An anti-recombinant canine interleukin-1β yolk antibody, and a preparation method and application thereof

Abstract

The present application relates to a kind of anti-recombination canine interleukin-1β yolk antibody and its preparation method and application, the preparation method utilizes genetic engineering prokaryotic expression preparation antigen recombination canine interleukin-1β, antigen recombination canine interleukin-1β is emulsified after first immunization egg chicken with first immunoadjuvant, again the recombination canine interleukin-1β antigen is emulsified to egg chicken with second immunoadjuvant, and egg chicken is strengthened immunization, collect egg, separate egg yolk and extract the antibody in the yolk of egg, successfully obtain anti-recombination canine interleukin-1β yolk antibody, the titer of the yolk antibody is 1:409600, and higher purity, with better specificity and stability, can provide stable antigen and antibody material basis for canine IL-1β related detection, antibody preparation and subsequent application research, with wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of egg yolk antibodies, specifically to an egg yolk antibody against recombinant canine interleukin-1β, its preparation method, and its application. Background Technology

[0002] Interleukin-1β (IL-1β) is an important pro-inflammatory cytokine in the inflammatory response, playing a key regulatory role in innate immune activation, inflammatory cascade amplification, and tissue damage progression. IL-1β is first synthesized in the form of a precursor protein, which is then activated by the inflammasome-related pathway, cleaved by caspase-1, and forms a mature active protein. This protein then mediates downstream inflammatory signal transduction through the IL-1 receptor. Therefore, IL-1β not only participates in the occurrence of inflammatory responses but is also closely related to the persistence of inflammation, tissue damage, and imbalance of the local immune microenvironment.

[0003] In canine studies, abnormal expression of IL-1β has been observed in various inflammatory diseases and tissue damage processes. For example, in enteritis, heart disease, and other inflammatory lesions, IL-1β levels in tissues or peripheral blood are often elevated to varying degrees, suggesting a close relationship with disease state changes and the progression of inflammatory responses. Therefore, research on canine IL-1β detection, antibody preparation, and subsequent applications is of significant research importance and practical value.

[0004] Currently, common methods for preparing antibodies against specific antigens mainly include mammalian-derived IgG antibodies and avian-derived IgY antibodies. Compared to traditional mammalian IgG antibodies, IgY is derived from egg yolks, eliminating the need for repeated blood sampling and causing less harm to animals. Furthermore, laying hens can continuously produce eggs, thus typically yielding a larger quantity of antibody material per unit time, offering advantages such as stable source, higher yield, and relatively lower cost. In addition, IgY exhibits less non-specific reaction with mammalian complement, rheumatoid factor, and Fc receptor-related components in some immunoassay systems, which helps reduce background interference. Therefore, IgY shows promising application prospects in antigen detection, immunoassay, and antibody preparation.

[0005] However, several technical challenges remain in preparing specific IgY antibodies against canine IL-1β. First, IL-1β is synthesized in its precursor form and processed and cleaved to form a mature active protein. If the antigen fragment is not selected appropriately, the resulting antibody may primarily recognize non-functionally relevant regions of the precursor, thus affecting the antibody's specificity and subsequent application value. Second, during prokaryotic expression of recombinant canine IL-1β protein, problems such as insufficient expression levels, low soluble expression ratios, or excessive impurities after purification may occur, affecting the quality of the immunogen. Third, the effectiveness of IgY preparation is also closely related to the immunization procedure, adjuvant selection, and egg yolk antibody extraction and purification methods. Improper condition control can easily lead to insufficient antibody titer, low purity, or unsatisfactory specificity.

[0006] Therefore, it is still necessary to establish a complete technical method for antigen design, recombinant expression, immunopreparation, and IgY extraction, purification, and identification of canine IL-1β, in order to obtain anti-canine IL-1β egg yolk antibodies with high titer, good specificity, and high purity, providing a stable antigen and antibody material basis for canine IL-1β-related detection and subsequent application research. Summary of the Invention

[0007] The purpose of this invention is to address at least one deficiency in the prior art by providing an anti-recombinant canine interleukin-1β egg yolk antibody, its preparation method, and its applications. This invention establishes a complete technical route around canine IL-1β, from target gene cloning, recombinant protein expression and purification, to the preparation and basic identification of specific egg yolk antibodies. It obtains relatively stable canine IL-1β recombinant antigen and specific antibody, and provides a material basis for subsequent research on the inflammatory regulation mechanism and related applications of canine IL-1β, thereby solving the problem of the lack of high-purity, high-titer, and high-specificity anti-canine IL-1β egg yolk antibodies in related technologies.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention is to provide a method for preparing an egg yolk antibody against recombinant canine interleukin-1β, comprising the following steps: Recombinant canine interleukin-1β antigen was prepared by prokaryotic expression using genetic engineering. The recombinant canine interleukin-1β antigen was emulsified with a first immunizing adjuvant and then used to immunize laying hens for the first time. The recombinant canine interleukin-1β antigen was then emulsified with a second immunizing adjuvant and used to boost the immunization of laying hens. Eggs were collected, egg yolks were separated, and egg yolk antibodies were extracted from the egg yolks to obtain the egg yolk antibodies against recombinant canine interleukin-1β.

[0009] Furthermore, the amino acid sequence of the recombinant canine interleukin-1β antigen is shown in SEQ ID NO: 1.

[0010] Furthermore, the preparation of recombinant canine interleukin-1β antigen using prokaryotic expression via genetic engineering specifically includes the following steps: Total RNA was extracted from canine spleen tissue, and cDNA was synthesized by reverse transcription. The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 was obtained by PCR amplification, cloned into the pMD-19T vector, and then subcloned into the pET-28a prokaryotic expression vector. The vector was transformed into host cells BL21(DE3), and the recombinant canine interleukin-1β antigen was obtained after induction of expression and purification.

[0011] Furthermore, the nucleotide sequence encoding the recombinant canine interleukin-1β protein is shown in SEQ ID NO: 2.

[0012] Furthermore, the PCR amplification program is as follows: pre-denaturation at 92~96℃ for 29~31 s; denaturation at 93~95℃ for 29~31 s, annealing at 52~54℃ for 29~31 s, extension at 70~72℃ for 1~1.5 min, for a total of 30 cycles; final extension at 70~72℃ for 4~5 min; storage at 4℃.

[0013] The preferred PCR amplification program is as follows: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ final extension for 5 min; storage at 4℃.

[0014] Furthermore, the PCR amplification reaction system is as follows: 2 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, 25 μL premixed solution, and deionized water to make up to 50 μL; wherein, the upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4.

[0015] Further, the induction expression conditions are as follows: induction at 16~37℃ with 0.1~2 mmol / L isopropyl thiogalactoside for 2~8 hours, preferably induction at 37℃ with 0.1 mmol / L isopropyl thiogalactoside (IPTG) for 6 hours.

[0016] Further, the first immune adjuvant is Freund's complete adjuvant, and the volume ratio of the first immune adjuvant to the antigen recombinant canine interleukin-1β is 1:1; the second immune adjuvant is Freund's incomplete adjuvant, and the volume ratio of the second immune adjuvant to the antigen recombinant canine interleukin-1β is 1:1.

[0017] Furthermore, the enhanced immunized laying hens include: Second immunization: 12-16 days after the first immunization, preferably 14 days; Third immunization: 26-30 days after the first immunization, preferably 28 days.

[0018] Furthermore, the injection dose of the recombinant canine interleukin-1β antigen is 125~235 μg / kg per single immunization dose based on the weight of the laying hen; wherein, the amount of the recombinant canine interleukin-1β antigen injected into each hen is 250~350 μg / time, preferably 350 μg / time.

[0019] Furthermore, both the initial immunization and the booster immunization are performed using a subcutaneous multi-point injection method, wherein the subcutaneous multi-point injection method involves setting 4 to 6 injection points under the skin of the neck or back of the laying hen.

[0020] Furthermore, the extraction of yolk antibodies from egg yolks includes the following steps: S1. Separate the egg yolk and egg white, collect the egg yolk liquid, add PBS buffer to the egg yolk liquid at a volume ratio of 1:2 and mix well; then add PEG-6000 to a final concentration of 3%~4% (W / V), stir until completely dissolved, let stand for 15~25 min and then centrifuge to collect the supernatant. S2. Filter the supernatant obtained in step S1, add PEG-6000 to the filtrate with a final concentration of 8%~9% (W / V), stir and let stand for 18~23 min, then centrifuge and discard the supernatant.

[0021] S3. Dissolve the precipitate obtained in step S2 in PBS, add PEG-6000 to a final concentration of 10%~14% (W / V), mix well, let stand for 8~12 min, centrifuge, discard the supernatant, and the resulting precipitate is the egg yolk antibody against recombinant canine interleukin-1β.

[0022] Further, in steps S1, S2, and S3, the centrifugation is performed at 3-5°C at 10,000-14,000 r / min for 28-35 min, preferably at 4°C at 12,000 r / min for 30 min.

[0023] Furthermore, the filtration described in step S2 is a double-layer filter paper filtration.

[0024] A second aspect of the present invention is to provide an egg yolk antibody against recombinant canine interleukin-1β, said egg yolk antibody being prepared using the preparation method described in the first aspect.

[0025] A third aspect of the present invention is to provide a preparation method as described in the first aspect and / or the use of egg yolk antibody as described in the second aspect in the preparation of a detection reagent for detecting canine interleukin-1β protein.

[0026] The present invention adopts the above technical solution and has the following technical effects compared with the prior art: This invention utilizes bioinformatics analysis of canine IL-1β precursor protein, selecting a 340-798 bp target fragment (after removing the immature precursor region) as the expression sequence. A recombinant expression plasmid, pET-28a-cIL-1β, was successfully constructed, transformed into host cells, and induced to express and be purified to obtain recombinant canine interleukin-1β. Immunization of SPF-grade laying hens with the obtained recombinant canine IL-1β protein successfully yielded anti-canine IL-1β specific egg yolk antibodies. Indirect ELISA results showed that the IgY titer extracted from the yolk of egg yolk No. 1 after three immunizations was the highest, with a maximum positive dilution of 1:409600. Furthermore, the obtained anti-recombinant canine IL-1β egg yolk antibody exhibited high purity, superior specificity, and stability. This invention has significant application value in canine interleukin-1β protein detection, antibody preparation, and research on canine IL-1β-related inflammatory mechanisms, providing a stable antigen and antibody material basis for canine IL-1β-related immunodetection and subsequent application research. Attached Figure Description

[0027] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a graph showing the results of protein hydrophilicity analysis in one embodiment of the present invention; Figure 2 This is a graph showing the analysis results of the transmembrane region of a protein in one embodiment of the present invention; Figure 3 This is a graph showing the prediction results of protein signal peptides in one embodiment of the present invention; Figure 4 This is a diagram showing the predicted secondary structure of a protein in one embodiment of the present invention; Figure 5 This is a diagram showing the amplification results of the canine IL-1β gene in one embodiment of the present invention; Figure 6 This is a diagram of bacterial culture PCR results in one embodiment of the present invention; Figure 7 This is a diagram showing the double enzyme digestion identification results of the recombinant plasmid in one embodiment of the present invention; Figure 8 This is a diagram showing the induced expression results of recombinant protein in one embodiment of the present invention; Figure 9 This is a graph showing the comparison results of different induction combinations for recombinant protein expression in one embodiment of the present invention; Figure 10 This is a graph showing the optimized concentration of the inducer for recombinant protein expression in one embodiment of the present invention; Figure 11 This is a Western blot identification result of recombinant protein in one embodiment of the present invention; Figure 12 This is an SDS-PAGE result of the purified and concentrated recombinant protein in one embodiment of the present invention; Figure 13 This is a graph showing the antibody titer results of egg yolk after the first and second immunizations in one embodiment of the present invention; Figure 14 This is a graph showing the antibody titer results of egg yolk after three immunizations in one embodiment of the present invention; Figure 15 This is an SDS-PAGE analysis result of IgY extracted and purified by PEG precipitation in one embodiment of the present invention; Figure 16 This is a Western blot analysis result of the reaction between purified IgY and recombinant canine IL-1β protein in one embodiment of the present invention; Figure 17 This is a graph showing the stability analysis results of anti-canine IL-1βIgY in one embodiment of the present invention. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0029] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0030] The sources of the reagents and test animals involved in the embodiments of this invention are as follows: Ex Taq enzyme, restriction enzymes BamHI, XhoI, and T4 DNA Ligase were all purchased from Takara. M-MLV reverse transcription polymerase was purchased from Aikerui Biotechnology Co., Ltd. N-Ampicillin, kanamycin, and IPTG were all purchased from Proteintech Biotechnology Co., Ltd. O-180 Protein Marker was purchased from Novizan Biotechnology Co., Ltd. P-gel recovery kit and plasmid mini-extraction kit were purchased from Tiangen Biotech Co., Ltd. Q-French complete adjuvant, French incomplete adjuvant, and polyethylene glycol (PEG-6000) were all purchased from Beyotime Biotechnology Co., Ltd. (Shanghai). The prokaryotic expression vector pET-28a, Escherichia coli DH5α, and BL21 were all preserved in our laboratory; SPF laying hens were purchased from Nanjing Tegeli Planting Professional Cooperative, and the canine spleen tissue was obtained from a spleen removal surgery at an animal hospital in Nanjing.

[0031] Other manufacturers not specifically indicated in the embodiments of this invention can be obtained through commercial purchases.

[0032] The bioinformatics websites required in the embodiments of this invention are shown in Table 1: Table 1 Required Bioinformatics Websites The present invention will be described below by way of example.

[0033] Example 1 - Preparation of Recombinant Canine IL-1β Protein This embodiment relates to a specific method for preparing recombinant canine IL-1β protein, which includes the following steps: 1. IL-1β protein sequence analysis Based on the canine IL-1β reference sequence published in GenBank, and using the bioinformatics software listed in Table 1, the structural characteristics of its precursor protein were analyzed, and the results are as follows: (1) Physicochemical property analysis of the protein: To clarify the basic physicochemical properties of the canine IL-1β precursor protein, its amino acid sequence was analyzed using ProtParam and ProtScale. The results showed that the protein consists of 265 amino acid residues, with a relative molecular mass of 30.14 kDa, a theoretical isoelectric point (pI) of 5.31, an instability coefficient of 41.91, and an adipose coefficient of 81.55. The hydrophilicity analysis is shown in Figure 1, with a GRAVY value of -0.327, indicating that the protein is generally hydrophilic and has relatively poor stability.

[0034] (2) Protein transmembrane structure analysis: Based on the prediction results of DeepTMHMM, such as Figure 2As shown, the predicted type of canine IL-1β precursor protein is Globular, and no obvious transmembrane region was found in the whole sequence, suggesting that it does not belong to transmembrane protein.

[0035] (3) Protein signal peptide sequence analysis: Based on the signal peptide prediction results, such as Figure 3 As shown, the canine IL-1β precursor protein did not contain a typical Sec / SPI type N-terminal signal peptide, nor was there a clear cleavage site, indicating that it is not a secreted protein transported via the classical secretory pathway.

[0036] (4) Protein structure prediction analysis: SOPMA prediction results are as follows Figure 4 As shown in the figure, the canine IL-1β precursor protein contains a total of 265 amino acid residues, of which α-helices account for 25.66%, β-sheets account for 25.28%, and random coils account for 49.06%. Overall, the protein is mainly composed of random coils, while also containing a certain proportion of α-helices and β-sheets, suggesting that it has a certain degree of structural flexibility.

[0037] The results showed that the full-length fragment was 265 amino acid residues, predominantly hydrophilic, lacking a typical N-terminal signal peptide and transmembrane region. Considering the characteristic of IL-1β being synthesized as a precursor protein and then processed and cleaved intracellularly to form a mature active protein, this study selected the 340–798 bp target fragment (after removing the immature precursor region) as the expression sequence and successfully constructed the recombinant expression plasmid pET-28a-cIL-1β. This fragment is 459 bp long and corresponds to the functionally relevant region of canine IL-1β. Using this fragment for prokaryotic expression helps reduce the interference of the immature precursor region on subsequent protein expression, purification, and antibody recognition, making the resulting recombinant protein closer to the functional antigen with application value, thereby improving the specificity of subsequent yolk antibody preparation.

[0038] As analyzed above, the canine IL-1β precursor protein is 265 amino acid residues in length, predominantly hydrophilic, lacks a typical N-terminal signal peptide, and has no transmembrane region. Based on the synthesis of canine IL-1β in precursor form, after intracellular processing and cleavage, a biologically active mature protein is formed. Considering the structural characteristics of the canine IL-1β precursor protein and the requirements for subsequent prokaryotic expression, this embodiment selects a 340-798 bp target fragment (after removing the immature precursor region) as the expression sequence encoding the functionally relevant region of canine IL-1β. The target fragment is 459 bp in length, corresponding to the functionally relevant region of canine IL-1β, and its encoded amino acid sequence is shown in SEQ ID NO: 1. Using this fragment for prokaryotic expression helps reduce the interference of the immature precursor region on subsequent protein expression, purification, and antibody recognition, making the resulting recombinant protein closer to the functional antigen with application value, thereby improving the specificity of subsequent specific yolk antibody preparation.

[0039] Based on the above-mentioned target fragment, cloning primers cIL-1β-1-F and cIL-1β-1-R, as well as expression primers cIL-1β-2-F and cIL-1β-2-R containing restriction enzyme sites, were further designed for amplification, as shown in Table 2; wherein, the amino acid sequence encoded by the target fragment SEQ ID NO: 1 is as follows: AAMQSVDCKLQDISHKYLVLSNSYELRALHLNGENVNKQVVFHMSFVHGDESNNKIPVVLGIKQKNLYLSCVMKDGKPTLQLEKVDPKVYPKRKMEKRFVFNKIEIKNTVEFESSQYPNWYISTSQVEGMPVFLGNTRGGQDITDFTMEFSS Table 2 PCR primer sequences 2. Gene extraction and recombinant plasmid construction Canine spleen tissue was used as the material for total RNA extraction. The tissue was obtained from clinical spleen surgical samples at an animal hospital in Nanjing and was used for subsequent total RNA extraction after standardized acquisition. Total RNA was extracted from the spleen using the TRIzol method, and the concentration and purity of RNA were determined using NanoDrop. cDNA was synthesized by reverse transcription using total RNA as a template under the action of M-MLV reverse transcriptase. The reverse transcription system was prepared according to Table 3, and the reverse transcription reaction was performed according to the procedure shown in Table 4. The target gene was amplified using cIL-1β-1-F and cIL-1β-1-R as primers. The PCR amplification system is shown in Table 5. The PCR reaction program was: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; final extension at 72℃ for 5 min; storage at 4℃. After identification by agarose gel electrophoresis, the PCR product was excised and purified to obtain the target fragment; the results are as follows. Figure 5 As shown.

[0040] The purified and recovered target fragment was ligated into the pMD-19T cloning vector. The ligation product was transformed into DH5α competent cells and plated on LB agar plates containing the appropriate antibiotics, then incubated overnight at 37°C with the plates inverted. The next day, single white colonies were picked and inoculated into 500 μL of LB liquid medium containing the appropriate antibiotics. After incubation at 37°C with shaking for 3 h, the bacterial culture was used as a template for PCR identification. The PCR-positive strains were further cultured, and recombinant plasmids were extracted and sequenced. The pMD-19T-IL-1β recombinant plasmid with the correct inserted sequence was obtained through screening.

[0041] The target gene fragment was then amplified using the correctly sequenced pMD-19T-IL-1β recombinant plasmid as a template. The nucleotide sequence of the target gene fragment is shown in SEQ ID NO: 2. Sequencing results showed that the target fragment was consistent with the expected design sequence. The target gene fragment and the expression vector pET-28a were then double-digested with BamHI and XhoI, respectively. The target gene digestion system is shown in Table 6, and the vector digestion system is shown in Table 7. The digestion products were purified by agarose gel electrophoresis, and then ligated with T4 DNA ligase to the linearized vector. The ligation system is shown in Table 8, and ligation was carried out at 16℃ for 12 h. The ligation product was transformed into DH5α competent cells, 1 mL of antibiotic-free LB liquid medium was added, and the cells were incubated at 37℃ for 1 h. The bacterial culture was then plated onto LB solid medium plates containing kanamycin resistance and incubated upside down at 37℃ for approximately 16 h.

[0042] The following day, a single colony was picked and inoculated into 500 μL of LB broth containing kanamycin resistance. After incubation at 37°C with shaking for 3 h, the bacterial culture was collected for PCR identification. The identification results are as follows: Figure 6 As shown in Table 9, the bacterial culture that was correctly identified by PCR was cultured overnight at 37°C, and then plasmids were extracted according to the Beyotime Plasmid Mini Preparation Kit instructions. The extracted plasmids were then subjected to double enzyme digestion as shown in Table 9, with digestion conditions of 37°C for 3 hours. The digestion products were analyzed by agarose gel electrophoresis, and the results are shown in Table 9. Figure 7 As shown in the figure; simultaneously, the recombinant plasmid was sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The recombinant plasmid that was correctly identified by sequencing was named pET-28a-cIL-1β.

[0043] Table 3 Reverse Transcription System Table 4 Reverse Transcription Procedure Table 5 PCR amplification system Table 6. Target gene enzyme digestion system Table 7. Vector Enzyme Digestion System Table 8 Connection System Table 9 Recombinant plasmid digestion system Results analysis: from Figure 5It can be seen that, using cDNA derived from canine spleen tissue as a template, a target gene fragment of approximately 459 bp was successfully amplified; where M: DL 5000 DNA Marker; 1: PCR product of canine IL-1β gene.

[0044] from Figure 6 It can be seen that the bacterial culture PCR identification results show that strains 1, 5, 6 and 8 are all positive clones. After sequencing the positive clones, it was found that the inserted fragment is consistent with the expected target sequence. Among them, M: DL 2000 DNA Marker; 10: negative control; 1-9: bacterial culture samples.

[0045] from Figure 7 The linearized vector band and the target gene band are clearly visible, indicating that the canine IL-1β prokaryotic expression vector was successfully constructed; where M: DL 5000 DNA Marker; 1: PET-28a-IL-1β enzyme digestion product; 2: PET-28a-IL-1β recombinant plasmid.

[0046] The nucleotide sequence of the target gene fragment, SEQ ID NO: 2, is shown below: GCAGCCATGCAATCGGTGGACTGCAAGTTACAGGACATAAGCCACAAATAACCTGGTGCTGTCTAACTCATATGAGCTTCGGGCTCTCCACCTCAATGGGGAAAATGTGAACAAACAAGTGGTGTTCCACATGAGCTTTGTGCACGGGGATGAAAGTAATAACAAGATACCTGTGGTCTTGGGCATCAAACAAAAGAATCTGTACCTGTCCTGTGTGATGAAGGATGGAA AGCCCACCCTACAGCTAGAGAAGGTAGACCCCAAAGTCTACCCAAAGAGGAAGATGGAAAAGCGATTTGTCTTCAACAAGATAGAAATCAAGAACACAGTGGAATTTGAGTCTCTCAGTACCCTAACTGGTACATCAGCACCTCTCAAGTCGAAGGAATGCCTGTCTTCCTAGGAAATACCAGAGGTGGCCAGGATATAACTGACTTCACCATGGAATTCTCTTCCTAG 3. Optimization of expression form and induction conditions of canine IL-1β protein To explore the optimal induction conditions for recombinant canine IL-1β protein expression and preliminarily confirm its expression form, the recombinant plasmid pET-28a-cIL-1β was transformed into competent BL21(DE3) cells, plated on LB agar plates containing kanamycin resistance, and incubated overnight at 37°C with the plates inverted. Single colonies were then picked and inoculated into LB liquid medium containing kanamycin for activation culture. Subsequently, a 2% inoculum was transferred to fresh LB liquid medium containing kanamycin and cultured at 37°C with shaking until the bacterial culture reached OD. 600 When the nm value was approximately 0.6, IPTG was added to induce expression, followed by ultrasonic disruption and separation. The expression pattern was then analyzed, and the results are as follows: Figure 8 As shown, M: protein molecular weight standard; 1: empty vector induction control; 2: induced whole bacteria; 3: induction supernatant; 4: induction precipitation.

[0047] The results showed a clear target band at approximately 22 kDa, and the recombinant protein was distributed in both the supernatant and the precipitate, indicating simultaneous soluble and insoluble expression. Considering that the soluble protein in the supernatant is more conducive to subsequent purification and does not require refolding, subsequent experiments will focus on the supernatant.

[0048] (1) Comparison of different induction combinations: Under the condition of a final IPTG concentration of 1 mmol / L, referring to the commonly used induction protocols in prokaryotic expression, three induction combinations were set up: 37℃ for 6 h, 28℃ for 12 h, and 16℃ for 24 h, to compare the expression of the target protein under different induction combinations. BL21(DE3) transformed with the empty pET-28a vector was set up as a control group. After induction, the bacterial cells were collected by centrifugation, resuspended in PBS, and lysed by sonication. The total bacteria, lysate, and bacterial pellet were separated by centrifugation. Each sample was added to 5× loading buffer and denatured in a metal bath at 95℃ for 10 min, followed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining analysis. The results are as follows: Figure 9 As shown, M: protein molecular weight standard; 1: 37℃, 6 h empty vector induction control; 2: 37℃, 6 h whole bacteria induction; 3: 37℃, 6 h induction supernatant; 4: 37℃, 6 h induction precipitation; 5: 28℃, 12 h empty vector induction control; 6: 28℃, 12 h whole bacteria induction; 7: 28℃, 12 h induction supernatant; 8: 28℃, 12 h induction precipitation; 9: 16℃, 24 h empty vector induction control; 10: 16℃, 24 h induction whole bacteria; 11: 16℃, 24 h induction supernatant; 12: 16℃, 24 h induction precipitation.

[0049] Depend on Figure 9As can be seen, the target protein band was detected under all three induction conditions compared. The target band was clearer under the 37℃ induction for 6 h condition, and the soluble target protein band in the supernatant was relatively more prominent. Considering that this condition resulted in a shorter induction time and better expression, 37℃ induction for 6 h was subsequently selected as the experimental condition for further comparison of IPTG concentration and preparation of recombinant proteins.

[0050] (2) Optimization of induction concentration: Based on the experimental conditions obtained from the above screening, different final IPTG concentrations (0, 0.1, 0.2, 0.5, 1.0 and 2.0 mmol / L) were further set for induction expression for 6 h, and SDS-PAGE analysis was performed using the same method. The results are as follows: Figure 10 As shown, M: protein molecular weight standard; 1: no inducer added; 2: expression induced by 0.1 mM IPTG; 3: expression induced by 0.2 mM IPTG; 4: expression induced by 0.5 mM IPTG; 5: expression induced by 1 mM IPTG; 6: expression induced by 2 mM IPTG.

[0051] Depend on Figure 10 As can be seen, a high expression level was achieved with 0.1 mmol / L IPTG, and no significant increase in band intensity was observed after further increasing the IPTG concentration. Therefore, 0.1 mmol / L IPTG was selected as the inducer concentration for subsequent experiments.

[0052] 4. Large-scale expression, identification, and purification of canine IL-1β protein 5 mL of activated bacterial culture was inoculated into 500 mL of LB medium containing kanamycin for large-scale expression of the recombinant protein, and the OD of the bacterial culture was measured. 600 When the saturation point (nm) is approximately 0.6, IPTG is added and induced under the same conditions. After induction, the bacterial cells are collected by centrifugation, resuspended in pre-cooled PBS, and sonicated to disrupt the cells. The supernatant is then collected by centrifugation as the soluble protein fraction. The supernatant is mixed with His-tagged protein purification packing material (Ni-NTA / His Resin) and incubated for 1 h. Purification is performed using affinity column chromatography, washing sequentially with a low concentration of imidazole wash buffer to remove non-specifically bound proteins, followed by elution with a higher concentration of imidazole elution buffer to elute the target protein. The elution fraction is then collected.

[0053] The eluted recombinant protein was placed in an MD44 dialysis bag (molecular weight cutoff 7000 D) and dialyzed at 4°C for 12 h. The dialysis buffer was prepared as follows: 3.15 g Tris and 29 g sodium chloride were dissolved in deionized water, followed by the addition of 50 mL glycerol and thorough stirring. The final volume was adjusted to 1 L and stored at room temperature. After dialysis, the protein sample was concentrated to a suitable volume using a 3 K ultrafiltration centrifuge tube. The purified and concentrated recombinant canine IL-1β protein was subjected to BCA protein concentration determination, SDS-PAGE electrophoresis, and Coomassie brilliant blue staining to detect protein concentration and purity. Western blot analysis was further used to verify the reaction specificity of the target band. The Western blot analysis results are shown below. Figure 11 As shown, 1: Induction of precipitation; 2: Induction of supernatant; 3: Induction of whole bacteria. The SDS-PAGE results of the purified and concentrated recombinant protein are shown below. Figure 12 As shown, M: protein molecular weight standard; 1: before purification; 2: recombinant protein after purification and concentration.

[0054] from Figure 11 A clear, specific reaction band was observed at approximately 22 kDa, indicating that the expressed protein was recombinant canine IL-1β protein; from Figure 12 It can be seen that after large-scale induction expression under optimized conditions, and after Ni-NTA affinity purification, dialysis and ultrafiltration concentration, the SDS-PAGE results showed that the target band was significantly enriched after purification, indicating that a recombinant protein with high purity was obtained.

[0055] Example 2 - Immunization of laying hens with recombinant canine IL-1β protein This embodiment uses the recombinant canine IL-1β protein obtained in Example 1 as an immunogen to prepare anti-recombinant canine IL-1β egg yolk antibody IgY by immunizing laying hens. The specific steps include: Three healthy SPF-grade laying hens from Nanjing Tegeli Planting Professional Cooperative were selected and randomly numbered after 7 days of acclimatization. Purified recombinant canine IL-1β protein was used as an immunogen for animal immunization.

[0056] For the initial immunization, recombinant canine IL-1β protein was thoroughly emulsified with an equal volume of Freund's complete adjuvant; for subsequent booster immunizations, the antigen was thoroughly emulsified with an equal volume of Freund's incomplete adjuvant. Each chicken received 300 μg of antigen per immunization, administered via subcutaneous multi-site injection. The immunization schedule was set as follows: initial immunization on day 0, booster immunizations on days 14 and 28, for a total of three immunizations. During the immunization process, the laying hens' mental state, feed intake, and local injection site reactions were closely observed. Observations showed no significant abnormalities in the experimental chickens' mental state, feed intake, or egg production during the immunization period, and no significant adverse reactions were observed at the injection sites.

[0057] Eggs were collected starting 7 days after the initial immunization, each egg was individually numbered, and stored at 4°C for later use. Eggs were subsequently collected at different time points for yolk separation and IgY extraction.

[0058] Example 3 - Indirect ELISA detection of IgY titer In this embodiment, indirect ELISA was used to detect the titer of egg yolk samples collected at different immunization stages in Example 2, dynamically monitor the changes in the level of anti-canine IL-1β specific IgY, and screen the egg collection time period with higher antibody titer to provide materials for subsequent IgY extraction, purification and identification.

[0059] To dynamically monitor changes in anti-canine IL-1β IgY levels in egg yolk after immunization and to screen for egg collection time periods with higher antibody titers, indirect ELISA was used to detect the titer of egg yolk samples collected at different time points in Example 2. Purified recombinant canine IL-1β protein was used as the coating antigen, diluted to an appropriate concentration with coating buffer, and added to 96-well high-adsorption ELISA plates, 100 μL per well, and incubated overnight (12–18 h) at 4°C. The next day, the plate was discarded, and washed three times with PBST, 300 μL per well, soaking for 1–2 min each time and then patted dry. 200 μL of 5% skim milk blocking buffer was added to each well, and the plate was blocked at room temperature for 1 h. The blocking buffer was discarded, and the plate was washed 2–3 more times. Egg yolk samples from different time points were pretreated appropriately and then serially diluted with PBST (1:3200–1:409600), 100 μL per well, with replicates, and incubated at room temperature for 1 h. After washing the plate three times, add 100 μL of HRP-labeled rabbit anti-chicken IgY secondary antibody diluted 1:5000 to each well and incubate at room temperature for 1 h. After washing the plate five more times, add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for 10–15 min. Stop the reaction by adding 50 μL of stop solution and immediately read the OD value at 450 nm. Use unimmunized egg yolk samples or pre-immunized egg yolk samples as negative controls. The average OD value of the sample wells and the negative control wells is calculated. 450 A p / N ratio greater than 2.1 is considered a positive result, and the highest dilution factor meeting this criterion is taken as the IgY titer. Indirect ELISA results are as follows: Figure 13 , Figure 14 As shown, where, Figure 13 In the table, 1-1, 1-2, and 1-3 represent IgY extracted from egg yolks of eggs 1, 2, and 3 after the first immunization, respectively; 2-1, 2-2, and 2-3 represent IgY extracted from egg yolks of eggs 1, 2, and 3 after the second immunization, respectively; negative: IgY extracted from unimmunized egg yolks. Figure 14 The titer represents the antibody titer of egg yolks after three immunizations. Among them, 3-1, 3-2, and 3-3 represent IgY extracted from egg yolks of eggs 1, 2, and 3 after three immunizations, respectively; negative represents IgY extracted from egg yolks of unimmunized eggs.

[0060] from Figure 13 As shown in Figure 14, compared with the negative control, the OD of egg yolk samples at each time point after immunization was significantly higher. 450 The values ​​all increased significantly, and generally increased with the number of immunizations. Among them, the IgY titer extracted from the yolk of egg yolk No. 1 after the third immunization was the highest, with the highest positive dilution being 1:409600. Based on this result, egg yolk samples collected after the third immunization were subsequently selected for IgY extraction and purification.

[0061] Example 4 - Extraction and purification of IgY Based on the indirect ELISA titer detection results of Example 3, this embodiment selected eggs collected during the period of high anti-canine IL-1β IgY titer. The yolk and egg white were separated, residual egg white on the yolk surface was absorbed with filter paper, the yolk membrane was punctured, and the yolk fluid was collected. IgY in the egg yolk was extracted and purified using the PEG precipitation method. The specific method includes the following steps: Add 2 parts PBS buffer (pH 7.5) to 1 part egg yolk solution at a volume ratio of 1:2, and mix thoroughly by shaking for 30 s. Then add PEG-6000 to a final concentration of 3.5% (w / v), stir thoroughly until completely dissolved, and let stand at room temperature for 20 min. Centrifuge at 12,000 r / min for 30 min at 4 °C, and filter the supernatant through double-layer filter paper. Add PEG-6000 to the filtrate to a final concentration of 8.5% (w / v), stir thoroughly to dissolve, let stand at room temperature for 20 min, and then centrifuge at 12,000 r / min for 30 min at 4 °C, discarding the supernatant. Dissolve the resulting precipitate in an appropriate amount of PBS, add PEG-6000 to a final concentration of 12% (w / v), mix thoroughly, let stand at room temperature for 10 min, and then centrifuge at 12,000 r / min for 30 min at 4 °C, discarding the supernatant. The resulting precipitate is the crude IgY extract. The precipitate was resuspended and dissolved in an appropriate amount of PBS, then aliquoted and stored at -20℃ for later use.

[0062] The purified IgY sample was analyzed for protein concentration using the BCA method, and its purification effect was analyzed by SDS-PAGE. The purity and integrity of the extracted IgY were evaluated based on the clarity of the IgY heavy and light chain bands and the presence of contaminating protein bands in the electrophoresis results. The SDS-PAGE results are shown below. Figure 15 As shown, M: protein molecular weight standard; 1: purified IgY sample.

[0063] Based on the titer test results, egg yolk samples collected after the third immunization were selected as the antibody preparation material. IgY was extracted and purified using the PEG precipitation method. The purified sample protein concentration was 10 mg / mL. Figure 15 Clear IgY heavy and light chain bands are visible, with relatively few other proteins, indicating that the obtained IgY has good purity and integrity.

[0064] Example 5 - IgY Specificity Analysis This embodiment is based on the recognition specificity of recombinant canine IL-1β protein by IgY with good purity and integrity obtained in Example 4.

[0065] The specificity of the prepared IgY was analyzed using Western blot. Purified recombinant canine IL-1β protein and the unrelated recombinant protein IL-31 were used as control proteins, mixed with 5× protein loading buffer, and then boiled for denaturation. The IL-31 control protein was used to evaluate whether the anti-recombinant canine IL-1β IgY exhibited non-specific binding to other recombinant proteins, thus aiding in determining the specificity of the prepared IgY for recognizing recombinant canine IL-1β protein. The IgY was separated by SDS-PAGE and transferred to a PVDF membrane. After blocking with 5% skim milk powder at room temperature, purified anti-recombinant canine IL-1β IgY was added as the primary antibody, and the membrane was incubated overnight at 4°C. The next day, the membrane was washed with TBST, and HRP-labeled rabbit anti-chicken IgY secondary antibody was added, followed by incubation at room temperature for 1 h. After washing again, ECL chemiluminescence solution was added for color development, and the band distribution was observed under a gel imaging system. The results are shown below. Figure 16 As shown (M: molecular weight standard of protein; 1: IL-1β protein; 2: IL-31 protein), the specificity of the prepared IgY for recognizing canine IL-1β was evaluated by comparing the difference in the response of IgY to recombinant canine IL-1β protein with that of the control protein.

[0066] from Figure 16 It can be seen that the purified IgY can specifically react with recombinant canine IL-1β protein and a clear band appears at about 22 kDa; while no corresponding reaction signal was observed in the recombinant protein IL-31 control group. This indicates that the IgY does not generally bind to other recombinant proteins in a non-specific manner, but can specifically recognize recombinant canine IL-1β protein.

[0067] Example 6 - Stability analysis of anti-canine IL-1β IgY This embodiment analyzes the stability of the anti-canine IL-1β egg yolk antibody with good purity and integrity obtained in Example 4.

[0068] The purified anti-canine IL-1β IgY obtained in Example 4 was prepared into a 1 mg / mL solution with PBS (pH 7.4) and aliquoted for storage. Different temperature storage groups, different pH treatment groups, and repeated freeze-thaw treatment groups were set up. The residual binding activity of the purified anti-canine IL-1β IgY under different treatment conditions was detected by indirect ELISA to analyze its stability. The results are as follows: Figure 17 As shown.

[0069] (1) Temperature stability test setup: 4℃ for 0, 7, 14, 21, and 28 days; 25℃ for 0, 3, 7, and 14 days; 37℃ for 0, 1, 3, and 5 days. Samples were taken at each time point, and the binding activity against recombinant canine IL-1β protein was detected by indirect ELISA. The results are as follows: Figure 17 As shown in a.

[0070] (2) pH stability test setup: IgY was placed in buffer systems at pH 2.0, pH 4.0, pH 7.0, pH 9.0 and pH 11.0 respectively, and incubated at 37℃ for 2 h. After treatment, the samples were immediately adjusted back to neutral with Tris-HCl buffer, and then its binding activity to recombinant canine IL-1β protein was detected by indirect ELISA. The results are as follows: Figure 17 As shown in b.

[0071] (3) Freeze-thaw stability test setup: IgY samples were repeatedly frozen and thawed between -20℃ and room temperature once, three times and five times. After treatment, the binding activity of IgY samples to recombinant canine IL-1β protein was detected by indirect ELISA.

[0072] Each treatment group had three parallel samples, with the untreated IgY sample serving as the control group. Its binding activity was defined as 100%. The residual binding activity of each treatment group was calculated, and the results are as follows: Figure 17 As shown in c.

[0073] Results analysis: Temperature stability test results (17a) show that the IgY can still maintain high antigen binding activity after being stored at 4 ℃ for 28 days; after short-term storage at 25 ℃, the binding activity decreased slightly, indicating that the antibody can still maintain good activity under low temperature conditions.

[0074] pH stability test results (17b) show that this IgY has the highest binding activity under neutral conditions and can still maintain good antigen binding ability under weak acid and weak alkaline conditions.

[0075] The results of repeated freeze-thaw stability tests (17c) showed that after 1, 3 and 5 freeze-thaw treatments, anti-canine IL-1βIgY still maintained superior binding activity to recombinant canine IL-1β protein.

[0076] The above results indicate that the anti-canine IL-1β IgY prepared in this application has good storage stability and superior environmental tolerance, which can provide favorable support for its subsequent preservation, transportation and application.

[0077] In summary, this invention, through bioinformatics analysis of canine IL-1β precursor protein and considering the characteristics of IL-1β being synthesized in precursor protein form and undergoing intracellular processing and cleavage to form mature active protein, selected the 340-798 bp target fragment (after removing the immature precursor region) as the expression sequence, and successfully constructed the recombinant expression plasmid pET-28a-cIL-1β. The optimal induction conditions were 37℃, 0.1 mmol / L IPTG, and induction time of 6... The recombinant canine IL-1β protein obtained exhibits both soluble and insoluble expression. The soluble protein in the supernatant does not require complex denaturation and renaturation treatments, thus maintaining the native or near-native conformational epitopes and simplifying the operation steps. The supernatant was used primarily for purification, and high-purity recombinant canine IL-1β protein was obtained through His-tag affinity chromatography. After immunizing SPF-grade laying hens with this protein, anti-canine IL-1β specific egg yolk antibodies were successfully obtained. Indirect ELISA results showed that the IgY titer extracted from the yolk of egg No. 1 after three immunizations was the highest, with a maximum positive dilution of 1:409600. The obtained anti-recombinant canine IL-1β egg yolk antibody exhibited high purity, superior specificity and stability, and significantly reduced preparation costs, demonstrating important application significance and research value.

[0078] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing an egg yolk antibody against recombinant canine interleukin-1β, characterized in that, Includes the following steps: Recombinant canine interleukin-1β antigen was prepared by prokaryotic expression using genetic engineering. The recombinant canine interleukin-1β antigen was emulsified with a first immunizing adjuvant and then used to immunize laying hens for the first time. The recombinant canine interleukin-1β antigen was then emulsified with a second immunizing adjuvant and used to boost the immunization of laying hens. Eggs were collected, egg yolks were separated, and egg yolk antibodies were extracted from the egg yolks to obtain the egg yolk antibodies against recombinant canine interleukin-1β.

2. The preparation method according to claim 1, characterized in that, The amino acid sequence of the recombinant canine interleukin-1β is shown in SEQ ID NO:

1.

3. The preparation method according to claim 1, characterized in that, The preparation of recombinant canine interleukin-1β antigen using prokaryotic expression via genetic engineering specifically includes the following steps: Total RNA was extracted from canine spleen tissue, and cDNA was synthesized by reverse transcription. The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 was obtained by PCR amplification, cloned into the pMD-19T vector, and then subcloned into the pET-28a prokaryotic expression vector. The vector was transformed into host cells BL21(DE3), and the recombinant canine interleukin-1β antigen was obtained after induction of expression and purification.

4. The preparation method according to claim 3, characterized in that, The nucleotide sequence encoding the recombinant canine interleukin-1β protein is shown in SEQ ID NO:

2.

5. The preparation method according to claim 3, characterized in that, The PCR amplification program is as follows: pre-denaturation at 92-96℃ for 29-31 s; denaturation at 93-95℃ for 29-31 s, annealing at 52-54℃ for 29-31 s, extension at 70-72℃ for 1-1.5 min, for a total of 30 cycles; final extension at 70-72℃ for 4-5 min; storage at 4℃; and / or The PCR amplification reaction system consisted of: 2 μL cDNA, 1 μL upstream primer, 1 μL downstream primer, 25 μL premix, and deionized water to a final volume of 50 μL; wherein the upstream primer sequence is shown in SEQ ID NO: 3, and the downstream primer sequence is shown in SEQ ID NO: 4; and / or The induction conditions were as follows: induction at 16–37°C with 0.1–2 mmol / L isopropyl thiogalactoside for 2–8 hours.

6. The preparation method according to claim 1, characterized in that, The first immune adjuvant is Freund's complete adjuvant, and the volume ratio of the first immune adjuvant to the recombinant canine interleukin-1β antigen is 1:1; the second immune adjuvant is Freund's incomplete adjuvant, and the volume ratio of the second immune adjuvant to the recombinant canine interleukin-1β antigen is 1:1; and / or The enhanced-immunization laying hens include: Second immunization: 12-16 days after the first immunization; Third immunization: 26-30 days after the first immunization.

7. The preparation method according to claim 1, characterized in that, The recombinant canine interleukin-1β antigen injection dose is 125-235 μg / kg per single immunization dose based on the weight of the laying hen; and / or Both the initial immunization and booster immunization were administered via subcutaneous multi-point injection.

8. The preparation method according to claim 1, characterized in that, The extraction of yolk antibodies from egg yolks includes the following steps: S1. Separate the egg yolk and egg white, collect the egg yolk liquid, add PBS buffer to the egg yolk liquid at a volume ratio of 1:2 and mix well; then add PEG-6000 to a final concentration of 3%~4% (W / V), stir until completely dissolved, let stand for 15~25 min and then centrifuge to collect the supernatant. S2. Filter the supernatant obtained in step S1, add PEG-6000 to the filtrate with a final concentration of 8%~9% (W / V), stir and let stand for 18~23 min, then centrifuge and discard the supernatant. S3. Dissolve the precipitate obtained in step S2 in PBS, add PEG-6000 to a final concentration of 10%~14% (W / V), mix well, let stand for 8~12 min, centrifuge, discard the supernatant, and the resulting precipitate is the egg yolk antibody against recombinant canine interleukin-1β.

9. An egg yolk antibody against recombinant canine interleukin-1β, characterized in that, The egg yolk antibody was prepared using the preparation method described in any one of claims 1 to 8.

10. The use of an anti-recombinant canine interleukin-1β egg yolk antibody prepared by any one of the preparation methods described in claims 1 to 8 and / or the egg yolk antibody described in claim 9 in the preparation of a detection reagent for detecting canine interleukin-1β protein.

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