Preparation method and application of specific anti-streptococcus bovis IgY antibody

Abstract

The invention relates to the technical field of biology, and provides a preparation method and application of a specific anti-streptococcus bovis IgY antibody, and the preparation method comprises the following steps: S1, designing and synthesizing a streptococcus bovis multi-epitope antigen, S2, constructing a recombinant plasmid pVAX1-CRR, and S3, preparing the antibody to obtain the specific anti-streptococcus bovis IgY antibody. A multi-epitope antigen eukaryotic expression vector of streptococcus bovis can be constructed to carry out DNA immunization on laying hens, so that the laying hens generate a high-titer specific streptococcus bovis killing antibody (anti-CRR-IgY), and the problems of drug resistance easily caused by traditional antibiotic treatment of streptococcus bovis infection and flora disorder caused by spectrum sterilization are effectively solved; and a novel material preparation method is provided for targeted therapy, disinfection and killing of streptococcus bovis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a preparation method and application of a specific anti-bovine streptococcus IgY antibody. Background Art

[0002] The Streptococcus bovis group (SBSEC) is a class of Gram-positive streptococci that are non-hemolytic or α-hemolytic. It has a variety of species and subspecies and is an important group of streptococci. Bovine Streptococcus is closely related to many diseases and is seriously harmful. Studies have shown that for humans, bovine Streptococcus is one of the important pathogens of infective endocarditis and bacteremia. Among them, about 18%-62% of patients with bovine Streptococcus endocarditis have colorectal tumors, and about 25%-80% of patients with bovine Streptococcus bacteremia also suffer from colorectal tumors. These diseases may not only cause patients to experience fever, pain, fatigue and other discomfort symptoms, seriously affecting the quality of life of patients, but when patients with colorectal cancer are infected with bovine Streptococcus, it may even aggravate the condition, affect the treatment effect of the tumor and the prognosis of the patient, and endanger life. For animals (especially cattle), Streptococcus bovis can not only cause infective endocarditis and bacteremia, but also cause infections in other parts of the body, such as meningitis, peritonitis, arthritis, cholecystitis, cholangitis, etc., which seriously affect animal health and breeding benefits. It is worth noting that animals infected with Streptococcus bovis are very likely to pose a threat to human health through the food chain.

[0003] At present, antibiotics are mainly used to treat bovine Streptococcus infection. However, traditional antibiotic treatment methods are uncertain before the results of drug sensitivity tests are obtained. Empirical medication may lead to treatment failure and aggravate drug resistance due to strain resistance; and medication based on drug sensitivity test results may miss the best treatment time, causing economic losses to farms. On the other hand, the widespread use of antibiotics themselves brings many hazards, such as flora imbalance, allergic reactions, and accelerated development of bacterial resistance. Therefore, when dealing with bovine Streptococcus infection, it is urgent to actively develop new antimicrobial drugs and alternative therapies on the basis of rational use of antibiotics, focus on comprehensive supportive treatment after animal infection, ensure animal health and human safety, and respond to the increasingly severe antibiotic resistance crisis. Summary of the invention

[0004] The first purpose of the present application is to provide a method for preparing specific anti-bovine Streptococcus IgY antibodies, by constructing a eukaryotic expression vector of bovine Streptococcus multi-epitope antigens to perform DNA immunization on laying hens, so that laying hens can produce high-titer and specific antibodies that kill bovine Streptococcus (anti-CRR-IgY), effectively overcoming the problems of traditional antibiotics that easily induce drug resistance and spectrum sterilization that cause bacterial flora disorders in the treatment of bovine Streptococcus infection, and providing new materials and new methods for the preparation of targeted treatment, disinfection and killing of bovine Streptococcus.

[0005] The second object of the present application is to provide a specific anti-bovine Streptococcus IgY antibody specific bactericidal solution.

[0006] The third object of the present application is to provide a specific anti-bovine Streptococcus IgY antibody biological disinfectant.

[0007] The fourth object of the present application is to provide a specific anti-bovine Streptococcus IgY antibody feed.

[0008] The above applications are used to prevent and treat bovine streptococcal infection, reduce the production of drug-resistant bacteria, and solve the problems of drug resistance, risk of bacterial imbalance, and additional burden on the body faced by antibiotic treatment in bovine streptococcal infection. The innovation lies in the design and formation of corresponding products for different application scenarios (environmental disinfection, feed additives, disease treatment).

[0009] In order to solve the above technical problems, the technical solution adopted in this application is: In a first aspect, the present application provides a method for preparing a specific anti-bovine Streptococcus IgY antibody, which comprises the following steps: S1. Design and synthesis of bovine Streptococcus multi-epitope antigens, comprising the following steps: ①. Target protein selection and epitope screening, taking the rod-shaped determinant protein (RodA) of bovine Streptococcus as the target protein, obtaining amino acids and gene sequences, performing T and B cell dominant epitope prediction and screening, connecting the screened epitope sequence in series twice to enhance immunogenicity, and connecting the mucosal immune adjuvant CTB gene sequence at the C-terminus to obtain a fusion protein sequence CTB-RodA-RodA (CRR); ②. Sequence optimization and synthesis, introducing a BamH I restriction site at the 5′ end of the CRR sequence and an EcoR I restriction site at the 3′ end. I restriction site and protective base sequence, the gene sequence is optimized according to the codon preference of poultry, and a CRR gene fragment with a length of 1080bp and a theoretical molecular weight of about 40 KD is synthesized; S2. The construction of the recombinant plasmid pVAX1-CRR includes the following steps: 1). Double restriction enzyme reaction, take the CRR gene fragment and the eukaryotic expression vector pVAX1, and use BamH I and EcoR I to perform double restriction enzyme reaction to obtain a reaction solution; 2). Enzyme digestion product purification, the reaction solution is subjected to agarose gel electrophoresis, the target band is cut out, and the target DNA fragment is purified using a gel recovery kit; 3). Ligation reaction, the purified target DNA fragment is placed in a sterile microcentrifuge tube to prepare a ligation reaction system, mixed and centrifuged, and placed at 16°C for ligation overnight to obtain a ligation product; 4). Transform competent cells, take E.coliDH5α competent cells, add the ligation product, mix and place in an ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and quickly transfer to ice for cooling for 2 minutes , add to LB liquid culture medium, shake and culture at 37°C, 200rpm for 1h to revive the bacterial colony, spread the revived bacterial solution on LB solid culture medium plate containing kanamycin, and invert and culture at 37°C overnight to obtain colonies; 5). Colony PCR identification, take the colony and culture it in LB liquid culture medium containing kanamycin, then perform PCR amplification, and obtain PCR products. Perform agarose gel electrophoresis on the PCR products. If a band with the expected size appears, it is preliminarily judged as a positive clone; 6). Enzyme digestion identification, extract plasmid from the positive clone, and use BamH I and EcoR I were double-digested to obtain a digestion product, and the digestion product was analyzed by agarose gel electrophoresis. If a band of the expected size appeared, it further indicated that the recombinant plasmid pVAX1-CRR was successfully constructed; 7). Sequencing identification, the recombinant plasmid pVAX1-CRR identified correctly by digestion was sequenced, and the sequencing primers were the universal primers on the pVAX1 vector. The sequencing results were compared with the designed CRR sequence. If the sequences were completely consistent, it was confirmed that the recombinant plasmid pVAX1-CRR was correctly constructed, and the recombinant plasmid pVAX1-CRR was obtained; S3.The antibody preparation comprises the following steps: I. Select laying hens, design an immunization program with CTB as an immune adjuvant, and immunize the laying hens with the recombinant plasmid pVAX1-CRR; II. Select the collected eggs, take out the egg yolk liquid and freeze it for later use; III. Extract the water-soluble components of the egg yolk to obtain a water-soluble component (WSF) containing IgY; IV. Separate and purify the water-soluble components to obtain an IgY precipitate; V. Dialyze the IgY precipitate to obtain a specific anti-bovine Streptococcus IgY antibody.

[0010] Compared with mammalian IgG, specific anti-bovine Streptococcus IgY has the advantages of being cheap, easy to obtain, stable and high in yield. Compared with traditional antibiotics, specific anti-bovine Streptococcus IgY is highly specific and can accurately identify and bind to specific antigens on the surface of bovine Streptococcus, effectively reducing common digestive tract discomfort and secondary infections during antibiotic treatment; secondly, IgY antibodies have good stability and a long half-life, and can remain active under certain temperature, pH and other conditions, and can continue to play a role in the body; not only that, IgY antibodies also have immunomodulatory effects, have the ability to directly neutralize bovine Streptococcus, and cooperate with the body's immune system to provide animals with more lasting protection; most importantly, the use of IgY antibodies can effectively avoid the development of antibiotic resistance. Since the mechanism of action of IgY antibodies is different from that of antibiotics, they will not induce bacterial resistance, which effectively inhibits the emergence of resistant strains and helps maintain the stability of global public health.

[0011] In the present application, a multi-epitope antigen of Streptococcus bovis was creatively designed and synthesized, the screened epitope sequence was concatenated twice to enhance the immunogenicity, and the mucosal immune adjuvant CTB gene sequence was connected to the C-terminus of the concatenated epitope sequence, laying the foundation for the subsequent use of CTB as an immune adjuvant for immunization of laying hens. At the same time, the gene sequence was optimized according to the codon preference of poultry, and the 1080bp sequence was optimized for expression in chickens, providing a basis for the subsequent preparation of CRR-IgY antibodies.

[0012] There are many advantages to using a eukaryotic expression vector constructed for multiple epitope antigens of Streptococcus bovis to immunize laying hens with DNA to obtain specific anti-bovine Streptococcus IgY: first, it has the ability to induce efficient immunogenicity, which can fully activate immunity and produce a large number of high-affinity specific antibodies; second, the production process is simple and stable, omitting complex antigen preparation and purification steps and reducing antigen quality fluctuations; third, it has safety advantages and does not use live bovine Streptococcus pathogens, reducing the risk of biological hazards; fourth, it is cost-effective, reducing the costs associated with antigen production, and can achieve low-cost large-scale antibody production; fifth, it has high specificity and affinity, ensuring accurate and reliable diagnosis and treatment, and efficient immune response helps to improve the effectiveness of antibodies in detection and prevention.

[0013] In some embodiments of the present application, in the above step I, the immunization program designed with CTB as the immune adjuvant is specifically as follows: 600 μg of CTB is injected before immunization (0 day); 150 μl of bovine Streptococcus (sp) is injected for the first immunization (7-9 days); 150 μl of bovine Streptococcus (sp) is injected for the second immunization (14-21 days); 150 μl of bovine Streptococcus (sp) is injected for the third immunization (28-52 days); 150 μl of bovine Streptococcus (sp) is injected for the fourth immunization (62-72 days).

[0014] In the present application, a set of immunization programs is creatively designed with CTB as an immune adjuvant, and 600 μg of CTB is injected into laying hens before immunization, so that the laying hens produce an inflammatory effect before the immunization program, which is conducive to the successful immunization.

[0015] In some embodiments of the present application, in the above step I, immunization is performed by multiple injections at the subcutaneous part of the wing base and the chest muscle.

[0016] In some embodiments of the present application, the specific method in the above step II is: select the collected eggs, soak them in 0.1% concentration of Chlorhexidine solution for 30 minutes, take them out and dry them, use an egg yolk separator to take out the egg yolk, pierce the yolk membrane of the egg yolk to take out the egg yolk liquid, put the egg yolk liquid into a -80°C refrigerator and freeze it for 2 hours, transfer it to a 4°C refrigerator and refrigerate it overnight to allow it to return to temperature.

[0017] In some embodiments of the present application, the specific method of the above step III is: placing the egg yolk liquid into ultrapure water with a pH of 5.2 and mixing evenly to obtain an egg yolk suspension; placing the egg yolk suspension in a 4°C refrigerator and letting it stand for 10 hours, placing it in a centrifuge, setting the speed to 10,000 rpm and the temperature to 4°C for centrifugation for 25 minutes, and the supernatant after centrifugation is the water-soluble component (WSF) containing IgY.

[0018] In some embodiments of the present application, the specific method of step IV above is: placing the water-soluble component in a centrifuge tube, adding a saturated ammonium sulfate solution, stirring so that the final saturation of ammonium sulfate is 50%, placing it in a 4°C refrigerator and letting it stand for 3 hours, then placing it in a centrifuge, setting the speed to 3000 rpm, centrifuging at 4°C for 20 minutes, discarding the supernatant, adding ultrapure water and a saturated ammonium sulfate solution, stirring so that the final saturation of ammonium sulfate is 33%, placing it in a 4°C refrigerator and letting it stand for 3 hours, placing it in a centrifuge, setting the speed to 3000 rpm, centrifuging at 4°C for 20 minutes, discarding the supernatant, and obtaining an IgY precipitate.

[0019] In some embodiments of the present application, the specific method of the above step V is: the IgY precipitate is placed in a dialysis bag for dialysis, and the dialysis external fluid is detected using a naphthol reagent to detect whether there is yellow, and the dialysis external fluid is detected using a 1% concentration of BaCl2 solution to detect whether there is a precipitate. When the dialysis external fluid is not yellow and has no precipitate, it indicates that the dialysis is completed and a specific anti-bovine Streptococcus IgY antibody is obtained.

[0020] In a second aspect, an embodiment of the present application provides a specific anti-bovine Streptococcus IgY antibody specific bactericidal liquid, the raw materials of which include specific anti-bovine Streptococcus IgY antibodies.

[0021] In some embodiments of the present application, the preparation method of the above-mentioned specific anti-bovine Streptococcus IgY antibody specific bactericidal solution is: filtering the specific anti-bovine Streptococcus IgY antibody through a filter membrane with a pore size of 0.22 μm, adjusting the concentration of the specific anti-bovine Streptococcus IgY antibody by adding PBS buffer, and preparing the specific bactericidal solution.

[0022] In some embodiments of the present application, the pH of the above PBS buffer is 7.2-7.4.

[0023] In some embodiments of the present application, the PBS buffer is composed of Na2HPO4, KH2PO4, NaCl, HCL and ultrapure water.

[0024] In a third aspect, an embodiment of the present application provides a specific anti-bovine Streptococcus IgY antibody biological disinfectant, the raw materials of which include specific anti-bovine Streptococcus IgY antibodies.

[0025] In some embodiments of the present application, the preparation method of the above-mentioned specific anti-bovine Streptococcus IgY antibody biological disinfectant is: weigh the specific anti-bovine Streptococcus IgY antibody, mix it with sterile water in a ratio of 1:9, gently stir and shake to prepare a disinfectant with a concentration of 10%.

[0026] In a fourth aspect, an embodiment of the present application provides a specific anti-bovine Streptococcus IgY antibody feed, wherein the raw materials of the feed include specific anti-bovine Streptococcus IgY antibodies.

[0027] In some embodiments of the present application, the preparation method of the above-mentioned specific anti-bovine Streptococcus IgY antibody feed is: weigh the specific anti-bovine Streptococcus IgY antibody, mix it with the feed in a ratio of 1:9 to mix them evenly, and pack the mixed specific anti-bovine Streptococcus IgY feed into sterile bags and seal them for storage at 4°C.

[0028] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. Specific anti-bovine Streptococcus IgY has the advantages of strong specificity, good stability, large yield, long half-life and immune regulation.

[0029] 2. Specific anti-bovine Streptococcus IgY solves the problems of lack of specificity and easy induction of drug resistance when traditional antibiotics are used to treat bovine Streptococcus infection.

[0030] 3. Enhance animals' immune defense against bovine streptococci and reduce the probability of diseases caused by bovine streptococci.

[0031] 4. The preparation cost is low, and the extraction and purification method achieves the purpose of economy and high efficiency.

[0032] 5. It can be prepared into injections, biological disinfectants, feed additives, etc. for use, with a wide range of applications and strong adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 A restriction enzyme digestion identification diagram of a recombinant plasmid pVAX1-CRR of a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Figure 2 An electrophoretic diagram of IgY purification of a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Figure 3 A diagram showing the transfection efficiency of a recombinant plasmid pVAX1-CRR of a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention in HEK293T cells; Figure 4 A detection diagram of CRR expression in cells of a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Figure 5 A trend chart of the production of IgY during the immunization cycle in Test Example 2 in a method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Figure 6 A diagram showing the antibacterial effect of IgY on a standard strain of Streptococcus bovis (ATCC33317) in Test Example 4 of a method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Figure 7A diagram showing the antibacterial effect of IgY on the clinical strain of Streptococcus bovis Naqu in Test Example 4 in a method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Figure 8 A diagram showing the antibacterial effect of IgY on Escherichia coli BL21 in Test Example 4 of a method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Fig. 9 A diagram showing the growth of bovine Streptococcus in the mouse bedding on the bovine Streptococcus selective medium in Example 3 of the method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Fig.10 A graph showing the colony count of bovine Streptococcus in the mouse bedding on the bovine Streptococcus selective medium in Example 3 of the method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Fig.11 A diagram showing the growth of bovine Streptococcus in the intestinal tissue homogenate on the bovine Streptococcus selective medium in Example 4 of the method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention; Fig.12 This is a graph showing the colony count of Streptococcus bovis in the intestinal tissue homogenate on the Streptococcus bovis selective culture medium in Example 4 of the method for preparing a specific anti-bovine Streptococcus IgY antibody provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to specific embodiments.

[0037] Required materials and reagents: standard strain of Streptococcus bovis ATCC 33317 (purchased from Ningbo Mingzhou Biotechnology Co., Ltd.), clinical isolate of Streptococcus bovis Naqu (isolated from yak feces sample in Naqu by the laboratory), Escherichia coli strains E. coli BL21 (DE3) and E. coli DH5α (preserved in the laboratory), human embryonic kidney cell line HEK293T (preserved in the laboratory), PEI transfection reagent, DMEM high glucose medium, endotoxin-free plasmid extraction kit, 4% paraformaldehyde solution, 0.3% TritonX-100 solution, 3% bovine serum albumin (BSA), anti-cholera toxin B subunit (CTB) antibody, anti-bovine Streptococcus antibody, goat anti-mouse IgG fluorescent secondary antibody, anti-BL21-IgY antibody, 4',6-diamidino-2-phenylindole (DAPI), anti-fluorescence quencher, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) related reagents (including acrylamide, SDS, Tris-HCl, glycine, ammonium persulfate, TEMED, etc.), protein lysis buffer (RIPA buffer and phenylmethylsulfonyl fluoride (PMSF) mixed at a ratio of 50:1), BCA protein quantification kit, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody, chemiluminescent substrate (ECL), restriction endonucleases BamH I and EcoR I, T4 DNA ligase, DNA molecular weight standard (Marker), protein molecular weight standard, kanamycin, LB liquid medium, and LB solid medium.

[0038] The relevant solutions are prepared as follows: Coating solution: PBS buffer (pH 7.3, 0.01M): weigh 107.5 g Na2HPO4, 0.9 g KH2PO4, 9 g NaCl and 1 mol / l HCL, adjust the pH to 7.3, and make up to 1000 ml with ddH20.

[0039] Blocking solution (1% BSA-PBS, pH 7.4): weigh 0.1 g BSA and dissolve it in 10 ml PBS.

[0040] Washing solution (0.05% Tween20-PBS, PBST, pH 7.2): Dissolve 0.5 ml Tween-20 in 1 L PBS.

[0041] Sample diluent (0.05% Tween20-1% BSA-PBS solution, pH 7.4): weigh 0.1 g BSA and dissolve it in 100 ml washing solution.

[0042] Terminator (1 mol / L H2SO4): Slowly add 100 ml of concentrated sulfuric acid to 600 ml of double distilled water while stirring continuously, and add double distilled water to 900 ml.

[0043] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0044] Example 1 A method for preparing a specific anti-bovine Streptococcus IgY antibody comprises the following steps: S1. The design and synthesis of multi-epitope antigens of Streptococcus bovis include the following steps: ①. Target protein selection and epitope screening. The rod-shaped determinant protein (RodA) of Streptococcus bovis was used as the target protein. Its amino acid sequence and corresponding gene sequence were obtained from the NCBI database. BepiPred 2.0 and IEDB website were used to predict and screen T cell and B cell dominant epitopes. Due to the limited number of RodA epitopes, the screened epitope sequences were concatenated twice to enhance immunogenicity. At the same time, the mucosal immune adjuvant CTB gene sequence was connected to the C-terminus of the concatenated epitope sequence to improve the body's immune responsiveness, and the fusion protein sequence CTB-RodA-RodA (CRR) was obtained.

[0045] ②. Sequence optimization and synthesis: add a BamH I restriction site at the 5′ end of the CRR sequence, introduce an EcoR I restriction site and a protective base sequence at the 3′ end, and then use the vectorbuilder online tool to optimize the gene sequence according to the codon preference of poultry. The optimized gene sequence was handed over to Sangon Biotechnology Co., Ltd. for chemical synthesis to obtain a CRR gene fragment with a length of 1080bp and a theoretical molecular weight of about 40 KD. Its gene sequence is as follows: ggatccatga ttaagttgaa gtttggagtt ttcttcacag ttctgctgtc ttcagcttat 60 gctcacggca caccgcagaa cattactgat ctgtgtgcag agtaccacaa cacacagatc 120 cacactctga acgataaaat cttgagttac actgagtcac ttgcaggtaa acgcgaaatg 180 gccatcatta cattcaagaa tggagaaact ttccaggtgg aggtacctgg gagccagcac 240 atcgattctc agagaaggc tatcgaacgt atgaagaca cacacggat tgcttatctg 300 acagaagcga aggtggagaa gctttgcgtc tggacaaca agacccctca cgcaatcgcg 360 gccattagta tggcaatgg cggcggaggc agcggtggag gtggtccgg gggaggaggt 420 agcaaaatg attggaact aaagaagggc attagttggt ggataatca aaagcacggc 480 aagatatat tctacagcct gggcatggat acgtatcaa ttataaaaa gtggctggac 540 cccttttctt acgcaaatc cattgcctac aagaaagcac tcatcacact gccagtcatg 600 aataagaat tgcaaaaga tctggcacg gcaatgaga agcatgggaa agacatattt 660 tattcgttaa agagtacca aattaatagg atcagtgctt ggagaaaca acagacccag 720 ggaatgatat cattagaa gaaaatgac tggagctga aaaagggat ctcttggtgg 780 attataaaaa aacatggcaa ggacatctc tatagcttgg gtatggacac ctaccagatc 840 aaaaaaaat ggttagatcc cttttcctat gccaaatcaa tcgcctataa gaagctctt 900 attaccctcc catgatgat tagaactg cagaaggacc tcgggactgc catgagaaa 960 catgggaagg atatctttta ctccctcaaa aaataccaga taaacagaat ttccgcctgg 1020 aagaaacagc agacccaagg aatgatatct atccatcacc accatcatca ttgagaattc 1080 S2. Construction of recombinant plasmid pVAX1-CRR, including the following steps: 1). Double enzyme digestion reaction, take the synthesized CRR fragment and the eukaryotic expression vector pVAX1, and perform double enzyme digestion reaction with BamH I and EcoR I respectively. The enzyme digestion system is as follows: 5 μL of 10× restriction endonuclease buffer, 1 μL of BamH I enzyme, 1 μL of EcoR I enzyme, 3 μL of CRR gene fragment, and sterile deionized water to 50 μL, gently mix, centrifuge, and place in a 37°C water bath for enzyme digestion for 3 hours to obtain a reaction solution.

[0046] 2). Purification of the digestion product: After the digestion reaction is completed, the reaction solution is subjected to agarose gel electrophoresis. The electrophoresis results are observed under ultraviolet light, and the target bands (CRR fragments and linearized pVAX1 vectors) are cut out. The target DNA fragment is recovered and purified using a gel recovery kit according to the instructions.

[0047] 3). Ligation reaction: prepare the ligation reaction system in a sterile microcentrifuge tube. The reaction system is as follows: 2 μL of 10×T4 DNA ligase buffer, 1 μL of purified pVAX1 vector, 3 μL of purified CRR fragment, 1 μL of T4 DNA ligase, and make up to 20 μL with sterile deionized water. Mix gently, centrifuge, and place at 16°C for ligation overnight to obtain a ligation product.

[0048] 4). Transform competent cells. Take 100 μL of E. coli DH5α competent cells (take out from -80°C refrigerator and thaw on ice), add 10 μL of ligation product, mix gently, ice bath for 30 minutes, heat shock at 42°C for 90 seconds, quickly transfer to ice and cool for 2 minutes, add 900 μL of LB liquid culture medium preheated to room temperature, shake and culture at 37°C and 200 rpm for 1 hour to revive the bacteria, take 200 μL of the revived bacterial liquid and spread it on LB solid culture medium plate containing kanamycin (final concentration of 50 μg / mL), and invert and culture at 37°C overnight to obtain colonies.

[0049] 5). Colony PCR identification: randomly select several single colonies from the plate and inoculate them into 5 mL of LB liquid culture medium containing kanamycin. Incubate at 37°C and 200 rpm overnight with shaking. Take 1 μL of bacterial solution as template for PCR amplification. The PCR reaction system is as follows: 5 μL of 10× PCR buffer, 4 μL of dNTPs mixture (2.5 mM each), 1 μL of upstream primer (upstream primer of CRR sequence), 1 μL of downstream primer (downstream primer of CRR sequence), 0.5 μL of Taq DNA polymerase, 1 μL of bacterial solution template, and make up to 50 μL with sterile deionized water. PCR reaction conditions: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, for a total of 35 cycles; extension at 72°C for 10 min, take 10 μL The PCR products were detected by agarose gel electrophoresis. If a band with the expected size appeared, it was preliminarily judged as a positive clone.

[0050] 6). Enzyme digestion identification: For clones that are initially judged to be positive, extract plasmids and operate according to the instructions of the endotoxin-free plasmid extraction kit. Take an appropriate amount of extracted plasmids and perform double enzyme digestion identification with BamH I and EcoR I. The enzyme digestion system and reaction conditions are the same as the double enzyme digestion step in step 1) above. The enzyme digestion products are analyzed by agarose gel electrophoresis. If the expected size bands (CRR fragment and linearized pVAX1 vector) appear, it further indicates that the recombinant plasmid pVAX1-CRR is successfully constructed. The enzyme digestion identification results of the recombinant plasmid pVAX1-CRR are as follows: Figure 1 shown.

[0051] 7). Sequencing identification: the recombinant plasmid pVAX1-CRR identified as correct by enzyme digestion was sent to a bioengineering company for DNA sequencing. The sequencing primers were the universal primers on the pVAX1 vector. The sequencing results were compared with the designed CRR sequence. If the sequences were completely consistent, it was confirmed that the recombinant plasmid pVAX1-CRR was correctly constructed and the recombinant plasmid pVAX1-CRR was obtained.

[0052] S3. Antibody preparation, including the following steps: I. Select 4 laying hens aged 120 days, two in each group and divide them into two groups A and B, design an immunization program with CTB as an immune adjuvant, Group A is immunized with CTB as an immune adjuvant, and Group B is immunized without CTB as an immune adjuvant. The immunization and egg collection procedures are shown in Table 1, and the immunization method is immunization by multiple injections at the subcutaneous part of the wing base and the chest muscle.

[0053]

[0054] II. Select the collected eggs and soak them in 0.1% concentration of Chlorhexidine solution for 30 minutes. Turn the eggs gently during the soaking process to ensure that the surface of the eggs is fully in contact with the solution. After the soaking, take out the eggs and put them in a well-ventilated place to dry. Use an egg yolk separator to separate the yolk from the eggs. Use a syringe needle to gently pierce the yolk membrane of the egg yolk to take out the egg yolk liquid, place it in a centrifuge tube, freeze the egg yolk liquid in a -80℃ refrigerator for 2 hours, and then transfer it to a 4℃ refrigerator and refrigerate overnight to allow it to return to temperature.

[0055] III. Take 5 ml of the warmed egg yolk liquid and place it in 40 ml of ultrapure water with a pH of 5.2. Use a vortex mixer to mix thoroughly to obtain an egg yolk suspension. During the mixing process, be careful to avoid excessive bubbles to avoid affecting the subsequent centrifugation effect. Place the egg yolk suspension in a 4°C refrigerator and let it stand for 10 hours to fully separate the water-soluble components and fat-soluble components in the egg yolk. Place the egg yolk suspension after standing in a centrifuge, set the speed to 10,000 rpm, and centrifuge for 25 minutes at 4°C. The supernatant after centrifugation is the water-soluble component (WSF) containing IgY. Carefully aspirate it and transfer it to a new centrifuge tube.

[0056] IV. Take 20 ml of WSF and place it in a 45 ml centrifuge tube. Use a dropper to add 20 ml of saturated ammonium sulfate solution drop by drop, stirring gently while adding, so that the final saturation of ammonium sulfate is 50%. When adding ammonium sulfate solution, pay attention to controlling the drop speed to avoid adding too quickly, which will cause excessive local concentration and affect the precipitation effect of the antibody. Place the centrifuge tube in a 4°C refrigerator and let it stand for more than 3 hours to allow the antibody to fully precipitate. Then place it in a centrifuge, set the speed to 3000 rpm, and centrifuge at 4°C for 20 minutes. After centrifugation, discard the supernatant and retain the precipitate. Place the precipitate back in the centrifuge tube, add 10 ml of ultrapure water, and use a dropper to add 5 ml of saturated ammonium sulfate solution drop by drop, stirring gently while adding, so that the final saturation of ammonium sulfate is 33%. Place it in a 4°C refrigerator and let it stand for 3 hours to allow the antibody to precipitate again. Place it in a centrifuge, set the speed to 3000 rpm, and centrifuge at 4°C for 20 minutes. After centrifugation, discard the supernatant and retain the precipitate. Repeat the above "making the final saturation of ammonium sulfate 33%" step twice. The final precipitate is the IgY precipitate.

[0057] V. The IgY precipitate was placed in a dialysis bag for dialysis. The dialysis fluid was tested for yellowness using Naphthol reagent. The 1% BaCl2 solution was used to test for precipitation. When the dialysis fluid was not yellow and had no precipitation, the dialysis was completed and specific anti-bovine Streptococcus IgY antibody was obtained. The molecular weight of the purified IgY was 66×10 3 Da of IgY heavy chain and 25×10 3 The purified IgY light chain is as follows: Figure 2 shown.

[0058] Test Example 1 Based on Example 1, Test Example 1 is to test the transfection rate of the cell slide by immunofluorescence analysis, and the steps are as follows: 1. Cell culture and preparation before transfection: One day before transfection, HEK293T cells in the logarithmic growth phase were taken and digested with 0.25% trypsin. The digestion was terminated with DMEM medium containing 10% fetal bovine serum (FBS). The cells were gently blown to form a single cell suspension. An appropriate amount of cell suspension was taken and the cell density was counted by trypan blue exclusion method. The cell concentration was adjusted to 0.5×10 5 / mL, inoculate the cell suspension into a 24-well plate at a volume of 0.5mL per well, gently shake the 24-well plate to evenly distribute the cells, and culture in a 37°C, 5% CO2 incubator. 2 hours before transfection, carefully remove the culture medium in the 24-well plate, gently wash the cells once with serum-free and antibiotic-free DMEM culture medium preheated to 37°C, then add 0.5mL of fresh serum-free and antibiotic-free DMEM culture medium to each well, and return to the incubator to continue culturing.

[0059] 2. Preparation of transfection complex: Calculate the required amount of DNA and PEI according to the instructions of PEI transfection reagent. For each well of cells, take 50 μL of serum-free culture medium, add 4 μL of recombinant plasmid pVAX1-CRR (concentration is 1 μg / μL) and 1 μL of PEI transfection reagent, mix gently, and incubate at 37°C for 5 min to obtain DNA dilution solution and PE dilution solution, respectively. Slowly add PEI dilution solution drop by drop into DNA dilution solution, mix gently while adding to avoid bubbles, and then incubate at 37°C for 15 min to form a transfection complex (DNA-PEI transfection working solution).

[0060] 3. For transfection, add 105 μL of the prepared transfection complex dropwise into the cell culture wells of the 24-well plate, and gently shake the 24-well plate to evenly distribute the transfection complex; at the same time, add 105 μL of serum-free culture medium to the blank control group wells, and place the 24-well plate in a 37°C, 5% CO2 incubator for culture.

[0061] 4. Change the culture medium. After 8 hours of transfection, carefully aspirate the transfection medium in the 24-well plate, add 0.5 mL of complete culture medium (DMEM medium containing 10% FBS and 1% penicillin-streptomycin) preheated to 37°C to each well, and continue to culture in a 37°C, 5% CO2 incubator for 40 hours.

[0062] 5. Cell fixation and permeabilization. 48 hours after transfection of HEK293T cells, remove the cell slides, gently wash the cells 3 times with pre-cooled PBS, 5 minutes each time, add 4% paraformaldehyde solution, fix the cells for 20 minutes, wash them 3 times with PBS, 5 minutes each time after fixation, add 0.3% Triton X-100 solution, permeabilize at room temperature for 15 minutes, wash them 3 times with PBS, 5 minutes each time after permeabilization.

[0063] 6. Blocking and incubation with primary antibody: add 3% BSA solution and block at room temperature for 15 minutes. After blocking, add diluted primary antibody (mouse anti-CTB antibody, 1:100 dilution; mouse anti-bovine Streptococcus antibody, 1:500 dilution) directly without washing, so that the antibody completely covers the cells and incubate at 4°C overnight.

[0064] 7. Secondary antibody incubation and fluorescent staining. After overnight incubation, wash the cells three times with pre-cooled PBS for 10 min each time, add fluorescent secondary antibody (Alexa Fluor® 488-labeled goat anti-mouse IgG (H + L), 1:200 dilution), incubate at room temperature in the dark for 1.5 h. After incubation, wash the cells three times with PBS for 10 min each time, add DAPI stain (final concentration of 1 μg / mL), incubate at room temperature in the dark for 15 min to stain the cell nuclei.

[0065] 8. Sealing and observation. After the incubation, wash the cells with PBS for 3 times, 5 minutes each time, add anti-fluorescence quencher to seal the slide, place the cell slide under a fluorescence microscope for observation, randomly select multiple fields of view to take pictures and record, use Image J software to analyze the proportion of green fluorescent cells (cells expressing CRR protein) to the total number of cells, calculate the transfection efficiency, and the transfection efficiency is about 30-40%, indicating that the constructed recombinant plasmid pVAX1-CRR can be effectively expressed in HEK293T cells. The results are shown in Figure 2. Figure 3 shown.

[0066] Test Example 2 Based on Example 1 and Test Example 1, this Test Example 2 is used for expression identification (Western blot) of the recombinant plasmid pVAX1-CRR. The steps of transfecting HEK293T cells with the recombinant plasmid pVAX1-CRR in the experimental steps are the same as those in Test Example 1 and are not described here. The steps after transfection are different from those in Test Example 1. The specific steps are as follows: 1. Protein extraction: 48 hours after transfection of HEK293T cells, discard the culture medium in the 24-well plate, wash the cells twice with pre-cooled PBS, add 200 μL pre-cooled protein lysis buffer (RIPA:PMSF = 50:1) to each well, and place on ice for 10 minutes. During this period, gently shake the cell culture plate to fully lyse the cells. Use a cell scraper to collect the cell lysate into a 1.5 mL centrifuge tube, centrifuge at 4°C, 14000 rpm for 15 minutes, and carefully aspirate the supernatant and transfer it to a new centrifuge tube, which is the total protein sample.

[0067] 2. Protein quantification: Use BCA protein quantification kit to determine the concentration of total protein samples according to the kit instructions.

[0068] 3. SDS-PAGE electrophoresis. According to the protein quantification results, calculate the required loading volume to make the loading protein amount of each sample consistent (30μg in this test example 3). Take an appropriate amount of protein sample, add 5×SDS-PAGE loading buffer (protein sample: loading buffer = 4:1), mix well, heat in a water bath at 100°C for 5 minutes to denature the protein, prepare SDS-PAGE gel (the separation gel concentration is selected according to the molecular weight of the target protein. In this experiment, 12% separation gel can be selected). Install the gel plate in the electrophoresis tank, add electrophoresis buffer (Tris-glycine buffer, pH8.3), load the denatured protein sample into the gel well, and the loading amount per well is determined according to the protein concentration and gel size. At the same time, add a protein molecular weight standard (Marker) to the well next to it. Turn on the power supply and start electrophoresis. The initial voltage is set to 80V. After the protein sample enters the separation gel, adjust the voltage to 120V, continue electrophoresis until the bromophenol blue indicator reaches the bottom of the gel, and turn off the power supply.

[0069] 4. Transfer. After the electrophoresis is completed, carefully remove the gel and assemble the transfer device according to the "sandwich" structure (sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad). Make sure there are no bubbles between the layers. The PVDF membrane needs to be activated by soaking in methanol for 30 seconds before use, and then soaked in transfer buffer (Tris-glycine buffer, containing 20% ​​methanol) for balance. Put the assembled transfer device into the transfer tank with the PVDF membrane facing the positive electrode and the gel facing the negative electrode. Add pre-cooled transfer buffer and transfer at a constant voltage of 100V for 1.5 hours under ice bath conditions.

[0070] 5. Membrane blocking and antibody incubation. After the transfer, take out the PVDF membrane and place it in TBST buffer (Tris-HCl buffer, containing 0.1% Tween-20 and 0.9% NaCl) containing 5% skimmed milk powder. Block it with slow shaking at room temperature for 1 hour. After the blocking is completed, place the PVDF membrane in the primary antibody solution diluted with TBST buffer (mouse anti-CTB antibody, 1:100 dilution; mouse anti-bovine Streptococcus antibody, 1:500 dilution) and incubate it with slow shaking at 4°C overnight.

[0071] 6. Secondary antibody incubation and color development: After overnight incubation, wash the PVDF membrane three times with TBST buffer, each time for 10 minutes, and place the PVDF membrane in the HRP-labeled goat anti-mouse IgG secondary antibody solution diluted with TBST buffer (1:5000 dilution), and incubate at room temperature with slow shaking for 1 hour. After the incubation, wash the PVDF membrane three times with TBST buffer, each time for 10 minutes. Finally, place the PVDF membrane in the ECL chemiluminescent substrate and incubate in the dark for 2 minutes to allow the protein bands to fully react with the substrate.

[0072] 7. Development and result analysis: After incubation, the PVDF membrane was taken out, wrapped with plastic wrap, and placed in a gel imaging system for exposure and development. The expression of the target protein was analyzed according to the exposure results. In this experiment, the recombinant fusion protein CRR showed a band at 42KD, which was consistent with the expected molecular weight, while the control had no corresponding band, indicating that the CRR protein was successfully and specifically expressed in HEK293T cells. The results are shown in Figure 2. Figure 4 shown.

[0073] Test Example 3 Based on Example 1 and Test Example 2, this Test Example 3 is used to determine the antibody titer using an indirect ELISA method, and the specific steps are as follows: 1. Protein concentration determination: Use spectrophotometer to determine the protein concentration of CRR protein and antibody stock solution respectively.

[0074] 2. Dilution and coating of CRR protein: dilute the CRR protein to 1:5 with coating solution, add the diluted CRR protein to the ELISA wells, 100 μl per well, and incubate the ELISA plate in a 37°C incubator for 2 hours.

[0075] 3. Washing: After incubation, discard the solution in the wells, pat the ELISA plate dry with absorbent paper, and then add washing solution, 250μl per well. Let it stand for 1 minute after each addition of washing solution, then discard the solution in the wells, and repeat the washing operation 3 times to ensure sufficient washing.

[0076] 4. Blocking operation: After washing, add 250 μl of blocking solution to each well and place the ELISA plate at room temperature for 2 hours.

[0077] 5. Sample dilution and addition: Dilute the sample (IgY) to 1:2000, 1:4000, 1:6000, 1:8000, 1:10000, 1:12000 and 1:14000 with sample diluent, place the diluted samples in the ELISA plate, add 100 μl to each well, and let the ELISA plate stand at room temperature for 2 hours.

[0078] 6. Add enzyme-labeled secondary antibody: Add 100 μl of rabbit anti-chicken IgG-HRP enzyme-labeled secondary antibody to each well, and then place the ELISA plate at room temperature for 2 hours.

[0079] 7. Wash the plate: Repeat the washing operation in step 3 5 times to ensure that the plate is washed thoroughly and the unbound enzyme-labeled secondary antibody is removed.

[0080] 8. Substrate color development: Add TMB substrate color development solution to each well, including 50μl of solution A and solution B respectively, and place the ELISA plate at room temperature in the dark for 25 minutes.

[0081] 9. Reaction termination: Add 1 mol / L H2SO4 to each well, the amount added to each well is 50μl, shake for a few seconds to mix, and terminate the color reaction.

[0082] 10. Determination of light absorption value: Use a dual-wavelength microplate reader to measure the light absorption value of each well at 450nm and 630nm (A 450 / 630 ), the highest dilution whose OD value was 2.1 times greater than the average value of the negative control was the antibody titer.

[0083] Specific antibodies appeared in laying hens about 2 weeks after immunization. After four DNA vaccine immunizations, the titer of anti-CRR-IgY gradually increased, reaching a peak around the 7th week after immunization, and then stabilized at 1:6400 for more than two weeks. The titer of group B without CTB as an immune adjuvant was lower than that of group A with CTB as an immune adjuvant, indicating that it is easier to successfully immunize laying hens after immunization with CTB as an immune adjuvant. The results are shown in Figure 5 shown.

[0084] Test Example 4 Based on Example 1, this test example 4 is used to detect the bactericidal effect of the antibody. The in vitro inhibition of bovine streptococcus activity by anti-CRR-IgY was detected by plate colony counting experiment. Fresh bovine streptococcus (ATCC33317) was recovered and adjusted to a concentration of 3×10 7CFU / mL of bacterial solution, the bacterial solution was incubated with specific antibody CRR-IgY (10mg / ml, 5mg / ml, 2mg / ml), nonspecific antibody BL21-IgY (10mg / ml, 5mg / ml, 2mg / ml), positive control kanamycin (50μg / mL), and control (Pbs) for 3h, and then diluted 10 3 times, take 100 μl to plate, three parallels in each group, culture at 37℃ for 72 h, observe and record the number of colony clones.

[0085] The concentration of Streptococcus bovis clinical strain Naqu and Escherichia coli BL21 was adjusted to 3×10 7 CFU / mL of bacterial solution, clinical strain Naqu was incubated with specific antibody CRR-IgY (10 mg / ml), nonspecific IgY (10 mg / ml), kanamycin (50 μg / mL), and control (Pbs) for 3 hours. Escherichia coli was incubated with specific antibody CRR-IgY (10 mg / ml), kanamycin (50 μg / mL), and control (Pbs) for 3 hours. Other steps were the same as above.

[0086] The antibacterial effect of standard strain of Streptococcus bovis is as follows Figure 6 As shown. Compared with the control group, kanamycin significantly inhibited the growth of bovine streptococci. 2mg / ml and 5mg / ml CRR-IgY had no inhibitory effect on the growth of bovine streptococci, and 10mg / ml CRR-IgY had a significant inhibitory effect on the growth of bovine streptococci. The non-specific BL21-IgY had no inhibitory effect on bovine streptococci. The antibacterial effect of clinical strain Naqu Figure 7 As shown in the figure, the control group, CRR-IgY and kanamycin can significantly inhibit the growth of Naqu, while the non-specific BL21-IgY has no inhibitory effect on Naqu. Figure 8 As shown, kanamycin can significantly inhibit the growth of E. coli, while CRR-IgY cannot.

[0087] Finally, it was shown that 10 mg / ml specific antibody CRR-IgY had obvious antibacterial effect against the standard strain of bovine Streptococcus and the clinical strain Naqu, but had no antibacterial effect against Escherichia coli. The non-specific antibody BL21-IgY had no antibacterial effect against the standard strain of bovine Streptococcus and the clinical strain Naqu, indicating that the CRR-IgY antibody is specific and effective against both the standard strain of bovine Streptococcus and the clinical strain.

[0088] Example 2 On the basis of Example 1, this Example 2 provides a specific anti-bovine Streptococcus IgY antibody specific bactericidal liquid, and the effect of the bactericidal liquid is tested, and the specific steps are as follows: 1. Experimental animals and strains Experimental animals: 60 healthy clean-grade BALB / c mice of similar weight (16±2 g) were selected, half of which were male and half were female; Experimental strains: Isolates of Streptococcus bovis, which were maintained in the laboratory and passaged and identified regularly.

[0089] 2. Preparation of bacterial culture Pick an appropriate amount of colonies from the preserved bovine Streptococcus strains, inoculate them into LB liquid medium, and culture them at 37°C with shaking for 24 hours to ensure that the strains are fully activated and proliferated. Then dilute to 5×10 8 CFU / ml for future use.

[0090] 3. Adaptive feeding of mice Before the experiment begins, the experimental mice need to be adaptively raised in an animal breeding environment control box for 5 days. The breeding conditions are a temperature of 25°C, a humidity of 60%, free access to food and water, and the feed is standard mouse feed.

[0091] 4. Mouse Grouping and Infection 60 mice were randomly divided into 3 groups, 20 mice in each group, namely antibiotic treatment group (group A), specific anti-bovine chain IgY antibody injection treatment group (group B) and saline control group (group C). The prepared bacterial solution was inoculated into the experimental and control mice by refraction, and the amount of bacterial solution inoculated into each mouse was 0.2 ml.

[0092] 5. Treatment measures Antibiotic treatment group: After mice were infected with S. bovis, antibiotics were given by intraperitoneal injection according to the prescribed dose (15 mg / kg body weight of kanamycin per mouse per injection) and time interval (one injection per day); IgY treatment group: After mice were infected with S. bovis, they were treated with specific anti-bovine chain IgY by intraperitoneal injection according to the prescribed dose (0.4 mg / kg body weight of specific anti-bovine chain IgY per mouse per injection) and time interval (one injection per day); Control group: After mice were infected with bovine Streptococcus, they were not given any therapeutic drugs and were only injected with an equal amount of normal saline.

[0093] 6. Observation indicators and records The mice were observed once at 9 am and 4 pm every day, and the clinical symptoms and deaths of the mice were recorded. Once a mouse was found dead, the time of death was recorded immediately and the dead mouse was removed from the cage.

[0094] 7. Survival analysis and analysis of clinical symptom improvement The survival rate of each group of mice was calculated, survival rate = number of surviving mice / total number of mice in each group × 100%. At the same time, according to the clinical symptoms of mice recorded every day, the time and degree of symptom improvement of mice in different treatment groups were analyzed, and the differences in treatment effects of different treatment groups were analyzed.

[0095] 8. Results The survival rate of mice in the antibiotic treatment group was 100%; the survival rate of mice in the IgY treatment group was 90%; and the survival rate of mice in the control group was 0%. In terms of improvement of clinical symptoms, the symptoms of mice in the antibiotic treatment group and the IgY treatment group gradually alleviated with the treatment time. Antibiotic treatment had a more obvious improvement on the clinical symptoms of mice in the early stage, and IgY treatment played an important role in the immune regulation and persistent recovery of mice in the later stage.

[0096] Example 3 On the basis of Example 1, this Example 3 provides a specific anti-bovine Streptococcus IgY antibody biological disinfectant, and performs an effect test on the biological disinfectant, and the specific steps are as follows: 1. Experimental Materials and Reagents In the previous experiments, mice infected with bovine streptomycin used some bedding (mixed with mouse feces).

[0097] Sterile saline, sterile water.

[0098] Specific anti-bovine Streptococcus IgY.

[0099] Antibiotic: Kanamycin.

[0100] 2. Preparation of Disinfectant and Antibiotic Solutions Specific anti-bovine Streptococcus IgY biological disinfectant: Accurately measure an appropriate amount of specific anti-bovine Streptococcus IgY, mix it with sterile tap water, gently stir evenly, and prepare a 1 mg / mL IgY disinfectant, which is ready for use.

[0101] Antibiotic solution: Weigh an appropriate amount of kanamycin, dissolve it in sterile water to prepare a 15 μg / mL solution, and store it at 4°C for later use.

[0102] 3. Group processing Experimental group (IgY group): Place 5g of litter sample (containing 2g of feces) in a sterile container and use a spray bottle to evenly spray 2ml of 10% IgY disinfectant on the litter in a cross-spraying manner.

[0103] Normal saline control group (Group C): Place 5 g of litter sample (containing 2 g of feces) in a sterile container and use a spray bottle to evenly spray 2 ml of normal saline on the litter in a cross-spraying manner.

[0104] Antibiotic control group (kan group): Place 5 g of litter sample (containing 2 g of feces) in a sterile container. Use a spray bottle to evenly spray 2 ml of antibiotic solution on the litter in a cross-spraying manner.

[0105] 4. Preparation of bacterial suspension and sample inoculation After spraying, place the litter of the experimental group and the control group in a 37°C incubator for 30 minutes. After standing, take the treated litter sample in a sterile centrifuge tube. Add 5 mL of LB liquid culture medium to the centrifuge tube and place it in an incubator at 37°C, 200 rpm, for 1 hour to fully release the bacteria in the litter into the culture medium, and then make a gradient dilution bacterial suspension. Take the appropriate concentration of bacterial suspension and spread it on the cattle chain selection medium plate.

[0106] 5. Culture and Sample Collection The inoculated plate was placed upside down in an incubator and cultured at 37°C for 36 hours.

[0107] 6. Experimental Results Experimental group (IgY group): In the same bacterial solution dilution gradient, the number of S. bovis colonies in the experimental group on the S. bovis selective medium plate was less than that in the saline control group.

[0108] Antibiotic control group (kan group): In the same bacterial solution dilution gradient, the number of bovine Streptococcus colonies on the antibiotic control group plate was also significantly less than that of the control group.

[0109] Normal saline control group (Group C): In the same bacterial solution dilution gradient, the number of bovine Streptococcus colonies on the plates of the normal saline control group was significantly higher than that of the experimental group and the antibiotic control group.

[0110] Colony plate map and data diagram Figure 9-10 As shown, Fig. 9 This is a graph showing the growth of Streptococcus bovis in mouse bedding on the Streptococcus bovis selective medium; Fig.10 The figure shows the colony count of Streptococcus bovis in mouse bedding on the Streptococcus bovis selective medium. Note: Group C is the saline control group, the IgY group is the experimental group, and the kan group is the antibiotic control group.

[0111] Example 4 On the basis of Example 1, this Example 4 provides a specific anti-bovine Streptococcus IgY antibody feed, and performs effect detection on the feed, and the specific steps are as follows: 1. Experimental Materials Experimental animals: 30 healthy clean-grade BALB / c mice of similar weight (16 ± 2 g) were selected; Experimental strains: S. bovis isolates, maintained in the laboratory and regularly passaged and identified; Standard mouse basic feed, meeting the normal growth nutritional needs of mice.

[0112] Antibiotic: Kanamycin.

[0113] 2. Preparation of bacterial culture Pick an appropriate amount of colonies from the preserved bovine Streptococcus strains, inoculate them into LB liquid medium, and culture them at 37°C with shaking for 24 hours to ensure that the strains are fully activated and proliferated. Then dilute to 1×10 6 CFU / ml for future use.

[0114] 3. Preparation of feed additives IgY additive group: weigh 1g of specific anti-bovine Streptococcus IgY and slowly add it to 100g of feed to ensure uniform mixing. Pack the mixed 1% additive feed into sterile bags or containers, seal them, and place them in a 4℃ refrigerator for future use to prevent the reduction of IgY activity and feed deterioration.

[0115] Kanamycin-added group: Weigh 20 mg of kanamycin and slowly add it to 100 g of feed to ensure uniform mixing. Pack the mixed kanamycin feed into sterile bags or containers, seal them, and place them in a 4°C refrigerator for later use to prevent the feed from spoiling.

[0116] 4. Grouping and Adaptive Feeding of Mice Before the experiment, all mice were adaptively raised in an animal breeding environment for 7 days, and healthy mice were randomly divided into 3 groups, with at least 15 mice in each group. The grouping is as follows: Infection control group: fed with standard mouse basal diet, and infected with bovine Streptococcus after 7 days of adaptive feeding; 1% IgY additive group: fed with 10% additive feed, and infected with bovine streptococcus after 7 days of adaptive feeding; Kanamycin-added group: The group was fed with kanamycin-added feed and infected with Streptococcus bovis after 7 days of adaptive feeding; 5. Feeding and bacterial infection After 7 days of feeding, all mice were infected with Streptococcus bovis. 6 CFU / mL bovine Streptococcus liquid was administered to mice by intraperitoneal injection, with each mouse being inoculated with 0.2 ml of liquid.

[0117] 6. Sample Collection and Processing After infection, the general condition of the mice was observed every day. On the 7th day after infection, at least 5 mice were randomly selected from each group for sample collection. The mice were euthanized by cervical dislocation. The abdominal cavity was opened with sterile scissors and forceps, the intestines were removed, both ends of the intestines were ligated, a section of the intestine (about 5 cm) was cut off, placed in a sterile centrifuge tube, 1 mL of sterile saline was added, and the intestinal contents were gently ground with a tissue grinder to make a homogenate, which was then graded and diluted for subsequent bacterial counting.

[0118] 7. Bacteria Count The dilution of the intestinal content tissue homogenate was spread on the bovine chain selective medium plate with a coating volume of 100 μL. The plate was inverted and placed in a 37 ℃ incubator for 24 h, and the colony growth was observed and counted.

[0119] 8. Experimental Results Infected control group: Mice developed mild to moderate symptoms, with large amounts of Streptococcus growing in their intestines; 1% IgY additive group: The mice had no obvious symptoms or only mild symptoms, and the number of streptococci in the intestine was significantly lower than that in the infection control group, and slightly higher than that in the kanamycin addition group; Kanamycin-added group: Mice had no obvious symptoms and the number of streptococci in the intestine was the lowest.

[0120] The experimental results are as follows Figure 11-12 As shown, Fig.11 This is a graph showing the growth of Streptococcus bovis in intestinal tissue homogenate on Streptococcus bovis selective medium. Fig.12 Figure 2 shows the colony count of Streptococcus bovis in intestinal tissue homogenate on the Streptococcus bovis selective medium. Note: The kan group is the kanamycin-added group, the IgY group is the 1% IgY additive group, and the C1 and C2 groups are the saline control groups. The dilution gradient of the Streptococcus bovis in the kan group, IgY group, and C1 group is 1×10 1 The dilution gradient of C2 group bovine chain was 1×10 2 .

Claims

1. A method for preparing a specific anti-bovine Streptococcus IgY antibody, characterized in that: The following steps are involved: S1. Design and synthesis of multi-epitope antigens of Streptococcus bovis, comprising the following steps: ① Target protein selection and epitope screening: rod-shaped determinant protein (RodA) of Streptococcus bovis was used as the target protein to obtain amino acid and gene sequences, and predict and screen T and B cell dominant epitopes. The screened epitope sequences were concatenated twice, and the mucosal immune adjuvant CTB gene sequence was connected at the C-terminus to obtain the fusion protein sequence CTB-RodA-RodA (CRR); ②, sequence optimization and synthesis, introducing a BamH I restriction site at the 5′ end of the CRR sequence, an EcoR I restriction site and a protective base sequence at the 3′ end, optimizing the gene sequence according to the codon preference of poultry, and synthesizing a CRR gene fragment with a length of 1080 bp; S2. Construction of recombinant plasmid pVAX1-CRR, including the following steps: 1) Double enzyme digestion reaction: take the CRR gene fragment and the eukaryotic expression vector pVAX1, and perform double enzyme digestion reaction with BamH I and EcoR I respectively to obtain a reaction solution; 2) Purification of the enzyme digestion product: subject the reaction solution to agarose gel electrophoresis, cut out the target band, and use a gel recovery kit to recover and purify the target DNA fragment; 3) Ligation reaction: put the purified target DNA fragment into a sterile microcentrifuge tube to prepare a ligation reaction system, mix well, centrifuge and place at 16°C for ligation overnight to obtain a ligation product; 4) Transform competent cells: take E. coli DH5α competent cells, add the ligation product, mix and place in an ice bath for 30 minutes, heat shock at 42°C for 90 seconds, quickly transfer to ice and cool for 2 minutes, add to LB liquid culture medium, shake and culture at 37°C and 200 rpm for 1 hour to revive the bacterial colony, spread the revived bacterial liquid on an LB solid culture medium plate containing kanamycin, and invert and culture at 37°C overnight to obtain colonies; 5) Colony PCR identification: the colony is cultured in LB liquid medium containing kanamycin and then PCR amplified to obtain PCR products. The PCR products are detected by agarose gel electrophoresis. If a band consistent with the expected size appears, it is preliminarily judged as a positive clone; 6) Enzyme digestion identification: extract plasmids from the positive clones, perform double enzyme digestion reaction with BamH I and EcoR I to obtain enzyme digestion products, and analyze the enzyme digestion products by agarose gel electrophoresis. If a band of the expected size appears, it further indicates that the recombinant plasmid pVAX1-CRR is successfully constructed; 7) Sequencing identification: DNA sequencing was performed on the recombinant plasmid pVAX1-CRR that was correctly identified by enzyme digestion. The sequencing primers were the universal primers on the pVAX1 vector. The sequencing results were compared with the designed CRR sequence. If the sequences were completely consistent, it was confirmed that the recombinant plasmid pVAX1-CRR was correctly constructed and the recombinant plasmid pVAX1-CRR was obtained; S3. Antibody preparation, comprising the following steps:

1. Select laying hens, design an immunization program with CTB as an immune adjuvant, and immunize the laying hens with the recombinant plasmid pVAX1-CRR; II. Select the collected eggs, take out the egg yolk liquid and freeze it for later use; III. extracting water-soluble components from the egg yolk to obtain a water-soluble component (WSF) containing IgY; IV, separating and purifying the water-soluble components to obtain an IgY precipitate; V. Dialyzing the IgY precipitate to obtain specific anti-bovine Streptococcus IgY antibodies.

2. The method for preparing a specific anti-bovine Streptococcus IgY antibody according to claim 1, characterized in that: In the step I, the immunization program designed with CTB as an immune adjuvant is specifically as follows: 600 μg of CTB is injected before immunization; the first immunization is performed at 7-9 days, and 150 μl of bovine Streptococcus (sp) is injected; the second immunization is performed at 14-21 days, and 150 μl of bovine Streptococcus (sp) is injected; the third immunization is performed at 28-52 days, and 150 μl of bovine Streptococcus (sp) is injected; the fourth immunization is performed at 62-72 days, and 150 μl of bovine Streptococcus (sp) is injected.

3. The method for preparing a specific anti-bovine Streptococcus IgY antibody according to claim 1, characterized in that: In the step I, immunization is performed by multiple injections at the subcutaneous part of the wing base and the chest muscle.

4. The method for preparing a specific anti-bovine Streptococcus IgY antibody according to claim 1, characterized in that: The specific method in step II is: select the collected eggs, soak them in 0.1% concentration of Chlorhexidine solution for 30 minutes, take them out and dry them, use an egg yolk separator to take out the egg yolk, pierce the yolk membrane of the egg yolk to take out the egg yolk liquid, put the egg yolk liquid into a -80°C refrigerator and freeze it for 2 hours, transfer it to a 4°C refrigerator and refrigerate it overnight to allow it to return to temperature.

5. The method for preparing a specific anti-bovine Streptococcus IgY antibody according to claim 4, characterized in that: The specific method of step III is: placing the egg yolk liquid into ultrapure water with a pH of 5.2 and mixing evenly to obtain an egg yolk suspension; placing the egg yolk suspension in a 4°C refrigerator and letting it stand for 10 hours, placing it in a centrifuge, setting the speed to 10,000 rpm and the temperature to 4°C for centrifugation for 25 minutes, and the supernatant after centrifugation is the water-soluble component (WSF) containing IgY.

6. The method for preparing a specific anti-bovine Streptococcus IgY antibody according to claim 5, characterized in that: The specific method of step IV is: placing the water-soluble component in a centrifuge tube, adding a saturated ammonium sulfate solution, stirring so that the final saturation of ammonium sulfate is 50%, placing it in a 4°C refrigerator and letting it stand for 3 hours, then placing it in a centrifuge, setting the speed to 3000 rpm, centrifuging at 4°C for 20 minutes, discarding the supernatant, adding ultrapure water and a saturated ammonium sulfate solution, stirring so that the final saturation of ammonium sulfate is 33%, placing it in a 4°C refrigerator and letting it stand for 3 hours, placing it in a centrifuge, setting the speed to 3000 rpm, centrifuging at 4°C for 20 minutes, discarding the supernatant, and obtaining an IgY precipitate.

7. The method for preparing a specific anti-bovine Streptococcus IgY antibody according to claim 6, characterized in that: The specific method of step V is: putting the IgY precipitate into a dialysis bag for dialysis, using Naphthol reagent to detect whether the dialysis external fluid is yellow, and using a 1% concentration of BaCl2 solution to detect whether the dialysis external fluid has precipitation. When the dialysis external fluid is not yellow and has no precipitation, it indicates that the dialysis is completed and specific anti-bovine Streptococcus IgY antibodies are obtained.

8. A specific anti-bovine Streptococcus IgY antibody specific bactericidal liquid, characterized in that: The raw material of the specific bactericidal liquid includes the specific anti-bovine Streptococcus IgY antibody prepared by the preparation method described in claim 1.

9. A specific anti-bovine Streptococcus IgY antibody biological disinfectant, characterized in that: The raw material of the biological disinfectant includes the specific anti-bovine Streptococcus IgY antibody prepared by the preparation method described in claim 1.

10. A specific anti-bovine Streptococcus IgY antibody feed, characterized in that: The raw material of the feed includes the specific anti-bovine Streptococcus IgY antibody prepared by the preparation method of claim 1.

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