Preparation method and application of micro-capsule coated egg yolk antibody IgY resistant to main pathogenic bacteria of dairy cow mastitis
A technology of dairy cow mastitis and yolk antibody, which is applied in the field of veterinary biological product preparation, can solve the problems of intestinal flora imbalance, unsatisfactory effect, and poor timeliness of traditional Chinese medicine treatment, and achieve high immunoglobulin activity and simple and practical technical solutions , Precipitate protein effect is good
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Embodiment 1
[0045] Preparation of the protein antigen complex of the main pathogenic bacteria:
[0046] (1) Select pathogenic Streptococcus agalactiae (S. agalactiae, CVCC55901, bovine source), Streptococcus dysgalactiae (S. dysgalactiae, CVCC55935 bovine source), Staphylococcus aureus (S. aurrus, CVCC) with specific antigens 8029 bovine origin) and Escherichia coli (E.coli, O157:K88, CVCC1489 bovine origin). The culture of the main pathogenic bacteria is preferably subcultured on blood agar slant medium, cultured at 37 °C for 24 hours, and there is no miscellaneous bacteria under the staining microscope to obtain pure species. The four main pathogenic bacteria were inoculated in broth medium (containing 5-10% calf serum) for expansion culture, placed in a constant temperature shaking incubator (150-200 r / min) at 37 ℃, and cultivated for 24 h , and the bacterial cells were collected by centrifugation.
[0047] (2) Preparation of concentrated protein antigen from single bacteria: Rinse t...
Embodiment 2
[0050] Preparation of multivalent bacterial protein antigen immunizer and animal immunization scheme:
[0051] (1) Selection of immune adjuvant: the preferred immune adjuvant is Freund's adjuvant, Freund's complete adjuvant (FCA) is used for the initial immunization, and Freund's incomplete adjuvant (FIA) is used for booster immunization (second and third immunizations). ), both have different efficacy in inducing neutralizing antibody in hen serum and egg yolk.
[0052] (2) Preparation of antigen immune material: for the first immunization, mix the prepared 10 mL complex bacterial protein antigen with an equal amount of Freund's complete adjuvant (FCA), use the rapid emulsification method, and use an ultrasonic pulverizer repeatedly under ice bath conditions Emulsify 3-4 times (10-15 s for each emulsification, place in a 4°C refrigerator with an interval of 1 min), until the antigen and adjuvant mixture is completely emulsified. After emulsification is complete, refrigerate ...
Embodiment 3
[0055] Isolation and purification of anti-DP-BM-IgY:
[0056]Anti-DP-BM-IgY was separated and purified by water dilution method-freezing method-polyethylene glycol (PEG-6000) precipitation method, and 4% and 12% PEG-6000 were used for purification to further remove egg yolk phospholipids and protein precipitation , the degreasing and protein precipitation effects are good, the average yield of the prepared anti-DP-BM-IgY can reach 50-60 mg / egg, and the purity of the antibody can reach more than 90%. Specifically, high-free eggs were sterilized with iodine tincture and alcohol cotton balls, egg yolks and egg whites were separated (as much as possible to remove albumin on the surface of clean egg yolks), yolk membranes were punctured, membrane-free egg yolk liquid was collected and volume was measured. Mix egg yolk liquid + deionized water at a ratio of 1: 6-10 (v / v, the average egg yolk liquid per egg is 15 mL), adjust the pH value to 5.0-6.0, and overnight at -20°C (about 8- ...
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