Immune antigen of streptococcus uberis, egg yolk antibody and preparation method and application thereof

Abstract

This invention discloses an immunogenic antigen for *Streptococcus mastitis*, an egg yolk antibody, and their preparation methods and applications, belonging to the field of biotechnology. This invention provides an immunogenic antigen for *Streptococcus mastitis*, which is a GapC protein with a common amino acid sequence as shown in SEQ ID NO.1, shared by *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis*. Egg yolk antibodies (IgY) are prepared by immunizing high-laying hens with this immunogenic antigen. The recombinant GapC protein prepared by this invention exhibits excellent immunogenicity. The egg yolk antibody prepared based on this protein is highly specific and precisely targeted, showing excellent binding ability against *Streptococcus agalactiae* and *Streptococcus dysgalactiae*, and exhibiting no cross-reactivity with *Staphylococcus aureus* and *Escherichia coli*. This invention provides a raw material basis for developing test strips or kits for the detection of *Streptococcus mastitis* in dairy cows.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an immunogenic antigen and egg yolk antibody based on the GapC protein of Streptococcus mastitis in dairy cows, their preparation method, and their application in the detection of pathogens causing mastitis in dairy cows. Background Technology

[0002] Bovine mastitis refers to an inflammatory condition of the mammary gland tissue in dairy cows caused by pathogenic microorganisms or external stimuli. Bovine mastitis can lead to decreased milk production, reduced productivity, and compromised dairy product quality and safety. The main pathogens causing bovine mastitis are Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus lactis. Treatment for bovine mastitis has traditionally relied on antibiotics. However, the widespread and continuous use of antibiotics has led to increasing antibiotic resistance in pathogens, rendering antibiotics ineffective or even useless. Therefore, developing simple, rapid, sensitive, and specific methods for detecting bovine mastitis pathogens is crucial for the prevention, rational drug use, and clinical diagnosis of bovine mastitis.

[0003] GapC protein is a protein on the surface of *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis* with glyceraldehyde-3-phosphate dehydrogenase activity. It also possesses transferrin and plasmin activity and is one of the key enzymes in the glycolysis pathway in prokaryotes and eukaryotes. It reversibly catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate, participating in DNA replication and repair, leading to apoptosis, and exhibiting a variety of biological activities. It is an important virulence and antigenic molecule shared by *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis*, and also has good immunomodulatory activity.

[0004] Immunoglobulin G (IgG) is the most abundant and dominant immunoglobulin in human serum, possessing antibacterial, antiviral, and antitoxin properties, and serving as the main antibody in the body's anti-infective immune response. However, IgG primarily relies on extraction from mammalian blood samples, which is limited by the live collection method, resulting in issues such as limited raw material sources, high production costs, and significant animal ethics controversies. Compared to IgG, egg yolk antibody (IgY) is a polyclonal antibody extracted from the yolk of laying hens after immunization, targeting a specific antigen. IgY is considered a functional alternative to mammalian IgG, possessing advantages such as high specificity, animal ethics friendliness, low cost, high yield, precise targeting, low cross-reactivity, and high safety. Therefore, this invention uses the GapC protein of *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis* as immunoantigens to prepare and apply egg yolk antibodies, laying the foundation for the development of test strips / reagents for the detection of *Streptococcus mastitis* in dairy cows. Summary of the Invention

[0005] To address the problems existing in the background art, the purpose of this invention is to provide a highly efficient immunogen based on the conserved GapC sequence of Streptococcus mastitis in dairy cows, and to use this antigen to prepare highly specific egg yolk antibodies for the detection of pathogens causing mastitis in dairy cows.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: An immunogenic antigen of *Streptococcus mastitis*, wherein the immunogenic antigen is the *Streptococcus mastitis* GapC protein, was screened using bioinformatics analysis to identify a conserved region of the GapC protein common to *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus masculinus*, the amino acid sequence of which is SEQ ID NO.1. The nucleotide sequence encoding this gene fragment is shown in SEQ ID NO.2. The encoding gene was constructed into the pET-32a(+) vector to obtain the recombinant plasmid pET-32a(+)-GapC; this plasmid was then transformed into BL21(DE3) strain and induced by IPTG at a temperature of 20-37℃ for 4-8 hours. The IPTG induction conditions were optimized (37℃, 6 hours) to obtain a highly expressed recombinant protein (approximately 30 kDa).

[0007] A bovine mastitis streptococcus egg yolk antibody was prepared by emulsifying the recombinant protein with an equal volume of water adjuvant and then immunizing high-laying hens via intramuscular injection. After three immunizations, eggs were collected and the yolks were separated. The yolks were then diluted with PBS, defatted with chloroform, precipitated with PEG6000, and resuspended in PBS to extract the egg yolk antibody (IgY).

[0008] ELISA results showed a serum titer as high as 1:27000 after three immunizations. Western blotting and fluorescence capture experiments confirmed that this IgY specifically binds to *Streptococcus agalactiae* and *Streptococcus dysgalactiae*, without cross-reacting with *Staphylococcus aureus* or *Escherichia coli*. Therefore, IgY can be used in the preparation of kits for detecting *Streptococcus mastitis* in dairy cows, or in the preparation of test strips for detecting *Streptococcus mastitis* in dairy cows.

[0009] Compared with the shortcomings and deficiencies of existing technologies, the present invention has the following beneficial effects: The immunoantigen for *Streptococcus mastitis* provided by this invention is the *Streptococcus mastitis* GapC protein, which is a conserved GapC protein sequence shared by *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis*. The prepared egg yolk antibody avoids cross-reaction with other bacteria, exhibits high specificity, and does not induce drug resistance. The antibody is obtained from chicken eggs, eliminating the need for blood sampling and complying with animal welfare requirements. This invention provides a core raw material for developing a rapid detection method to differentiate streptococci from other common mastitis cases and has broad application prospects. Attached Figure Description

[0010] Figure 1This is a schematic diagram of the common conserved sequence of GapC protein in Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus lactis provided in Embodiment 1 of the present invention; Figure 2 These are the results of amino acid sequence homology analysis of the three Streptococcus GapC proteins provided in Example 1 of this invention; Figure 3 The results of bioinformatics analysis of GapC protein provided in Example 1 of this invention are shown in Figure A, which shows the analysis results of GapC domains, Figure B shows the analysis results of GapC transmembrane regions, and Figure C shows the analysis results of GapC protein antigenicity. Figure 4 This is the sequencing peak diagram of the recombinant plasmid pET-32a(+)-GapC provided in Example 1 of this invention; Figure 5 This is the SDS-PAGE identification pattern of the recombinant protein provided in Example 2 of the present invention; Figure 6 The SDS-PAGE map of the purified and refolded recombinant protein provided in Example 2 of this invention; Figure 7 This is the Western blotting identification spectrum of the GapC-IgY antibody provided in Example 4 of this invention; Figure 8 The results are from the fluorescence capture experiment of the GapC-IgY antibody provided in Example 4 of this invention. Detailed Implementation

[0011] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0012] Example 1: Bioinformatics and Gene Synthesis of GapC Protein Based on the nucleic acid and amino acid sequences of *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis* published by NCBI, a conserved amino acid sequence of the GapC protein shared by the three bacteria, SEQ ID NO.1, was selected, as follows: Figure 1 As shown.

[0013] For the conserved amino acid sequence SEQ ID NO:1 of the GapC protein shared by the three bacteria, homology was first analyzed using NCBI BLAST, and the results are as follows: Figure 2 As shown, the conserved amino acid sequence shared by GapC proteins from *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, and *Streptococcus lactis* showed 100% homology. Then, CCD analysis was used to analyze the GapC domain, Topcon analysis was used to analyze the GapC transmembrane region, and DNAstar software was used to predict the antigenicity of the GapC protein. The results are as follows: Figure 3 As shown, Figure 3 A indicates that this gene segment has GAPDH activity. Figure 3 B indicates that this gene segment lacks a transmembrane region. Figure 3 C indicates that this gene segment has a favorable antigenic epitope.

[0014] The gene encoding the GapC protein (its nucleotide sequence is shown in SEQ ID NO.2) was cloned into the multiple cloning site of the prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET-32a(+)-GapC. After successful construction, sequencing was performed using universal primers T7 / T7 ter. The sequencing results are shown below. Figure 4 As shown, the target gene was completely constructed in the pET-32a(+) vector.

[0015] Example 2: Induction and purification of recombinant protein The correctly sequenced recombinant plasmid pET-32a(+)-GapC was transformed into BL21(DE3) competent cells. Single colonies were picked, and the cells were shaken to obtain a positive bacterial culture. The positive bacterial culture was inoculated into LB (ampicillin-resistant) liquid medium at a ratio of 1:100 and cultured at 37°C and 200 r / min until the bacterial culture OD... 600 When the expression value was 0.6, IPTG was added in different proportions to induce expression. The induction temperature was 20-37℃ and the induction time was 4-8 h to obtain GapC recombinant protein. The induction time (4 h, 6 h, 8 h) and induction temperature (20℃, 37℃) were optimized.

[0016] After identifying the predicted protein size and expression, bacterial cells were collected, ultrasonically pulverized, and centrifuged. The supernatant and precipitate were then subjected to SDS-PAGE analysis. The results are as follows: Figure 5 As shown in the figure, 1 represents pET-32a(+) without induction, 2 represents pET-32a(+) induced at 37℃ for 6 h, 3 represents recombinant protein without induction, 4 represents supernatant of recombinant protein induced at 37℃ for 2 h, 5 represents recombinant protein precipitate induced at 37℃ for 2 h, 6 represents recombinant protein supernatant induced at 37℃ for 4 h, 7 represents recombinant protein precipitate induced at 37℃ for 4 h, 8 represents recombinant protein supernatant induced at 37℃ for 6 h, 9 represents recombinant protein precipitate induced at 37℃ for 6 h, 10 represents recombinant protein supernatant induced at 37℃ for 8 h, and 11 represents recombinant protein precipitate induced at 37℃ for 8 h. It can be seen that after IPTG induction, a band appears at approximately 30 kDa, and the target protein expression level is highest after 6 hours of induction at 37℃. pET-32a(+)-GapC represents precipitate expression, and the obtained bands are consistent with the theoretical expected values, indicating that the pET-32a(+)-GapC recombinant plasmid expresses the target protein.

[0017] The protein induced at 37℃ for 6 hours was purified, and the flow-through, washing, and elution buffers were collected for SDS-PAGE analysis. The elution buffer was then placed in a dialysis bag, and 6 M, 4 M, 2 M, 1.5 M, 1 M, and 0 M urea refolding solutions (volume ratio 1:500) were prepared. Dialysis was performed at 4℃ for 12 hours using a magnetic stirrer, followed by SDS-PAGE analysis after dialysis. Results are as follows: Figure 6 As shown in the figure, M represents the standard mass of the protein molecule, 1 represents the unpurified protein, 2 represents the effluent, 3 represents the washing buffer, 4 represents the elution buffer, and 5 represents the refolded protein. It can be seen that the size of each protein purification product is consistent with the expected result, and there are no impurities.

[0018] Example 3: Preparation and titer determination of egg yolk antibodies The purified and refolded GapC recombinant protein was emulsified with an equal volume of water adjuvant. High-producing laying hens were injected intramuscularly into the thigh. A second immunization was administered 21 days after the first immunization, and a third immunization was administered 42 days after the first immunization. Serum was collected before the first, second, and third immunizations, and 10 days after the third immunization, along with eggs. Ten days after the third immunization, the eggs were wiped with 75% alcohol, the yolks were separated, diluted with 3 times the volume of PBS, and then chloroform was added at a 1:1 volume ratio. The mixture was incubated at 37°C for 20 min, centrifuged at 3000 g for 30 min, and the supernatant was discarded. The precipitate was resuspended in PBS and stored at -20°C for later use. The antibody titer of serum collected before and after the third immunization was determined using an indirect ELISA method. The results are shown in Table 1. The prepared antibody titer reached 1:27000, indicating successful immunization.

[0019] Table 1 Results of serum titer determination for GapC chicken triple vaccination .

[0020] Example 4: Antibody Specificity Verification The purified GapC recombinant protein was transferred onto a PEDV membrane after 12% SDS-PAGE. After blocking with blocking buffer at room temperature for 1 hour, purified anti-GapC-IgY (1:500) was used as the primary antibody and incubated overnight at 4°C. After washing three times with TBST, the membrane was incubated with anti-chicken secondary antibody (1:5000) at room temperature for 1 hour, washed three times with TBST, and then exposed to ECL development solution. Results are as follows: Figure 7 As shown in the figure, M represents the standard mass of the protein molecule, and 1 represents the GapC recombinant protein; it can be seen that a single specific band appears only at 30kDa.

[0021] Weigh 5 mg of FITC and dissolve it in 5 ml of DMSO. Add 0.5 mg of GapC-IgY and react overnight at room temperature in the dark. Then, place the reaction mixture in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyze for 24 hours to obtain FITC-GapC IgY. Pipette 500 μL of 1.0 × 10⁻⁶ mol / L DMSO solution. 8 CFU mL -1 Cultures of *Streptococcus agalactiae*, *Streptococcus dysgalactiae*, *Staphylococcus aureus*, and *Escherichia coli* were prepared. 50 μL of FITC-GapC IgY was added to each culture, and the cultures were incubated at 25°C in the dark for 15 minutes. After centrifugation at 3500 rpm for 5 minutes, the cultures were washed with PBS and resuspended in 200 μL of sterile PBS buffer. 10 μL of each suspension was added to a glass slide, covered with a coverslip, and the fluorescent images of the stained bacteria were observed and captured under a laser confocal microscope. The results are as follows: Figure 8 As shown, only the streptococcus group emitted obvious green fluorescence, indicating that the stained GapC-IgY can specifically capture agalactiae and dysgalactiae, but has no ability to capture Staphylococcus aureus and Escherichia coli. This shows that the GapC-IgY antibody can specifically capture antigens with good specificity. Therefore, IgY can be used to prepare kits for detecting Streptococcus mastitis in dairy cows, or to prepare test strips for detecting Streptococcus mastitis in dairy cows.

[0022] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An immunogenic antigen of Streptococcus mastitis in dairy cows, characterized in that, The immunoantigen is the GapC protein of Streptococcus bovis mastitis, and the amino acid sequence of the GapC protein of Streptococcus bovis mastitis is the same as that of the GapC protein shared by Streptococcus agalactiae, Streptococcus dysgalactiae, and Streptococcus masculinus, as shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that, The nucleotide sequence comprising encoding the GapC protein of Streptococcus bovis mastitis is shown in SEQ ID NO.

2.

3. A method for preparing the immunogenic antigen of *Streptococcus mastitis* as described in claim 1, characterized in that, Includes the following steps: (1) The gene encoding the GapC protein of the bovine mastitis streptococcus was cloned into the multiple cloning site of the prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET-32a(+)-GapC. (2) The verified recombinant plasmid pET-32a(+)-GapC was transformed into competent BL21(DE3) cells, and positive clones were screened. (3) GapC recombinant protein was obtained by IPTG induction expression and purification. GapC recombinant protein is used as an immunogenic antigen of Streptococcus mastitis in dairy cows.

4. The preparation method according to claim 3, characterized in that, In step (3), IPTG is used for induction at a temperature of 20-37℃ for 4-8 hours.

5. An egg yolk antibody against Streptococcus mastitis in dairy cows, characterized in that, The egg yolk antibody is obtained by emulsifying the GapC recombinant protein prepared according to claim 3 with an equal volume of water adjuvant and then immunizing high-producing laying hens via intramuscular injection.

6. The egg yolk antibody as described in claim 5, characterized in that, The method for extracting the egg yolk antibody includes: separating the egg yolk from eggs of immunized high-producing hens, diluting with PBS, defatting with chloroform, precipitating with PEG 6000, and resuspending in PBS to obtain the egg yolk antibody.

7. The use of the egg yolk antibody as described in claim 5 or 6 in the preparation of a detection product for Streptococcus mastitis in dairy cows.

8. A kit for detecting Streptococcus mastitis in dairy cows, characterized in that, It contains the egg yolk antibody as described in claim 5 or 6.

9. A test strip for detecting Streptococcus mastitis in dairy cows, characterized in that, It contains the egg yolk antibody as described in claim 5 or 6.

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