Anti-helicobacter pylori egg yolk antibody as well as preparation method and application thereof

A technology of Helicobacter pylori and egg yolk antibody, which is applied in the field of biomedicine, can solve the problems of weak activity, insufficient purity, and low abundance of ingredients, and achieve the effect of strong neutralization of Helicobacter pylori activity and high purity

Inactive Publication Date: 2019-03-22
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The problems and shortcomings of the prior art are mainly that the anti-Helicobacter pylori yolk antibody prepared by it has weak neutralizing activity against Hp, thus reducing the effect of preventing and treating gastrointestinal diseases caused by Hp infection
The main reason for this problem is mainly reflected in two aspects. On the one hand, the specific pathogenic components of Helicobacter pylori in the immunogen used for immunization of laying hens are weak or have low abundance, which leads to the production of anti-Hp yolk antibodies. The resultant force is small. On the other hand, the purity of the egg yolk antibody is not enough when extracting and purifying the egg yolk antibody from eggs, and the anti-Helicobacter pylori egg yolk antibody used to prevent and treat Hp infection requires a higher purity

Method used

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  • Anti-helicobacter pylori egg yolk antibody as well as preparation method and application thereof
  • Anti-helicobacter pylori egg yolk antibody as well as preparation method and application thereof
  • Anti-helicobacter pylori egg yolk antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Immune laying hen and antiserum titer determination

[0044] Select 3 healthy laying hens that are laying eggs and raise them separately, numbering #1, #2, #3 respectively. For the first immunization, the immunogen was fully mixed with an equal volume of Freund's complete adjuvant, and the phacoemulsification was complete. Two subcutaneous points on the back of the neck and two points on the pectoralis major muscle were used for injection. ml. The second immunization was carried out at an interval of 30 days. The difference between this immunization and the first immunization was that Freund's complete adjuvant was changed to Freund's incomplete adjuvant, and the immunization dose and injection volume were the same as the first immunization. The third immunization was carried out at an interval of 30 days after the second immunization. This immunization did not use adjuvants and was injected through the tail vein of chicken wings. The immunization dose was 50...

Embodiment 2

[0048] Example 2 Extraction and purification of anti-Hp yolk antibody

[0049] combined with figure 1 The yolk antibody preparation flow chart and the immunized eggs in Example 1 were collected, and the antibody was further extracted and purified by water dilution method, sodium chloride salting-out method and thiophile affinity chromatography.

[0050] Preparations: All experimental equipment (2 250ml beakers, 2 250ml glass reagent bottles, 1 250ml measuring cylinder, 1 egg yolk separator, 1 glass rod, 2 filter papers, 1 magnetic stirrer) and reagents (super Pure water, PBS) were sterilized by high temperature and high pressure one day in advance, and set aside.

[0051] Water dilution method to extract yolk antibody: wash the surface of the egg with water, soak it in bromogeramine for 1-3 minutes, take it out and dry it with gauze, then wipe it with 75% alcohol, and let it dry naturally, try to ensure that it is under sterile conditions Remove the egg whites. Use egg yolk...

Embodiment 3

[0055] Gained anti-Hp yolk antibody concentration determination and purity detection of embodiment 2

[0056] After the anti-Hp yolk antibody was purified, its A280 and A260 were detected with an ultra-micro spectrophotometer, and the PBS blank was used for detection. When A280 / A260<1.5, use the Lowry-Kalokar formula: sample protein content (mg / ml) = 1.45A280-0.74A260, calculate the yolk antibody concentration, generally one egg can extract and purify more than 80mg of yolk antibody according to the method in Example 2 .

[0057] SDS-PAGE and Coomassie Brilliant Blue staining were used to detect the purity of the above-mentioned purified antibodies, and the results were as follows image 3 As shown, where M is the protein molecular weight standard, lanes 1 and 2 are the denatured and reduced yolk antibodies extracted and purified from eggs laid by #3 laying hen at two time points of 2018 / 8 / 18 and 2018 / 8 / 11 respectively Electrophoresis bands, heavy chain about 70KD, light cha...

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Abstract

The invention discloses an anti-helicobacter pylori egg yolk antibody, which is prepared by immunizing an egg laying hen by immunizing an egg laying hen by injecting a mixture rich in three proteins,i.e., Ure, VacA and CagA separated from Hp lysate, wherein the mixture is used as an immunogen, and then extracting the purified antibody from egg yolk of the immunized hen by using a water dilution method, a sodium chloride salting-out method and a sulfurophilic affinity chromatography method. The egg yolk antibody prepared by the method is high in purity, has a stronger activity of neutralizinghelicobacter pylori, and can be applied to preparation of medicines for preventing and treating gastrointestinal diseases caused by infection of the helicobacter pylori.

Description

technical field [0001] The invention relates to biomedical technology, in particular to a specific anti-helicobacter pylori egg yolk antibody and its preparation method and application. Background technique [0002] Helicobacter pylori (Hp) is a helical, slightly anaerobic, Gram-negative bacillus that requires very harsh growth conditions. In 1983, it was successfully isolated from the gastric mucosal biopsy tissue of patients with chronic active gastritis for the first time. It is the only microbial species known to be able to survive in the human stomach. Helicobacter pylori disease includes gastritis, peptic ulcer, and lymphoproliferative gastric lymphoma caused by Hp infection. The poor prognosis of Helicobacter pylori disease is gastric cancer. Hp parasitizes in gastric mucosal tissue, 67%-80% of gastric ulcers and 95% of duodenal ulcers are caused by Hp. [0003] Hp is infected in people of different races and regions in the world, and it can be said to be the most ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/02C07K16/12C07K1/36C07K1/22C07K1/30A61K39/395A61P31/04A61P1/00
CPCA61K2039/505A61P1/00A61P31/04C07K1/22C07K1/30C07K1/36C07K16/02C07K16/121
Inventor 黄子祥郭志刚
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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