed by sonication and resuspending. 300 μL of lysozyme (100 mg / mL) was added and the mixture was incubated at 4℃ for 2 h. After centrifugation at 12000 rpm / 2 min, 10 μL of the supernatant and precipitate (remixed with PBS) were taken for protein electrophoresis to determine whether the protein was a high-density, insoluble inclusion body protein.

[0086] 16. Solubility of inclusion body proteins When the fragmented protein sample presents as a precipitate, it can be determined that the protein is a high-density, insoluble inclusion body protein. Then, 30 mL of inclusion body washing buffer is added to the precipitate, and the mixture is washed with a magnetic stirrer at 4°C for 25 min, centrifuged at 4000 rpm for 25 min, the supernatant is discarded, and the washing process is repeated once. An equal volume of inclusion body lysis buffer is added, and the mixture is stirred at room temperature for 30 min, allowed to stand for 2 h, and then electrophoresis is performed to verify whether the inclusion bodies have been fully lysed.

[0087] 17. Nickel column purification of recombinant proteins (1) Take all the ultrasonically disrupted bacterial solution (40 mL) and dispense it into 20 2 mL EP tubes. Centrifuge at 4℃ and 12000 rpm for 20 min, discard the supernatant and keep the precipitate.

[0088] (2) Add 40 mL of inclusion body washing solution to 20 EP tubes respectively, resuspend the precipitate, put them all into the same container, wash with a magnetic stirrer at 4℃ for 20 min, and then centrifuge at 4℃ and 12000 rpm for 20 min.

[0089] (3) Add 40 mL of inclusion body solution to 20 EP tubes respectively, resuspend the precipitate, and put them all into the same container. Dissolve them at 4°C with the help of a magnetic stirrer (12 h).

[0090] (4) Dispense the overnight solution (40 mL) into 20 2 mL EP tubes, centrifuge at 4 °C and 12,000 rpm for 20 min, and collect the supernatant. Finally, filter all the supernatant through an organic filter (0.45 μm).

[0091] (5) Equilibration: Equilibrate the protein purification column with 8 mL of equilibration buffer.

[0092] (6) Loading: Add the supernatant after filtration to the protein purification column (3-5 mL each time).

[0093] (7) Washing: Add 10 mL of washing solution to the protein purification column for washing.

[0094] (8) Elution: To determine the optimal imidazole concentration in the eluent, the protein was eluted with 1 mL of eluent containing different imidazole concentrations (100 mM, 200 mM, 300 mM, 400 mM, 500 mM), and 10 μL of each eluent was then subjected to SDS-PAGE to determine the optimal imidazole concentration.

[0095] (9) Preservation of the purification column: Wash the purification column with 10 mL of equilibration buffer, then wash the purification column with 10 mL of double-distilled water, then wash the purification column with 10 mL of 20% ethanol, and finally add 5 mL of 20% ethanol to the column and store it at 4℃.

[0096] (10) Take 10-15 μL of the purified protein solution for vertical electrophoresis and observe the results of the above experiment.

[0097] 18. Refolding and Concentration of Recombinant Proteins Cut the dialysis bag into strips approximately 15 cm long (pre-made dialysis bags do not require processing). Pour the target protein into the sealed transparent dialysis bag and place it in dialysis solution A (urea concentration: 6 M-1 M) with different urea concentrations, stirring evenly at low temperature, and dialyze for 8 hours sequentially from high to low concentration. Then, transfer it to dialysis solution B and dialysis solution C sequentially and dialyze at low temperature for 8 hours. Cover the entire surface of the dialysis bag with an appropriate amount of polyethylene glycol-6000 for concentration, and take 10 μL of the concentrated recombinant protein for protein electrophoresis.

[0098] Experimental Results and Analysis: like Figure 1 As shown, online analysis using TMHMM-2.0 revealed no obvious transmembrane region ( Figure 1 A in the text); IRF8 signal peptide prediction showed the presence of a signal peptide ( Figure 1 (B in the text); IRF8 protein overall average hydrophilicity (GRAVY): -2.778 is a hydrophilic protein ( Figure 1 The secondary structure of the IRF8 protein was analyzed, revealing that it possesses α-helical, β-sheet, and random coil structures, and is rich in antigenic epitopes. Figure 1 (D in the middle).

[0099] PCR amplification of the IRF8 gene revealed a clear target band at approximately 714 bp. Figure 2 (A) The selected positive colonies were subjected to culture PCR and gene sequencing, and the results confirmed the successful construction of the recombinant cloning plasmid. Figure 2 (B in the original text). The recombinant expression plasmid was identified by PCR in the amplified bacterial culture and verified by digestion with two restriction endonucleases, confirming the successful construction of the pET32a-IRF8 (714 bp) expression vector. Figure 2 C in Figure 2 (D in the middle).

[0100] like Figure 3 As shown, the nucleotide and amino acid sequences of the coding region of the recombinant IRF8 protein were completely consistent through gene sequencing, with no mutations or missing bases.

[0101] In the 10% SDS-PAGE electrophoresis verification, IPTG, a protein inducer, was used to act on the His-IRF8 fusion protein, and a clear target protein expression band was observed at 45 kDa. Figure 4 A in the text). Identification of the protein expression pattern revealed that the recombinant IRF8 protein was expressed as numerous high-density, insoluble inclusion bodies. Figure 4 (B in the original text). A target protein band of the same size as expected was obtained in the supernatant of the dissolved inclusion bodies. Figure 4 (C in the text). Preliminary studies have found that dialysis and concentration of the supernatant after dissolution of inclusion bodies yields a darker, wider target band, indicating a higher concentration of the protein. Figure 4 (D in the middle).

[0102] Example 2: Preparation of IRF8 polyclonal antibody in broilers Main test materials Table 12 Main Instruments and Equipment

[0103] Table 13 Main Reagents

[0104] Preparation of main test reagents (1) Preparation of ELISA-related reagents Antigen coating solution: Add 2.93 g of powdered sodium bicarbonate and 1.95 g of sodium carbonate to a container, dissolve in 900 mL of double-distilled water, and then add more double-distilled water to reach 1000 mL.

[0105] Phosphate buffer: Add 0.2 g of powdered potassium dihydrogen phosphate, 3 g of disodium hydrogen phosphate dodecahydrate, 0.2 g of potassium chloride, and 8.0 g of sodium chloride to a container, add double-distilled water to dissolve them, and then add double-distilled water to make up to 1 L.

[0106] Preparation of PBS-T: Add Tween-20 to the prepared PBS to make its concentration 0.05%.

[0107] Preparation of skim milk blocking solution (5%): Add 2.5 g of skim milk powder to a container, and then add 50 mL of PBS to dissolve it completely.

[0108] Preparation of the stop solution: Slowly add 11.1 mL of sulfuric acid to 88.9 mL of double-distilled water to prepare 2 M sulfuric acid solution.

[0109] (2) Preparation of Western blotting reagent Preparation of SDS-PAGE electrophoresis buffer: Add 18.4 g of Gly, 3 g of Tris, and 1 g of SDS to a container, then add double-distilled water to completely dissolve them, and bring the volume to 1 L.

[0110] Preparation of transfer buffer: Add 100 mL of methanol to 400 mL of prepared electrophoresis buffer to prepare transfer buffer.

[0111] Preparation of PBS-T: Add Tween-20 to the prepared PBS to make its concentration 0.05%.

[0112] Preparation of blocking solution: Add 2.5 g of skim milk powder to a container, then add 50 mL of PBS to dissolve it completely.

[0113] 1. Feeding and Management of New Zealand White Rabbits Healthy, mature, and appropriately sized New Zealand white rabbits were purchased from the Animal Experiment Center of Jiangxi Agricultural University and raised for one week to allow them to adapt before being vaccinated.

[0114] 2. Animal immunization and serum collection Serum was collected via the ear artery as a negative control before immunization. Recombinant IRF8 protein was emulsified at a ratio of 0.05 mg / mL (with 5 mg / mL of Poria cocos polysaccharide added) with Freund's complete adjuvant at a 1:1 ratio (complete emulsification was indicated by no dispersion after 1 minute when dropped into water), and injected subcutaneously into each New Zealand white rabbit multiple times. Recombinant IRF8 protein was then mixed 1:1 with Freund's incomplete adjuvant, and booster immunizations were performed at different sites every 7 days, for a total of 4 booster immunizations (multiple subcutaneous injections on the back, with 500-700 μg of antigen per rabbit per injection, and a volume not exceeding 100 μL per injection site). Blood was collected from the rabbit's ear artery at 28 and 35 days later, centrifuged at 3000×g for 5 min, and antiserum was separated.

[0115] 3. IRF8 protein antibody titer assay (ELISA) First, the recombinant protein at the optimal concentration (1.25 μg / mL) was coated onto 96-well plates. Then, the plates were sealed with skim milk, and the primary antibody IRF8 serum and pre-immunization serum were serially diluted (1:1000–1:4096000). The plates were incubated at 37°C for 1 h. HRP-labeled Goat Anti-Rabbit IgG (H+L) was prepared according to the reagent instructions at a dilution of 1:8000. After several washes, the reaction stop solution was added. Finally, the polyclonal antibody titer was determined using a multi-mode microplate reader based on the absorbance at 450 nm.

[0116] Based on the structural characteristics of traditional Chinese medicines, two medicinal materials, Poria cocos polysaccharide and Ganoderma lucidum polysaccharide, were screened for preliminary experiments. Rabbits were selected as immunization animals and randomly divided into two groups: a control group (without any injection treatment) and an experimental group. The experimental group was further divided into a recombinant protein IRF8 immunization group, a recombinant protein IRF8 combined with Poria cocos polysaccharide immunization group, and a recombinant protein IRF8 combined with Ganoderma lucidum polysaccharide immunization group. All groups underwent immunization according to a pre-defined procedure. After the immunization cycle, blood was collected from all rabbits, serum was separated, and the titer of IRF8-specific antibodies in the serum was determined using enzyme-linked immunosorbent assay (ELISA).

[0117] 4. Western blot (protein blotting) The prokaryotic expression product of IRF8 protein was subjected to SDS-PAGE of the appropriate size. 30 μg of protein sample was transferred to a PVDF membrane via wet transfer. The antiserum was diluted 1:200. After blocking, the membrane and rabbit anti-chicken IRF8 serum were incubated overnight at 4°C. The membrane and HRP-labeled Goat Anti-Rabbit IgG (H+L) were incubated at 37°C for 40 min, followed by washing with PBS-T 3–5 times. Finally, visualization was performed using the ChemiDoc chemiluminescent gel imaging system.

[0118] Experimental Results and Analysis Results of IRF8 antiserum titer assay: The titer of rabbit anti-chicken IRF8 protein antiserum was determined using enzyme-linked immunosorbent assay (ELISA). The experiment showed that when the antigen coating concentration was 1.25 μg / mL, the antibody level in the group immunized with Poria cocos polysaccharide was significantly higher than that in the group immunized without Poria cocos polysaccharide. The results showed that the titer of the serum immunized with Poria cocos polysaccharide was 1:2048000, while the titer of the serum immunized without Poria cocos polysaccharide was 1:1024000. Figure 5 ).

[0119] Rabbits were used as immunization animals in the preliminary experiment and randomly divided into two groups: a control group (without any injection treatment) and an experimental group. The experimental group was further divided into three subgroups: a recombinant IRF8 protein immunization group, a recombinant IRF8 protein combined with Poria cocos polysaccharide immunization group, and a recombinant IRF8 protein combined with Ganoderma lucidum polysaccharide immunization group. All groups underwent immunization according to a pre-defined procedure. After the immunization cycle, blood was collected from all rabbits, serum was separated, and the titer of IRF8-specific antibodies in the serum was determined using enzyme-linked immunosorbent assay (ELISA). The results showed that the serum-specific antibody titer in the recombinant IRF8 protein combined with Poria cocos polysaccharide immunization group was significantly higher than that in the recombinant IRF8 protein combined with Ganoderma lucidum polysaccharide immunization group. Figure 6 ).

[0120] Western blot results of IRF8 protein expressed in prokaryotes: (See attached image) Figure 7As shown, a clear and uniform band of the same size as the target protein appeared on the PVDF membrane, indicating that the antiserum has significant specificity for the IRF8 protein expressed in prokaryotic cells.

[0121] Example 3: IRF8 protein localization analysis and expression changes in BAS broiler chickens Main test materials Table 14 Main Reagents

[0122] Table 15 Main Instruments and Equipment

[0123] Preparation of main test reagents EDTA repair solution: Add 10 mL of EDTA repair solution (50×) to a container, then add 490 mL of ultrapure water, mix thoroughly, and store in a refrigerator at 4℃.

[0124] Phosphate buffer: Weigh 8 g sodium chloride, 0.2 g potassium dihydrogen phosphate, 0.2 g potassium chloride, and 3 g disodium hydrogen phosphate dodecahydrate into a flask containing 900 mL of double-distilled water. Shake to dissolve completely, then add more double-distilled water to reach 1000 mL.

[0125] Preparation of 3% H2O2PBS buffer: Add 10 mL of hydrogen peroxide (30%) to the container and bring the volume up to 100 mL with the prepared PBS buffer.

[0126] PVDF transfer solution: C4H 11 NO3 5.8 g, Gly 2.9 g, C 12 H 25 Mix 0.37 g of SO4Na and 200 mL of CH3OH, then add an appropriate amount of ultrapure water to a final volume of 1000 mL. After thorough mixing, store in a refrigerator at 4°C.

[0127] Blocking solution: Weigh 2.5 g of skim milk powder and mix thoroughly with 50 mL of PBS solution.

[0128] 1. Animal model establishment and sample collection Twenty 3-day-old white-feathered broiler chickens were randomly divided into a normal group (n=10, raised in a normal environment) and an ascites group (n=10, fed 0.12% NaCl water and a high-nutrient feed containing 3% lard and 4% fishmeal in a cold and humid environment). After 30 days, the broiler chickens in the ascites group developed obvious ascites. All broiler chickens were euthanized, and pulmonary artery, liver, kidney, heart, and lung tissue samples were prepared from 5 chickens in each group for 4% paraformaldehyde. Molecular samples were prepared from the corresponding tissues of the remaining 5 chickens and stored at -80℃. Five healthy and mature broiler chickens, ducks, pigeons, mice, goats, and rabbits were purchased from each group, raised in a stable environment for 7 days, and then euthanized. Pulmonary artery, liver, kidney, heart, and lung samples were collected. Molecular samples were stored in liquid nitrogen or at -80℃, and tissue samples were stored at room temperature in 4% paraformaldehyde.

[0129] 2. Extraction and concentration determination of total protein from pulmonary artery, heart, lung, liver and kidney tissues of broilers. A suitable amount of tissue was cut into a 1.5 mL centrifuge tube, and 100 μL of lysis buffer containing RIPA solution and protease inhibitor in a specific ratio was added. After homogenization at low temperature, the mixture was incubated on ice for 30 min, followed by centrifugation at 13000×g for 10 min. The obtained total protein was stored at -80℃. The absolute quantification of protein samples extracted from the pulmonary artery, heart, lung, liver, and kidney was performed using the BCA concentration detection method, referring to the detailed instruction manual of the kit produced by Solarbio Science & Technology Co., Ltd.

[0130] 3. Western blotting The specific operation is the same as described above.

[0131] 4. Immunohistochemical test Formalin-fixed tissue was prepared into paraffin sections using standard methods. The paraffin sections were then cut to a thickness of 3-5 μm. Dewaxing was performed by immersing the sections in xylene-anhydrous ethanol-90%-80%-70%-50% ethanol in that order. After antigen retrieval, the sections were incubated with 3% H2O2 at room temperature for 10 min, followed by washing with PBS for 5 min per wash, for a total of 4 washes. The sections were then incubated with goat serum blocking solution at 37°C for 30 min, and excess liquid was removed. The slides were incubated with positive serum against IRF8 / HIF1a and non-immunized serum (both diluted 1:200) at 37°C for 1.5 h. The slides were washed with PBS for 5 min per wash, for a total of 4 washes. Poly-HRP-Anti-Rabbit IgG incubation solution was added, and the sections were incubated at 37°C for 30 min. The sections were then washed with PBS for 5 min per wash, for a total of 4 washes. Finally, an appropriate amount of DAB chromogenic solution was added for 1-3 min of chromogenic development, followed by washing with PBS to terminate the chromogenic process. Finally, the slides were counterstained, dehydrated, cleared, and mounted using the standard slide preparation method. They were then observed, mounted, and photographed using a BM2000 biological microscope.

[0132] 5. Data Analysis The obtained data were analyzed using independent samples t-test and one-way ANOVA in SPSS 25 software. All results are shown in the statistical graph in the form of mean ± SE. Among them, "*" (P<0.05), "**" (P<0.01), "***" (P<0.001), and "****" (P<0.0001) are considered.

[0133] Experimental Results and Analysis To verify the specificity of the IRF8 polyclonal antibody, the effect of broiler ascites syndrome on IRF8 protein expression was further investigated. Western blot was used to detect IRF8 protein in total protein from key broiler tissues. Figure 8 As shown, the expression level of IRF8 protein increased in BAS, and the expression differences were most significant in pulmonary artery, kidney and lung (P<0.01).

[0134] The expression of IRF8 protein in the pulmonary artery, lung, heart, liver, and kidney of normal and ascites chickens was detected using IRF8 antibody. The results showed that, compared with normal chickens, the expression of IRF8 protein in these organs of ascites chickens was significantly upregulated. Figure 9 ).

[0135] Immunohistochemistry was used to locate the expression and distribution of IRF8 protein in key tissues of different animals. The results showed that IRF8 protein was mainly found in the cell membranes of pulmonary artery, heart, lung, liver, and kidney tissues of various animals. Figure 10 ).

[0136] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they are not intended to limit the present invention. It should be noted that various changes and modifications can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the concept and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing an IRF8 polyclonal antibody, characterized by, It comprises the following steps: Step one, insert the IRF8 target gene into T3 cloning vector, transform the ligation mixture into DH5α competent cells, screen to obtain positive strains, extract plasmid to obtain recombinant cloning plasmid; Step two, double enzyme digestion of the obtained recombinant cloning plasmid and PET empty carrier to insert the target gene into the PET empty carrier, transform the ligation product into E. coli competent cells, screen to obtain positive strains, extract plasmid to obtain recombinant expression plasmid, induce expression, collect recombinant protein and purify; Step three, mix the purified recombinant protein and Pachyman as an antigen mixture to immunize rabbits, and isolate serum to obtain rabbit anti-chicken IRF8 polyclonal antibody.

2. The method of claim 1, wherein the IRF8 polyclonal antibody is prepared by, The addition amount of Pachyman in step three is 80-120 times of the recombinant protein.

3. The method of claim 1, wherein the IRF8 polyclonal antibody is prepared by, In step three, the recombinant protein is mixed with Freund's complete adjuvant at an equal mass ratio as an antigen mixture.

4. The method of claim 1, wherein the IRF8 polyclonal antibody is prepared by, The IRF8 target gene has the nucleotide sequence shown in SEQ ID NO. 1; the recombinant protein has the amino acid sequence shown in SEQ ID NO.

2.

5. The method of claim 1, wherein the IRF8 polyclonal antibody is prepared by, In step one, the method for obtaining the target gene is: taking broiler lung artery tissue to extract RNA, reverse transcribing to obtain cDNA, taking the obtained cDNA as a template, and amplifying the IRF8 target gene by PCR; The primers used for amplifying the IRF8 target gene are shown in SEQ ID NO. 3 and SEQ ID NO.

4.

6. The method of claim 5, wherein the IRF8 polyclonal antibody is prepared by, The program for PCR amplification is: 95℃ for 3 min, 95℃ for 15 s, 68℃ for 15 s, 72℃ for 1 min, repeat for 35 cycles; finally 72℃ for 5 min.

7. The method of claim 1, wherein the IRF8 polyclonal antibody is prepared by, In step one, the specific operation for transforming the ligation mixture into DH5α competent cells is: mix the ligation mixture with 100 μL of DH5α competent cells, and stand on ice for 30 min; 42℃ water bath heat shock for 45 s, quickly transfer to ice bath, stand for 2 min; add 700 μL of sterile liquid medium without antibiotics to the centrifuge tube, mix evenly, then recover at 37℃, 200 rpm for 60 min; then evenly spread on the liquid medium solid culture plate, and place the plate in a 37℃ incubator overnight culture.

8. The method of claim 1, wherein the IRF8 polyclonal antibody is prepared by, The double digestion in step two is performed using BamH I and Hind III endonuclease; the specific operation for transforming the ligation product into the competent cells of E. coli is as follows: the ligation product is placed at 4°C for 12-16 h, and then is expanded in the competent cells of E. coli, and the specific operation is consistent with that of transforming into the competent cells of DH5α.

9. The IRF8 polyclonal antibody obtained by the preparation method of any one of claims 1-8.

10. The use of the IRF8 polyclonal antibody of claim 9 in the preparation of a drug for specifically recognizing broiler ascites syndrome or IRF8 protein.

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