A cell line with high yield of chicken igy heavy chain polypeptide and a construction method thereof

Abstract

This invention relates to the field of genetic engineering technology, specifically to a cell line that produces a high-yield chicken IgY heavy chain polypeptide and its construction method. Specifically, this invention utilizes genetic engineering technology to stably integrate the chicken IgY heavy chain encoding gene into the genome of mammalian cells (e.g., CHO-K1 cells), thereby constructing a cell line that stably expresses the chicken IgY heavy chain. This suspension cell line can stably and highly express the chicken IgY heavy chain, and the expressed chicken IgY heavy chain exhibits high activity. Specifically, the titer of polyclonal antibodies prepared using this chicken IgY heavy chain can reach over 1:256000, which is significantly superior to the titer of commercially available polyclonal antibodies. Therefore, when applied to chicken IgY immunoassays (such as ELISA, WB, IHC, etc.), it can exhibit higher sensitivity and specificity, and is also more economical.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a cell line for high-yield chicken IgY heavy chain polypeptide and its construction method. Background Technology

[0002] Immunoglobulin Y (IgY) is a major class of serum antibodies found in birds, amphibians, and reptiles. IgY is an avian homolog of human immunoglobulin G (IgG) and its evolutionary ancestor, exhibiting significant sequence conservation. Both IgG and IgY consist of two heavy chains and two light chains, each with a constant region and a variable region. The variable region is crucial for antigen recognition, while the constant region provides effector function. The Fc region of IgY includes two carbohydrate side chains, unlike immunoglobulins which have only one chain. IgY possesses unique structural and molecular characteristics in immunological and antibody research. Due to this structural advantage, using IgY antibodies in immunoassays may result in less background noise, fewer false positives, and reduced antibody aggregation—common phenomena observed in both monoclonal and polyclonal mammalian antibodies. Compared to mammalian immunoglobulins, IgY exhibits a higher degree of glycosylation, significantly influencing protein stability, proteolytic enzyme sensitivity, immunogenicity, and biological activity. Due to the phylogenetic distance between birds and mammals, avian IgY antibodies are more efficient than mammalian IgG antibodies in recognizing highly conserved proteins or epitopes in mammals. The use of avian IgY antibodies has significant implications for a wide range of clinical applications in humans and veterinary medicine. Furthermore, IgY antibodies exhibit high binding specificity and low cross-reactivity with other antigens, potentially offering greater value in various pathogen detection applications.

[0003] The industrial production of chicken IgY heavy chains mainly relies on extracting natural IgY from the yolk of immunized hens and then isolating the heavy chain, as well as synthesizing the heavy chain through recombinant expression systems. Natural extraction methods (isolation of heavy chains from yolk IgY) include water dilution-salting-out (the mainstream industrial method), membrane separation-ultrafiltration, and organic solvent extraction (traditional processes). Recombinant expression methods primarily involve prokaryotic expression system preparation, cloning the heavy chain gene into a pET vector, transforming it into BL21(DE3), and inducing expression via IPTG (isopropyl-β-D-thiogalactoside). The target protein exists in inclusion body form and requires subsequent denaturation, renaturation (gradient dialysis), and ion exchange chromatography purification to obtain the active protein. This approach to IgY heavy chain production suffers from cumbersome purification steps and high production costs.

[0004] Chinese hamster ovary (CHO) cells, isolated by Theodore Puck from Chinese hamster ovaries in 1957, are fibroblasts with unlimited proliferative capacity. They are among the most widely used mammalian cells in the production of biotherapeutic drugs. Compared to other expression systems, mammalian cell expression systems can correctly guide various post-translational processes during protein translation, including folding, phosphorylation, glycosylation, and disulfide bond modification. This allows recombinant proteins to be as similar as possible to their natural structures in terms of molecular structure, physicochemical properties, and biological functions. In addition to these advantages, CHO cell expression systems also offer benefits such as low endogenous protein secretion, efficient amplification and expression of recombinant genes, stable integration of exogenous proteins, and the ability to be cultured in both adherent and suspension forms.

[0005] However, there are few reports of using mammalian cell expression systems to stably produce and express avian IgY heavy chains. Summary of the Invention

[0006] Based on existing technologies, this invention utilizes genetic engineering techniques to modify the gene encoding the chicken IgY heavy chain polypeptide (specifically, by linking the 5' end to the coding sequence of the mouse IgGκ chain signal peptide (SEQ.ID NO: 2); and by adding the KOZAK sequence (SEQ.ID NO: 3) before the start codon ATG) and stably integrate it into mammalian cells (e.g., CHO-K1 cells), thereby constructing a cell line that stably expresses the chicken IgY heavy chain polypeptide. This production method is of great significance for the efficient and stable production of chicken IgY heavy chain polypeptide, its application in diagnosis, and the research and development of vaccines.

[0007] Therefore, in one aspect, the present invention provides a chicken IgY heavy chain recombinant polypeptide, particularly, the heavy chain recombinant polypeptide is composed of a mouse IgGκ chain signal peptide and a chicken IgY heavy chain polypeptide, the signal peptide enabling the chicken IgY heavy chain polypeptide to be secreted by cells into the cell culture supernatant, facilitating subsequent purification, and improving the correct folding rate of the chicken IgY heavy chain polypeptide. Specifically, in the constructed chicken IgY heavy chain polypeptide expression cell line, the mouse IgGκ chain signal peptide guides the target chicken IgY heavy chain polypeptide into the secretion pathway: the signal peptide is recognized by ribosomes, guiding the nascent polypeptide chain into the endoplasmic reticulum and Golgi apparatus, ultimately resulting in the secretion of IgY into the extracellular supernatant. The chicken IgY heavy chain polypeptide can be directly harvested from the supernatant without cell lysis, simplifying the purification process. Furthermore, the mouse IgGκ chain signal peptide can also improve the correct folding and post-translational modification of the chicken IgY heavy chain polypeptide: the secretion pathway contains molecular chaperones and disulfide isomerases, which help the chicken IgY heavy chain polypeptide to correctly form disulfide bonds, fold correctly, and undergo glycosylation modification. In addition, after the signal peptide enters the endoplasmic reticulum, it is cleaved by signal peptidase, so the mature chicken IgY heavy chain polypeptide secreted into the supernatant does not carry the signal peptide sequence at the N-terminus, thus not affecting the structure, antigenicity, or subsequent immune effect of the chicken IgY heavy chain polypeptide.

[0008] In another aspect, the present invention provides a method for producing chicken IgY heavy chain polypeptide, particularly comprising: a method for constructing a mammalian cell line (CHO-K1) stably expressing chicken IgY heavy chain polypeptide, for example comprising: introducing a recombinant plasmid containing a gene encoding chicken IgY heavy chain polypeptide into mammalian cells, followed by drug (e.g., G418) resistance screening, screening for cell lines that highly express chicken IgY heavy chain polypeptide, and suspension acclimatization.

[0009] In another aspect, the present invention provides a method for preparing chicken IgY heavy chain peptides using the constructed cell line. In yet another aspect, the present invention also provides the use of the chicken IgY heavy chain peptides described herein in the preparation of polyclonal antibodies, and so on.

[0010] In this regard, embodiments of the present invention include, but are not limited to, the following: In some embodiments, the present invention provides a chicken IgY heavy chain recombinant polypeptide, the amino acid sequence of which is shown in SEQ ID NO: 7.

[0011] In some embodiments, the C-terminus of the amino acid sequence of the chicken IgY heavy chain polypeptide or chicken IgY heavy chain recombinant polypeptide of the present invention is linked to a linker and a purification tag.

[0012] In some embodiments, the linker is (GGGGS)2 (i.e., the amino acid sequence of the linker is GGGGSGGGGS).

[0013] In some embodiments, the purification tag is a 6×His tag (i.e., the amino acid sequence of the purification tag is HHHHHH).

[0014] In some embodiments, the present invention provides a polynucleotide encoding the chicken IgY heavy chain recombinant polypeptide described herein, preferably, the polynucleotide sequence is shown in SEQ ID NO: 8.

[0015] In some embodiments, the present invention also provides a recombinant vector comprising the polynucleotides described herein.

[0016] In some embodiments, the present invention provides a cell line for the efficient production of the chicken IgY heavy chain polypeptide described herein, comprising the polynucleotides described herein and / or the recombinant vectors described herein.

[0017] In some embodiments, the present invention also provides the application of the chicken IgY heavy chain recombinant polypeptide, polynucleotide, recombinant vector or cell line described herein in the preparation of chicken IgY heavy chain polyclonal antibodies.

[0018] In some embodiments, chicken IgY heavy chain polypeptides produced using the cell line described in this invention are used to immunize animals (e.g., mammals (rats, mice, rabbits, etc.)) and polyclonal antibodies are extracted from the serum of the immunized animals.

[0019] In some embodiments, the present invention also provides a method for constructing a mammalian cell line stably expressing chicken IgY heavy chain polypeptide, comprising the following steps: (1) The polynucleotide sequence described in this invention is ligated into a eukaryotic expression vector to obtain a recombinant vector; (2) Transfect the recombinant vector obtained in (1) into a mammalian cell line; (3) The mammalian cell lines obtained in step (2) are subjected to suspension acclimatization.

[0020] In some embodiments, in the construction method of the present invention, step (2) includes: transfecting mammalian cells with a recombinant vector, screening mammalian cells containing the recombinant vector by using an anti-drug, and screening cell lines that highly express chicken IgY heavy chain polypeptide. And / or, the suspension acclimatization includes: using a suspension medium (e.g., CD CHO 031 suspension medium (purchased from Jianshun Biotechnology (Nantong) Co., Ltd.)) to perform multi-stage suspension culture on the mammalian cell line obtained in step (2), gradually reducing the fetal bovine serum (FBS) in the medium at each stage until the amount of FBS is reduced to 0, and then successfully passaged 3 times in a row to obtain a suspension mammalian cell line.

[0021] In some embodiments, the multi-stage suspension culture conditions of the present invention are as follows: the cells are cultured in a shaking incubator at 37°C, 5% CO2, and 100-120 rpm. The cell density and viability are detected every 24 hours. The cell density is required to increase exponentially within 3 days and the viability must reach more than 90% before proceeding to the next gradient. Otherwise, the cells are passaged.

[0022] In some embodiments, the multi-stage suspension culture of the present invention includes: the suspension medium used in the first stage has an FBS content of 10 wt%, the suspension medium used in the second stage has an FBS content of 8 wt%, the suspension medium used in the third stage has an FBS content of 5 wt%, the suspension medium used in the fourth stage has an FBS content of 2.5 wt%, the suspension medium used in the fifth stage has an FBS content of 1 wt%, the suspension medium used in the sixth stage has an FBS content of 0.1 wt%, and the suspension medium used in the seventh stage has an FBS content of 0 wt%.

[0023] In some specific embodiments, the sequence of the recombinant vector of the present invention is shown in SEQ ID NO: 9.

[0024] In some specific implementations, in step (2) of this invention, transfecting the recombinant vector into mammalian cells includes: digesting mammalian cells in the logarithmic growth phase with trypsin, and then culturing them at 37°C and 5% CO2 until the cell confluence reaches approximately 80% before starting transfection. The specific steps are as follows: 1) After washing the cells twice with PBS, add 1.5 mL of Opti-MEM medium; 2) Prepare solution A: Add 3 μg of plasmid to 250 μL of Opti-MEM medium and vortex to mix. 3) Prepare solution B: Dilute 6 μL of transfection reagent with 250 μL of Opti-MEM medium. Nulen PlusTrans TM Incubate at room temperature for 2-5 minutes; 4) Mix solution A and solution B, incubate at room temperature for 20 min, then add the mixture to a 6-well plate and gently shake to mix. 5) After incubation at 37℃ and 5% CO2 for 6 h, the culture medium was replaced with basal medium containing 5% newborn fetal bovine serum (FBS) to detect the expression of chicken IgY heavy chain peptides; And / or, screening mammalian cells containing recombinant vectors by resistance drugs includes: starting drug screening with resistance drugs 48 h after transfection with plasmids: discarding cell culture supernatant, adding 2 mL of complete culture medium (e.g., Ham's F-12K + 10% FBS + resistance drug), observing cell pathogenesis and growth daily, and changing the complete culture medium every 2 days due to cell metabolism, until mammalian cells containing recombinant vectors clump together, while the negative control cells not transfected with recombinant vectors die completely; And / or, screening for cell lines that highly express chicken IgY heavy chain peptides includes: seeding surviving cells into 96-well plates using a limiting dilution method for screening monoclonal cell lines; transferring selected cells to 24-well plates when a clear monoclonal cell community is observed in the 96-well plates; transferring to 6-well plates after confluence; at this point, the culture medium does not contain any drug used for resistance screening (e.g., G418); repeating the above steps for two monoclonal cell screenings; after one week of single-cell culture, the cells are cultured at the same cell concentration (e.g., approximately 4.0 × 10⁻⁶). 5 (cells / mL) were seeded into 24-well plates and cultured for 48 h. A portion of the supernatant was taken for Western blot analysis to quantify the expression level of secreted chicken IgY heavy chain peptide in the obtained cell lines. All positive cell lines were cryopreserved for seeding. The monoclonal cell line with the highest expression level of chicken IgY heavy chain peptide was further cultured and cryopreserved.

[0025] In some embodiments, the present invention also provides a monoclonal cell line for producing chicken IgY heavy chain polypeptides, which is obtained by using the construction method described in the present invention.

[0026] In some embodiments, the 3' end of the polynucleotide sequence of the present invention further contains a sequence encoding a linker and a purification tag; preferably, the linker is (GGGGS)2 and / or the purification tag is a 6×His tag.

[0027] In some specific implementations, the sequence of the encoded connector of the present invention is ggcggcggaggctccggcggaggaggatct.

[0028] In some specific implementations, the sequence for encoding the 6×His tag described in this invention is caccatcaccaccaccat.

[0029] In some embodiments, the vector described in this invention is a pcDNA3.1 vector.

[0030] In some embodiments, the mammalian cells described in this invention are CHO-K1 cells.

[0031] Compared with the prior art, the beneficial effects of the present invention include at least the following: This invention integrates the encoding gene of a specific chicken IgY heavy chain polypeptide into the genome of mammalian cells (e.g., CHO-K1) to obtain a cell line stably expressing the chicken IgY heavy chain polypeptide. This cell line can be easily domesticated, resulting in a suspension-type cell line with advantages such as short domestication time and good cell condition. In addition, the chicken IgY heavy chain polypeptide of this invention has high biological activity. Specifically, the titer of polyclonal antibodies prepared using this chicken IgY heavy chain polypeptide can reach over 1:256000, which is significantly better than the titer of commercially available polyclonal antibodies. Therefore, when applied to chicken IgY immunoassay (such as ELISA, WB, IHC, etc.), it can exhibit higher sensitivity and specificity, and is also more economical. Attached Figure Description

[0032] Figure 1 In Example 1, the recombinant plasmid Chiken IgYheavy chain_pcDNA3.1(-) synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0033] Figure 2 Example 2: Western Blot analysis of IgY protein expression in CHO-K1 cells 48 h after transfection with recombinant expression plasmid: 1 is the negative control, 2 is the Chiken IgY heavy chain_pcDNA3.1(-) plasmid transfected, and 3 is the Chiken IgY heavy chain_pcDNA3.1(-) plasmid precipitate transfected.

[0034] Figure 3 : Cytopathic effect diagram of CHO-K1 cells determined by the optimal concentration of G418 in Example 2.

[0035] Figure 4 Example 2: CHO-K1 cells after 10 days of G418 selection: A is a picture of the lesions of CHO-K1 cells without plasmid transfection, and B is a picture of the growth of CHO-K1 cells transfected with Chiken IgY heavy chain_pcDNA3.1(-).

[0036] Figure 5 Screening diagram of CHO-K1 monoclonal cell line in Example 3.

[0037] Figure 6 Example 2: The results of the first limiting dilution method for screening different monoclonal cell lines by comparing the gray values ​​of Western Blot. M is the 180 kDa Prestained Protein Marker. 3, 4, 5, 6, 19, 20, 23, 24, 25 and 26 are the supernatants of 10 monoclonal cell lines, respectively.

[0038] Figure 7 Example 2: Comparison of Western Blot grayscale values ​​of different monoclonal cell lines screened by the second limiting dilution method. M is the 180 kDa Prestained Protein Marker, and 3, 8, 12, 16, 23 and 26 are the supernatants of the 6 monoclonal cell lines, respectively.

[0039] Figure 8 Western blot analysis results of the supernatant of recombinant suspension IgY CHO-K1 cells from generation 0 to 15 after culturing for 48 h in Example 2: M is a 180 kDa Prestained Protein Marker, and P1 to P4 represent generation 0, 5, 10, and 15 of IgY CHO-K1 cells.

[0040] Figure 9 Example 2: Western blot results of cell supernatants from suspension acclimatization stages 1 to 7. M is a 180 kDa Prestained Protein Marker. Lane 1 represents the blank control. Lanes 2 to 8 represent the suspension acclimatization stages of IgY CHO-K1 cells: Stage 1 (10% FBS), Stage 2 (8% FBS), Stage 3 (5% FBS), Stage 4 (2.5% FBS), Stage 5 (1% FBS), Stage 6 (0.1% FBS), and Stage 7 (0% FBS).

[0041] Figure 10 Schematic diagram of the titer determination results of polyclonal antibodies prepared from chicken IgY heavy chain peptides.

[0042] Figure 11 : A schematic diagram comparing the titer determination results of the polyclonal antibody prepared in this application with those of commercial polyclonal antibodies. Detailed Implementation

[0043] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] The main experimental materials and reagents used in the examples are as follows: Plasmids and cells: pcDNA3.1 was preserved in our laboratory; CHO-K1 cells were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; Trans10 competent cells were purchased from Beijing TransGen Biotech Co., Ltd.; the gene synthesis involved in this invention was completed by Sangon Biotech (Shanghai) Co., Ltd.

[0045] Main reagents: Plasmid mini-prep kit purchased from Tiangen Biotech Co., Ltd.; Ham's F-12K medium purchased from Wuhan Pronosei Life Sciences Co., Ltd.; Transfection reagent Nulen PlusTrans TM All reagents were purchased from Shanghai Nolai Biotechnology Co., Ltd.; DNA marker was purchased from Beijing TransGen Biotech Co., Ltd.; Fetal Bovine Serum (FBS) and trypsin (0.25% Trypsin-EDTA) were purchased from Gibco; Anti-His Tag monoclonal antibody was purchased from Wuhan Sanying Biotechnology Co., Ltd.; Phosphate buffer saline (PBS) was purchased from Wuhan Sewell Biotechnology Co., Ltd.; CD CHO 031 suspension medium was purchased from Jianshun Biotechnology (Nantong) Co., Ltd.; G418 was purchased from Biosharp; Freund's complete / incomplete adjuvant (sigma-F5881, F5506); goat anti-mouse IgG H&L, Coomassie Brilliant Blue rapid staining solution, and BCA protein concentration assay kit were all purchased from Beyotime Biotechnology Co., Ltd.

[0046] Example 1: Construction of recombinant expression plasmid Chiken IgY heavy chain_pcDNA3.1(-) The chicken IgY heavy chain gene sequence was analyzed, and the designed amino acid sequence was optimized according to the codon preference of CHO cells, as shown in SEQ.ID NO: 1. A mouse IgGκ chain signal peptide coding sequence (SEQ.ID NO: 2) was ligated to the 5' end of the chicken IgY heavy chain gene sequence. Adding the KOZAK sequence (SEQ.ID NO: 3) before the start codon ATG improved the ribosome-mRNA binding efficiency and significantly increased the expression level of the chicken IgY heavy chain polypeptide in eukaryotic cells. Adding the (GGGGS)2 tag (SEQ.ID NO: 4) and 6×His (SEQ.ID NO: 5) coding sequences to the 3' end of the sequence allowed the fused functional domains (IgY heavy chain and His tag) to fold independently without interference, reducing the risk of fusion protein aggregation and improving the soluble expression level of the recombinant protein. Finally, the modified gene sequence was ligated into the eukaryotic expression vector pcDNA3.1(-), and the newly synthesized recombinant plasmid was named Chiken IgY heavy chain. chain_pcDNA3.1(-), plasmid map as follows Figure 1 As shown in SEQ ID NO: 8, the sequence contains the G418 resistance gene Neo.

[0047] Example 2: Expression of Chiken IgY heavy chain protein in CHO-K1 cells 1. Recombinant expression plasmid transfected into CHO-K1 cells CHO-K1 cells in logarithmic growth phase were digested with trypsin and seeded into 6-well plates. The cells were cultured at 37°C in a 5% CO2 incubator until approximately 80% confluence was achieved, at which point transfection began. The specific steps are as follows: 1) Discard the culture medium in the 6-well plate, wash twice with PBS, and then add 1.5 mL of Opti-MEM medium (Gibco, CAT: 31985070). 2) Prepare solution A: Add 3 μg of plasmid to 250 μL of Opti-MEM medium and vortex to mix. 3) Prepare solution B: Dilute 6 μL of transfection reagent with 250 μL of Opti-MEM medium. Nulen PlusTrans TM Incubate at room temperature for 2-5 minutes; 4) Mix solution A and solution B, incubate at room temperature for 20 min, then add the mixture to a 6-well plate and gently shake to mix. 5) After incubating at 37℃ and 5% CO2 for 6 hours, the culture medium was replaced with basal medium containing 5% newborn fetal bovine serum (FBS), with one well reserved as a negative control.

[0048] The expression of IgY heavy chain protein in CHO-K1 cells was detected by Western blotting 48 h after transfection. The primary antibody was 1:5000 dilution of Anti-His Tag monoclonal antibody, and the secondary antibody was 1:2000 dilution of goat anti-mouse IgG H&L. Imaging was performed using an Azure Biosystems C600 multifunctional molecular imaging system. The results are as follows: Figure 2 As shown in the WB diagram, IgY heavy chain protein is expressed in the supernatant, i.e., secretory expression, with a size of 40 kDa.

[0049] 2. Determination of the optimal concentration of G418 in CHO-K1 cells Normally growing CHO-K1 cells were digested into single-cell suspensions and seeded into 24-well plates. G418 was added to each well at concentrations of 300, 400, 500, 600, 700, 800, and 900 μg / mL, with a control group included. Each concentration was repeated in triplicate, and cell growth was observed. Results: After 6 days of G418 addition, 700 μg / mL was found to be the lowest concentration at which all CHO-K1 cells died. Therefore, the optimal concentration of G418 for CHO-K1 cell selection was 700 μg / mL. Figure 3 As shown in (100×).

[0050] 3. Drug sieve The previous section confirmed that the IgY gene can be secreted in CHO-K1 cells. Therefore, 48 hours after plasmid transfection, drug screening was performed using G418: the supernatant was discarded, and 2 mL of complete culture medium (Ham's F-12K + 10% FBS + 700 μg / mL G418) was added. Cell pathogenesis and growth were observed daily. Due to the influence of cell metabolism, the complete culture medium needed to be changed every 2 days until the IgY group showed clumped CHO-K1 cells, while the untransfected negative control group cells died completely. This process took approximately 2 weeks. The results are as follows: Figure 4 As shown.

[0051] 4. Screening for monoclonal cell lines with high expression The surviving cells were seeded into 96-well plates using a limiting dilution method for screening monoclonal cell lines. The screened cells showed a clear monoclonal cell community in the 96-well plates. Figure 5 Cells were transferred to 24-well plates, and after confluence, they were transferred to 6-well plates. At this point, the culture medium did not contain G418. The above steps were repeated for two monoclonal cell selections. After one week of single-cell culture, the selected cells were seeded into 24-well plates for further culture. A portion of the supernatant and cell samples were taken for Western blot analysis. Cell lines showing specific target protein bands were further expanded and cryopreserved, and named CHO-IgY cells.

[0052] After the cells in the 6-well plates reached confluence, the supernatant was collected. Ten monoclonal cell lines were selected through the first subcloning screening. IgY heavy chain protein expression was detected by Western blotting. ImageJ software was used for semi-quantitative analysis of different monoclonal cell lines using a reverse colorimetric method. Monoclonal cell line 26 showed the highest IgY heavy chain protein expression. The results are as follows: Figure 6 As shown in the figure; monoclonal cell line No. 26 was cloned again, and six monoclonal cell lines were selected through the second subcloning. The expression of IgY heavy chain protein was detected by Western blotting. ImageJ software was used to perform semi-quantitative analysis of different monoclonal cell lines using the inverse colorimetric method. Among them, monoclonal cell line No. 3 had the highest expression level of IgY heavy chain protein. The results are shown in the figure. Figure 7 As shown.

[0053] 5. Stability identification of CHO-K1 cell lines with high expression of IgY heavy chain protein The stability of the CHO-IgY monoclonal cell line No. 3 from step 4 of Example 2 was tested: Western blotting was performed on the supernatants of cells from passages 0, 5, 10, and 15. The results showed that IgY heavy chain protein was stably expressed at all passages. Figure 8 As shown.

[0054] 6. Suspension acclimatization of monoclonal cell lines IgY CHO-K1 adherent cells were acclimatized using a continuous adaptation method with gradient serum reduction. The culture medium was CD CHO 031 suspension medium from JS Bio (Nantong Biotechnology Co., Ltd.). A certain amount of anti-clustering agent was added for each passage based on the cell clumping situation.

[0055] The first stage involves seeding cells into a 125 mL shake flask with 10 wt% FBS suspension medium, then adding medium to a final concentration of 0.6–1.2 × 10⁻⁶ cells / mL to a final concentration of 30 mL. 6 Cells were cultured at 37℃, 5% CO2, and 120 rpm in a shaking incubator. Cell density and viability were monitored every 24 hours. Cell density was required to increase exponentially within 3 days, and viability was required to reach 90% or higher before proceeding to the next gradient; otherwise, passage was continued. The FBS concentrations in the culture media for stages two through seven were 8 wt%, 5 wt%, 2.5 wt%, 1 wt%, 0.1 wt%, and 0 wt%, respectively. After discontinuing FBS addition, three more passages were performed to confirm that cell viability and density remained stable, indicating successful acclimatization. Cell density and viability were monitored during suspension acclimatization stages 1 through 7. Cells showed good growth and relatively stable viability throughout the suspension acclimatization process, with minimal impact from the process; viability remained above 90%.

[0056] A small amount of cell supernatant from stages 1 to 7 of the suspension acclimation process was collected and subjected to Western blot analysis to observe whether there were any abnormalities in protein expression during the acclimation process. The results are as follows: Figure 9 As shown, the protein bands are of uniform size, with no abnormalities such as attenuation or non-expression.

[0057] Example 3: Preparation of Chicken IgY Heavy Chain Polyclonal Antibody 1. Animal preparation for immunization New Zealand White rabbits (2-3 kg, female) are purchased and kept in a laboratory for one week to confirm their health (no fever, diarrhea, or weight loss) before being vaccinated.

[0058] 2. Preparation of Immunoassay Reagents The purified IgY heavy chain protein was diluted with PBS to a protein concentration of 1 mg / ml. Freund's complete adjuvant (CFA) (for initial immunization to induce a strong immune response) and Freund's incomplete adjuvant (IFA) (for booster immunization to maintain the immune response) were both original Sigma products and stored at 4°C.

[0059] 3. Immunization regimen Take equal volumes of CFA and 1 mg / mL antigen, emulsify them by pushing with a syringe. The emulsion that does not spread when dropped into water is qualified. When performing booster immunization, replace CFA with IFA and perform multiple subcutaneous injections on the back. The specific plan is as follows in the table:

[0060] 4. Collection of antiserum Perform blood testing 7 days (the 52nd day) after the last booster immunization. Collect 1 - 2 mL of blood from the marginal vein of the rabbit ear, separate the serum, and detect the antibody titer by ELISA.

[0061] Place the collected blood in a sterile centrifuge tube, let it stand at room temperature for 1 - 2 h until the blood coagulates to form a blood clot; centrifuge at 4℃ and 3000 rpm for 20 min, collect the upper light yellow clear serum, discard the lower blood clot and precipitate; add gentamicin (final concentration 50 μg / mL) to prevent bacterial contamination, add protease inhibitors to prevent antibody degradation, then perform sub - packaging and store at - 80℃.

[0062] 5. Detection of polyclonal antibody titer by indirect ELISA Use the determined optimal reaction conditions for the rabbit serum after the 3rd booster immunization and negative rabbit serum, with three replicates in each group. Calculate the antibody titer according to P / N (P / N≥2.1). The specific experimental steps are as follows: Coating: Coat a 96 - well plate with IgY heavy - chain protein (1 μg / mL) diluted with PBS, 100 μL / well, and coat overnight at 4℃; Blocking: Discard the coating solution, block with 5% skim milk / PBS - T (0.05% Tween - 20), 200 μL / well, and block at 37℃ for 1 h; Primary antibody incubation: Dilute the antiserum in a serial dilution with PBST (1:1000, 1:2000, 1:4000, 1:8000, 1:16000……1:1024000), 100 μL / well, and incubate at 37℃ for 1 h; at the same time, set negative control (serum from non - immunized rabbits) and blank control (PBST); Secondary antibody incubation: Add HRP - labeled goat anti - rabbit IgG (diluted 1:5000), 100 μL / well, and incubate at 37℃ for 1 h; Color development and reading: Add TMB color - developing solution, 100 μL / well, develop color at 37℃ in the dark for 10 - 15 min, add 2 M H2SO4 to terminate color development, and read the absorbance at 450 nm (OD450) with an enzyme - labeled instrument.

[0063] The results showed that the ratio of rabbit polyclonal antibody D450 value to negative rabbit D450 value (P / N) remained greater than 2.1 even at a serum dilution of 1:102400, indicating that the polyclonal antibody titer could reach over 1:1024000 (see [link to relevant documentation]). Figure 10 ).

[0064] Example 4: Comparison of polyclonal antibody titers against chicken IgY heavy chain protein 1. HRP labeling of purified chicken IgY heavy chain protein polyclonal antibody using the periodate method. (1) Weigh 5 mg of HRP (Sigma-Aldrich, Cat: P6782) and dissolve it in 1 mL of distilled water; (2) Add 0.2 mL of freshly prepared 0.1 M NaIO4 solution to the solution obtained in (1), stir for 20 min at room temperature in the dark, add 0.5 mL of 0.16 M ethylene glycol aqueous solution, mix well, and let stand for 30 min; (3) The solution obtained in (2) is placed into a dialysis bag and dialyzed with 1 mM pH 4.4 sodium acetate buffer overnight at 4°C; (4) Add 20 μL of 0.2 M pH 9.5 carbonate buffer to raise the pH of the above aldehyde HRP to 9.0~9.5, and then immediately add it to 1 mL of 0.01 M carbonate buffer containing 2 mg of antibody. Stir gently at room temperature in the dark for 2 h. (5) Add 0.1 mL of NaBH4 solution (4 mg / mL), mix well, and let stand at 4℃ for 2 h; and (6) Dialyze the solution obtained in (5) with PBS and incubate overnight at 4°C.

[0065] 2. Antibody titer detection (indirect ELISA, for detecting secondary antibody titer) Antigen coating: Dilute the target antigen (IBV S1) to 0.5 μg / mL with coating buffer (pH 9.6 carbonate buffer: Na2CO3 1.59g, NaHCO3 2.93g, diluted with distilled water to 1000mL, stored at 4℃), add 100 μL to each well, and coat overnight at 4℃; add 100 μL of coating buffer to each blank control well. Blocking: Discard the coating solution and block with 5% skim milk / PBS-T (0.05% Tween-20), 200 μL / well, at 37℃ for 1 h; Primary antibody incubation: Dilute the self-made IBV polyclonal antibody with PBST (1:5000), 100 μL / well, and incubate at 37℃ for 1 h; negative control (PBST) and blank control (PBST) are set up at the same time. Secondary antibody incubation: The self-made IgY secondary antibody and the commercial IgY secondary antibody with the same initial concentration were diluted with PBST (1:1000, 1:2000, 1:4000, ..., 1:1024000), with 3 replicate wells for each dilution gradient. 100 μL of the diluted secondary antibody was added to each well and incubated at 37°C for 1 h. A blank control (PBST) was also provided. Color development and reading: Add 100 μL of TMB color development solution (purchased from Beyotime) to each well, and develop the color at 37℃ in the dark for 10-15 min. Add 2 M H2SO4 to stop the color development and read the absorbance at 450 nm (OD450) using a microplate reader.

[0066] The results showed that the titer of the self-made rabbit anti-chicken IgY-HRP antibody in this application reached over 1:256000, which was significantly better than that of commercially available antibodies (only over 1:128000). This indicates that the polyclonal antibody prepared using the recombinant chicken IgY heavy chain protein of this application has a higher titer, thus exhibiting higher sensitivity and specificity when applied to chicken IgY immunoassays (such as ELISA, WB, IHC, etc.), and is also more economical.

[0067] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0068] sequence list SEQ.ID NO: 1 Chicken IgY heavy chain polypeptide gene sequence cctgaagtgcaggtgctgcacgcctcttcctgcaccccctcccagtccgagtccgtagagctgctgtgcctggtgaccggcttttctcctgccagcgccgaggtggaatggctggtggacggcgtgggcggcctgctggtggcctctcagtcccctgctgtgcggtctggctctacatactctctgtcttcgagagtgaacgtgtctggcaccgactggcgggagggcaaatcctactcctgcagggttcggcacccagctaccaataccgtggtggaagaccacgtgaaaggctgtcctgacggcgctcaatcttgctcccctatccagctgtacgccatccctccaagccccggcgaactgtacatctccctggacgccaagctgagatgcctggtcgtgaacttgccttccgactcctctctgagcgtgacttggaccagagagaagtctggcaacctgcggcccgatcctatggtgctgcaagagcacttcaacggcacctactccgctagctctgctgtgcctgtgtccacccaggactggctctccggcgagagattcacctgtaccgtgcagcacgaggaactgcctctgcctctctccaagtccgtgtaccggaacacaggccctaccacccctccactgatctacccctttgcccctcatcctgaggaactgagcctgtctcgcgtgacactgtcttgtctggtcagaggcttcagacctagagacatcgagatcagatggctgagagatcacagagccgtgcccgctaccgagttcgtgaccacagccgtgctgcctgaagagagaaccgccaacggcgccggcggcgatggggacaccttcttcgtgtattccaagatgtccgttgagacagctaagtggaacggcggcaccgtcttcgcctgcatggccgtgcacgaggctctgcccatgcggttctcccagcggaccctgcagaagcaggctggtaag SEQ.ID NO: 2 Mouse IgGκ chain signal peptide coding sequence Atggaaactgatactctactactatgggtgctgctgctgtgggtgcctggctccaccgga (The encoded amino acid sequence is METDTLLLWVLLLWVPGSTG) SEQ.ID NO: 3 KOZAK sequence gccacc SEQ.ID NO: 4 (GGGGS)2 connector coding sequence ggcggcggaggctccggcggaggaggatct SEQ.ID NO: 5 6×His tag encoding sequence caccatcaccaccaccat SEQ.ID NO: 6 Chicken IgY Heavy Chain Peptide PEVQVLHASSCTPSQSESVELLCLVTGFSPASAEVEWLVDGVGGLLVASQSPAVRSGSTYSLSSRVNVSGTDWREGKSYSCRVRHPATNTVVEDHVKGCPDGAQSCSPIQLYAIPPSPGELYISLDAKLRCLVVNLPSDSSLSVTWTREKSGNLRPDPMVLQE HFNGTYSASSAVPVSTQDWLSGERFTCTVQHEELPLPLSKSVYRNTGPTTPPLIYPFAPHPEELSLSRVTLSCLVRGFRPRDIEIRWLRDHRAVPATEFVTTAVLPEERTANGAGGDGDTFFVYSKMSVETAKWNGGTVFACMAVHEALPMRFSQRTLQKQAGK SEQ.ID NO: 7 Chicken IgY heavy chain recombinant polypeptide (mouse IgGκ chain signal peptide + chicken IgY heavy chain polypeptide) METDTLLLWVLLLWVPGSTGPEVQVLHASSCTPSQSESVELLCLVTGFSPASAEVEWLVDGVGGLLVASQSPAVRSGSTYSLSSRVNVSGTDWREGKSYSCRVRHPATNTVVEDHVKGCPDGAQSCSPIQLYAIPPSPGELYISLDAKLRCLVVNLPSDSSLSVTWTREKSGNLRPDPMVLQE HFNGTYSASSAVPVSTQDWLSGERFTCTVQHEELPLPLSKSVYRNTGPTTPPLIYPFAPHPEELSLSRVTLSCLVRGFRPRDIEIRWLRDHRAVPATEFVTTAVLPEERTANGAGGDGDTFFVYSKMSVETAKWNGGTVFACMAVHEALPMRFSQRTLQKQAGK SEQ.ID NO: 8 Chicken IgY heavy chain recombinant polypeptide coding sequence (KOZAK sequence + mouse IgGκ chain signal peptide coding sequence + chicken IgY heavy chain polypeptide gene sequence) The sequence of SEQ ID NO: 9 Chiken IgY heavy chain_pcDNA3.1(-)

Claims

1. A chicken IgY heavy chain recombinant polypeptide, characterized in that, The amino acid sequence is as shown in SEQ ID NO:

7.

2. A polynucleotide, characterized in that, The chicken IgY heavy chain recombinant polypeptide according to claim 1 is preferably encoded as shown in SEQ ID NO:

8.

3. A recombinant vector, characterized in that, It contains the polynucleotide as described in claim 2.

4. A cell line for efficient production of chicken IgY heavy chain polypeptides, characterized in that, It contains the polynucleotide according to claim 2 and / or the recombinant vector according to claim 3.

5. The use of the chicken IgY heavy chain recombinant polypeptide according to claim 1, the polynucleotide according to claim 2, the recombinant vector according to claim 3, or the cell line according to claim 4 in the preparation of chicken IgY heavy chain polyclonal antibodies.

6. A method for constructing a mammalian cell line stably expressing chicken IgY heavy chain polypeptide, characterized in that, Includes the following steps: (1) The polynucleotide sequence according to claim 2 is ligated into a eukaryotic expression vector to obtain a recombinant vector; (2) Transfect the recombinant vector obtained in (1) into a mammalian cell line; (3) The mammalian cell lines obtained in step (2) are subjected to suspension acclimatization.

7. The construction method according to claim 6, characterized in that, Step (2) includes: transfecting mammalian cells with the recombinant vector, screening mammalian cells containing the recombinant vector by using an antibiotic resistance drug, and screening cell lines that highly express chicken IgY heavy chain polypeptide. And / or, the suspension domestication includes: using a suspension culture medium to perform multi-stage suspension culture on the mammalian cell line obtained in step (2), gradually reducing the fetal bovine serum (FBS) in the culture medium in each stage until the amount of FBS is reduced to 0, and then successfully passaged 3 times in a row to obtain a suspension mammalian cell line.

8. The polynucleotide according to claim 2 or the construction method according to claim 6 or 7, characterized in that, The 3' end of the polynucleotide sequence also contains a sequence encoding a linker and a purification tag; preferably, the linker is (GGGGS)2 and / or the purification tag is a 6×His tag.

9. The recombinant vector according to claim 3 or the construction method according to claim 6 or 7, characterized in that, The vector is pcDNA3.1 vector.

10. The recombinant vector according to claim 4 or the construction method according to claim 6 or 7, characterized in that, The cell line in question is the CHO-K1 cell line.

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