Preparation method of egg yolk antibody containing helicobacter pylori resisting IgY and application

A technology of Helicobacter pylori and egg yolk antibody, which is applied in the direction of antibodies, antibacterial drugs, antibacterial immunoglobulin, etc., can solve the problems of easy drug resistance, difficult acceptance by patients, and high cost of combination therapy, and achieve significant curative effect

Inactive Publication Date: 2017-05-10
徐州安替生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The current reliable anti-H. pylori treatment is mainly combination therapy, but due to the high cost of combination therapy and easy drug resistance, it is difficult for patients to accept it.

Method used

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  • Preparation method of egg yolk antibody containing helicobacter pylori resisting IgY and application
  • Preparation method of egg yolk antibody containing helicobacter pylori resisting IgY and application
  • Preparation method of egg yolk antibody containing helicobacter pylori resisting IgY and application

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Embodiment 1

[0032] Embodiment 1: Eggs containing yolk antibodies against Helicobacter pylori IgY are eggs produced by immunization with CagA gene-positive virulent Helicobacter pylori; preparation method of eggs containing yolk antibodies against Helicobacter pylori IgY:

[0033] 1) Including the cultivation of virulent Helicobacter pylori positive for the CagA gene: After thawing in a 37-degree water bath, the virulent Helicobacter pylori strain was inoculated on a sheep blood agar plate, and the plate was cultured in a 37-degree microaerobic environment for 48 hours. Helicobacter pylori was harvested from the plate and inoculated with brain heart infusion medium for expansion culture. Helicobacter pylori was harvested by centrifugation for 48-72 hours.

[0034] 2) Immune laying hens: 40-week-old healthy laying hens were selected and raised in isolation, and subcutaneously injected, and the first injection was immunized with 1.0x10 9 cfu / only; immunized once a week, and collected eggs a...

Embodiment 2

[0035] Embodiment 2: the preparation method of the yolk antibody containing anti-Helicobacter pylori IgY, specifically comprises the following steps:

[0036] (1) After washing and disinfecting eggs containing yolk antibodies against Helicobacter pylori IgY, break them, and carefully separate the yolks with an egg yolk separator to ensure that the yolk membrane is not broken;

[0037] (2) Roll the egg yolk several times on ordinary filter paper, absorb the egg white as much as possible, and collect the egg yolk by breaking the membrane;

[0038] (3) Dilute the egg yolk with 10mM PBS of 2 times the volume of the egg yolk, pH7.4, and stir at room temperature; precipitate the diluted egg yolk with a final concentration of 3.5% PEG6000. The centrifugation conditions are: speed 10000rpm / min, temperature 4°C, for 10 min; after centrifugation, collect the supernatant, and filter through ordinary filter paper to remove insoluble matter;

[0039](4) Add final concentration of 8.5% PEG...

Embodiment 3

[0044] Embodiment 3: Determination of titer of immunized egg and egg yolk antibody: adopt indirect method ELISA to measure the titer of serum and egg yolk antibody; Concrete steps include:

[0045] S1: Ultrasonic disruption of Helicobacter pylori, quantification of protein concentration by Bradford method;

[0046] S2: Dilute bacterial protein with pH9.6 carbonic acid buffer solution, coat the microtiter plate at 100ug / well, overnight at 4°C; shake off the coating solution, soak and rinse 3 times with PBS containing 0.05% Tween 20 for 60s each time ;

[0047] S3: Block with 1% bovine serum albumin at 37°C for 60 minutes, wash 2 to 5 times; add double-diluted serum or egg yolk, incubate at 37°C for 2 hours, wash the microtiter plate 5 times;

[0048] S4: Add 1:10000 diluted enzyme-labeled secondary antibody, react at 37°C for 30-60 minutes, wash the strips 4-6 times; TMB color development, room temperature for 15 minutes, 2M sulfuric acid to stop the color reaction, and read t...

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Abstract

The invention discloses a preparation method of an egg yolk antibody containing helicobacter pylori resisting IgY and an application of the egg yolk antibody. By the aid of a virulent helicobacter pylori immune laying hen with positive CagA genes, the egg yolk antibody is extracted from eggs laid by the laying hen and/or egg yolk powder is prepared. The egg yolk powder with the egg yolk antibody is added into food or prepared into health care food and can be used for preventing related diseases caused by infection of helicobacter pylori in an assisting manner, and the antibody can be prepared into medicines after being purified and is used for preventing the related diseases caused by infection of the helicobacter pylori.

Description

technical field [0001] The invention relates to the technical field of medical antibodies, in particular to a preparation method and application of an egg yolk antibody containing anti-helicobacter pylori IgY. Background technique [0002] Helicobacter pylori (Helicobacter Pylori, hereinafter referred to as HP) is one of the most common pathogens in the world. Helicobacter pylori is infectious. It has been listed as the first carcinogen of gastric cancer by the World Health Organization (WHO). Helicobacter pylori can invade gastric and duodenal mucosal epithelial cells through the oral cavity and cause gastrointestinal lesions. Not all H. pylori infection will develop into gastrointestinal lesions. Clinical research results show that Helicobacter pylori strains can be divided into two types, one is the virulent Helicobacter pylori containing the CagA gene related to cytotoxin expression, (type I Helicobacter pylori, also known as cytotoxin-producing Helicobacter pylori ), a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12C07K16/02A23L33/17A23L15/00A61K39/40A61P31/04
Inventor 石韦安益强高青
Owner 徐州安替生物科技有限公司
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