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

Inactive Publication Date: 2017-03-15
LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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AI Technical Summary

Problems solved by technology

At present, antibiotics, traditional Chinese medicine, and vaccines are mainly used for prevention and treatment of dairy cow mastitis, but there are the following main disadvantages in the way of medication and treatment methods: (1) Antibiotics are the first choice for the treatment of dairy cow mastitis, but there are single drugs with insufficient antibacterial spectrum and resistance. Drug properties, drug residue accumulation, intestinal flora imbalance
(2) The timeliness of traditional Chinese medicine treatment is poor, and the effect is not ideal, and the residual traditional Chinese medicine in milk affects the milk flavor, so it is mostly used as an auxiliary treatment for cow mastitis
(3) The most common problem in vaccine prevention and control is the variation of bacterial antigens, which makes the immune system always lag behind the bacteria when producing new antibodies, so it is difficult to prevent and treat clinically diseased groups and mixed-infected cow mastitis Effect
However, the specific egg yolk antibody IgY is easily degraded and inactivated in an extremely strong gastric acid environment, which restricts its practical application in oral preparations. In addition, the structure and function of the digestive tract of ruminants is relatively complex, and microorganisms, enzymes in the internal micro-ecological environment The types and functions of the yolk antibody are more diverse, so that if the conventional oral preparations are not processed by the preparation process, the egg yolk antibody IgY is easily degraded in the stomach, thereby weakening its biological activity or even disappearing completely

Method used

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  • Preparation method and application of micro-capsule coated egg yolk antibody IgY resistant to main pathogenic bacteria of dairy cow mastitis
  • Preparation method and application of micro-capsule coated egg yolk antibody IgY resistant to main pathogenic bacteria of dairy cow mastitis
  • Preparation method and application of micro-capsule coated egg yolk antibody IgY resistant to main pathogenic bacteria of dairy cow mastitis

Examples

Experimental program
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Effect test

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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Abstract

The invention discloses a preparation method and application of a micro-capsule coated egg yolk antibody IgY resistant to main pathogenic bacteria of dairy cow mastitis. A compound mycoprotein antigen is prepared by selecting streptococcus agalactiae, streptococcus dysgalactiae, staphylococcus aureus and escherichia coli and is mixed with an equal quantity of freund's adjuvant to prepare a multivalence antigen immune complex, laying hens are immune by adopting repeatedly alternative immune procedures and a muscular and subcutaneous multipoint injection means, a specific yolk immunoglobulin IgY resistant to the main pathogenic bacteria of dairy cow mastitis, namely DP-BM-IgY, is separated and purified from collected egg yolk, then homogeneous emulsification is conducted on the specific yolk immunoglobulin and a sodium alginate water solution and an emulsifying agent soya bean salad oil containing span-80, the emulsified solution is dropwise added into an encystation solution prepared from CaCl2 and chitosan for coating and curing, and the coated micro-capsules can effectively protect the activity of the DP-BM-IgY and have enteric solubility. The preparation method utilizes a micro-capsule technology and selects the chitosan and sodium alginate as coating materials (natural polysaccharides), and the egg yolk antibody is internally taken and free of any toxic or side effect.

Description

technical field [0001] The invention relates to the technical field of preparation of veterinary biological products, in particular to a preparation method and application of a microcapsule-coated anti-milk cow mastitis main pathogen yolk antibody IgY. Background technique [0002] Cow mastitis is an inflammatory response triggered by physical, chemical or microbial stimulation of cow mammary glands, among which the infection of various pathogenic microorganisms is the main cause of cow mastitis. At present, antibiotics, traditional Chinese medicine, and vaccines are mainly used for prevention and treatment of dairy cow mastitis, but there are the following main disadvantages in the way of medication and treatment methods: (1) Antibiotics are the first choice for the treatment of dairy cow mastitis, but there are single drugs with insufficient antibacterial spectrum and resistance. Drug properties, drug residue accumulation, and intestinal flora imbalance. (2) The timelines...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02A61K9/50A61K47/36A61K39/40A61P31/04A61P15/14
CPCA61K9/5036A61K39/40C07K16/02C07K16/1232C07K16/1271C07K16/1275
Inventor 王玲杨峰刘宇魏小娟罗永江苗小楼郭志廷郭文柱张彬罗金印
Owner LANZHOU INST OF ANIMAL SCI & VETERINARY PHARMA OF CAAS
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