Anti-Hafnia alvei egg yolk antibody and preparation method and application thereof

A technology of Hafnia haffonia and egg yolk antibody, which is applied in the preparation method of peptides, antibodies, antibacterial drugs, etc., can solve the problems that have not been reported in the application, and achieve the effect of high purity, high potency, and good specificity

Pending Publication Date: 2021-04-09
重庆君同生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, yolk antibody has been widely concerned and applied in animal husbandry, but its application in the treatment of bee-related diseases has not been reported

Method used

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  • Anti-Hafnia alvei egg yolk antibody and preparation method and application thereof
  • Anti-Hafnia alvei egg yolk antibody and preparation method and application thereof
  • Anti-Hafnia alvei egg yolk antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Preparation of Anti-Hafnia alvei Egg Yolk Antibody

[0034] 1.1 Cultivate Hafnia alvei and inactivate it to prepare antigen

[0035] Inoculate Hafnia alvei on the nutrient agar solid medium, and after inverting at 37°C for 12 hours, pick a single colony and inoculate it in the nutrient broth medium, shake the shaker at 37°C and 180rpm for 12 hours, then adjust the nutrition The amount of Hafnia alvei in the broth medium was 4×10 10 CFU / ml, and then centrifuged to obtain the Hafnia alvei precipitate. After the precipitate was resuspended and washed twice with normal saline, the amount of Hafnia alvei was 4×10 10 CFU / ml bacterial suspension. Inactivate in a constant temperature water bath at 70°C for 1.5 hours to obtain the inactivated Hafnia alveoli antigen, and store it in a 4°C refrigerator for later use.

[0036] 1.2 Immune laying hens and collect eggs

[0037] The laying hens were immunized for the first time and 2 to 3 times for booster immunization. ...

Embodiment 2

[0056] Example 2 Titer Detection of Egg Yolk Antibody

[0057] The titer was detected by indirect enzyme-linked immunosorbent assay (ELISA), with 2×10 8 CFU / ml Hafnia alvei-coated ELISA plate, 100 μL per well, coated overnight at 4°C, washed 5 times with PBST, then added blocking solution containing 1% BSA, blocked at 37°C for 4 hours. After washing 5 times, add the anti-Hafnia alvei egg yolk antibody in two-fold dilution, set blank control and negative control at the same time, and incubate at 37°C for 1h. After washing 5 times, horseradish peroxidase-labeled goat anti-chicken secondary antibody was added and incubated at 37°C for 1 h. After washing 5 times, TMB color developing solution was added, and the color was developed at 37°C in the dark for 10 min. Then 2M H 2 SO 4 After the reaction was terminated, the OD value at 450 nm was read with a microplate reader.

[0058] If the ratio of the absorbance value of the test sample to the negative control is greater than 2....

Embodiment 3

[0060] Embodiment 3 identifies egg yolk antibody concentration and purity

[0061] Take 10 mL of the purified protein solution, dilute it 10 times, and measure the concentration with the BCA protein concentration assay kit. The measured protein concentration is 9.862 mg / mL. Take 15 μL of the protein solution and mix it with an equal amount of 2×loading Buffer, then bathe in boiling water for 10 minutes, and perform 12% reducing SDS-PAGE electrophoresis.

[0062] The result is as figure 1 As shown, the heavy chain and light chain of the antibody IgY are located at 65KD and 30KD respectively, and the total molecular weight is about 180KD.

[0063] IgY antibody was separated and purified from egg yolk extract with YMC-BioPro Q75 anion exchange packing material and YMC-Pack Diol-200 size exclusion chromatography column. Specifically: first use YMC-BioPro Q75 anion exchange packing to separate and enrich; then use YMC-Pack Diol-200 size exclusion chromatography column to separate...

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Abstract

The invention belongs to the field of biological pharmacy, and relates to an anti-Hafnia alvei egg yolk antibody and a preparation method and application thereof. The anti-Hafnia alvei egg yolk antibody is obtained by immunizing laying hens by using inactivated Hafnia alvei as an antigen, collecting eggs of the immunized laying hens, and separating and purifying the eggs from egg yolks of the immunized laying hens. The anti-Hafnia alvei egg yolk antibody has a remarkable prevention effect and a remarkable treatment effect on Chinese bees infected with Hafnia alvei; and the purified antibody can be prepared into a medicine for treating related diseases caused by the bees infected with Hafnia alvei.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an anti-Hafnia alvei egg yolk antibody and its preparation method and application. Background technique [0002] Hafnia alvei is a Gram-negative rod-shaped pathogen closely related to food spoilage, and it is the dominant spoilage strain in food spoilage such as refrigerated meat products, dairy products and aquatic products. Hive hafniasis is an infectious disease that seriously threatens bee populations, and has been proven to cause bee sepsis. It is often found in bee colonies. It has the characteristics of fast transmission, serious damage, long duration of disease, and contamination of bee products. Features. Existing technology isolates Hafnia alveolus from the digestive tract and tissues of bees, and inoculates the chest of bees with Hafnia alveolus through experiments, and infects the bees. It is found that this bacteria causes bee sepsis and mortality More...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/02C07K1/36C07K1/34C07K1/30C07K1/18C07K1/16A61K39/40A61P31/04G01N33/569
CPCC07K16/1228C07K16/02A61P31/04G01N33/56911C07K2317/23A61K2039/505G01N2333/275
Inventor 吕凤林潘玉竹曹政杨军陈彩宇
Owner 重庆君同生物技术有限公司
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