Anti-paenibacillus larvae yolk immunoglobulin and preparation method and use thereof

A technology of honeybee larvae and bacillus, which is applied in the preparation methods of peptides, antibodies, antibacterial drugs, etc., and can solve problems such as applications that have not been reported.

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-paenibacillus larvae yolk immunoglobulin and preparation method and use thereof
  • Anti-paenibacillus larvae yolk immunoglobulin and preparation method and use thereof
  • Anti-paenibacillus larvae yolk immunoglobulin and preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Preparation of anti-bee larva bacillus yolk antibody

[0037] 1.1 Cultivate and inactivate Bacillus in bee larvae to prepare antigen

[0038] Inoculate honeybee larvae Bacillus on nutrient agar medium, culture upside down at 37°C for 12 hours, pick a single colony and inoculate it in nutrient broth medium, shake the shaker at 37°C and 180rpm for 8 hours, adjust the nutrient broth The amount of bee larva bacillus in the culture medium is 2×10 8 CFU / ml, after centrifuging to remove the supernatant, mix it with normal saline to obtain the amount of Bacillus in bee larvae, which is 2×10 8 CFU / ml bacterial suspension, centrifuged, replaced with normal saline with the same volume of 0.4% formaldehyde, and then inactivated at 4°C for 48 hours to obtain the inactivated Bacillus antigen of honeybee larvae.

[0039] 1.2 Immune laying hens and collect eggs

[0040] The laying hens were immunized for the first time and 2 to 3 times for booster immunization.

[0041] T...

Embodiment 2

[0057] Example 2 Identification of anti-bee larval Bacillus yolk antibody

[0058] 2.1 Concentration determination and purity determination

[0059] After the egg yolk antibody freeze-dried powder was dissolved in PBS, the concentration was measured with a BSA kit. The measured protein concentration was 13.426mg / ml, and the total protein amount was about 180mg, which was within the normal range. 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.

[0060] The result is as figure 1 As shown, the heavy chain and light chain are located at 66KD and 28KD respectively, and the total molecular weight is about 180KD.

[0061] 2.2 Indirect enzyme-linked immunosorbent assay (ELISA) detection titer

[0062] Take 2×10 8 CFU / ml inactivated Bacillus larvae of honey bee larvae antigen-coated ELISA plate, 100 μL per well, coated overnight at 4°C, washed 5 tim...

Embodiment 3

[0067] Embodiment 3 in vitro antibacterial experiment

[0068] Inactivate the bee larvae Bacillus and dilute to 1×10 3 CFU / ml, take 150 μL anti-bee larvae yolk antibody and common yolk antibody solution respectively, add the same amount of bacterial solution and mix them, and then combine at 37°C for 20 minutes, take 100 μL to coat the nutrient agar plate, set 2 replicates for each, and invert at 37°C After 24 hours of cultivation, the cells were photographed and counted.

[0069] Antibacterial experiments showed that the group added with yolk antibody could significantly inhibit the growth of bacillus in bee larvae, and the amount of bacteria was 1 / 5-2 / 5 of that of the group with ordinary yolk antibody.

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PUM

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Abstract

The invention belongs to the field of biological pharmacy and relates to anti-paenibacillus larvae yolk immunoglobulin and a preparation method and use thereof. The anti-paenibacillus larvae yolk immunoglobulin is obtained by taking inactivated paenibacillus larvae as an antigen to immunize laying hens, collecting eggs of the immunized laying hens, and separating and purifying egg yolk of the immunized hens. The high-purity anti-paenibacillus larvae yolk immunoglobulin obtained through separation and purification has high activity of neutralizing paenibacillus larvae and can be applied to prevent and treat bee diseases caused by paenibacillus larvae infection.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to an anti-bee larva bacillus yolk antibody and its preparation method and application. Background technique [0002] Paenibacillus larvae is a Gram-positive bee pathogen that causes American larval rot (AFB). AFB is an acute, high-mortality infectious disease that can cause bee larvae and bee pupae. It only occurs in bee larvae. Once the young bees are infected, if there is no proper treatment, the infected bees will die soon. And it will spread to the entire bee colony, causing the death of the entire bee colony, and even infecting other bee colonies that have not been in contact. The pathogenicity of Bacillus mellifera to 1-day-old larvae of Apis mellifera is very high, and the pathogenicity to 2-day-old larvae is greatly reduced, and the larvae and adult bees above 3-day-old are not infected. Oriental honeybees are resistant to this disease. Bacillus larvae are u...

Claims

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Application Information

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IPC IPC(8): C07K16/12C07K16/02C07K1/30A61K39/40A61P31/04
CPCC07K16/1267C07K16/02A61P31/04C07K2317/11A61K2039/505
Inventor 吕凤林杨军陈彩宇曹政潘玉竹
Owner 重庆君同生物技术有限公司
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