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Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof

A technology of egg yolk antibody and egg yolk powder, applied in chemical instruments and methods, animal feed, animal husbandry, etc., can solve the problems of high price, negative effects on livestock and poultry growth, and not approved for use, and can improve feed intake, The effect of inhibiting urease activity and reducing loss

Active Publication Date: 2010-06-16
胡文锋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These inhibitors are either expensive, or have negative effects on the growth of livestock and poultry itself, or are not approved for livestock and poultry farming, etc., so it is necessary to develop a safe and new urease inhibitor

Method used

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  • Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof
  • Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof
  • Yolk powder containing double-titer yolk antibody of anti-porcine CCK/Urease, and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The expression of embodiment 1CCK39 / UreB fusion protein and the preparation of vaccine

[0023] (1) Template preparation

[0024] (1) Preparation of CCK39 cDNA: Collect fresh porcine duodenum, extract total RNA from porcine duodenum according to the RNA extraction kit, and after testing to confirm its integrity, reverse transcribe the total RNA according to the requirements of the cDNA synthesis kit The corresponding cDNA was stored at -20°C for future use.

[0025] (2) Preparation of urease-producing coliform plasmid DNA: isolate and cultivate a single colony of urease-producing coliform from fresh pig manure, expand the culture overnight, then collect the fresh culture, and extract the coliform plasmid according to the plasmid recovery kit , dissolved in TE buffer and stored at -20°C for later use

[0026] (2) Acquisition of CCK39 gene

[0027] According to the sequence published on GenBank [gi:47523555], use Primer Premier5.0 to design primers to design a pair of ...

Embodiment 3

[0050] The processing of embodiment 3 high free eggs

[0051] After the first immunization, the eggs were collected and numbered daily, and stored at 4°C for later use. When taking the egg yolk, crack the egg shell first, separate the egg yolk and the egg white, rinse the surface of the egg yolk repeatedly with sterilized distilled water, remove the egg white as much as possible, pierce the egg yolk membrane with a needle, suck out the egg yolk, measure the volume of the egg yolk, add 9 times the sterilized distilled water to dilute, and use Adjust the pH value to 5.0-5.5 with HCl, let the diluted solution stand at 4°C for more than 6 hours, absorb the supernatant after obvious stratification occurs; centrifuge at 4°C, 12000r / min for 30min, take the supernatant, measure the volume, add 0.01 % mercury sulfide, in small aliquots, stored at -20°C.

Embodiment 4

[0052] Embodiment 4 Indirect ELISA detection

[0053] 1. Selection of enzyme-labeled antibody working concentration determination

[0054] Chicken anti-CCK39 / UreB positive IgY was prepared as 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 with 0.05moL / L pH9.6 carbonate buffer solution , 1:1280 dilution, coated with 1-8 columns of the enzyme-labeled plate, 100 μL / well, kept at 37°C for 1 hour, then transferred to 4°C overnight; after washing and blocking, the enzyme-labeled antibody was made 1:200 with pH7.4 PBS , 1:400, 1:800, 1:1000 diluted, added to the 1st-4th line of the microplate respectively, after 37 ° C water bath for 1 hour, detected by indirect ELISA method, and measured the OD of each well 490 value, when OD 490 The enzyme-labeled antibody concentration corresponding to the value of about 1.0 and the chicken anti-CCK39 / UreB positive IgY dilution is the largest, as the optimal working concentration of the enzyme-labeled antibody. The optimal enzyme-labeled antibody ...

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Abstract

The invention discloses yolk powder containing double-titer yolk antibody (Immunoglobulin yolk, IgY) of cholecystokinin / intestinal bacterial urease. A preparation method of the yolk powder comprises the following steps: 1) preparing vaccine by mixing purified cholecystokinin 39 peptide (CCK39) / urease B subunit (UreB) fusion protein with Freund's adjuvant well, immunizing healthy laying hens, determining yolk IgY titer through enzyme-linked immunosorbent assay and collecting hyperimmune eggs; and 2) separating yolk from hyperimmune eggs and preparing the yolk into yolk powder. The yolk powder containing double-titer IgY, which is prepared by the method, is added to feed for feeding pigs, and the satiety of the pigs is not produced or delayed due to CCK in the IgY and secreted by porcine intestinal canals so as to increase feed intake. The IgY can also neutralize urease secreted by intestinal bacteria so as to reduce the production of ammonia, protect porcine intestinal canals and respiratory tract, reduce the waste of feed protein and improve culture environment.

Description

technical field [0001] The invention relates to the field of biological preparations, in particular to a yolk powder containing a bivalent yolk antibody (Immunoglublin yolk, IgY) against porcine cholecystokinin (CCK) / intestinal bacteria urease (Urease) and a preparation method thereof. Background technique [0002] How to increase the feed intake of livestock and poultry, improve the breeding environment, and reduce the impact of ammonia on the environment has always been an urgent problem to be solved. At present, there are several common ways to increase the feed intake of livestock and poultry: (1) select high-quality and easily digestible raw materials; (2) determine the appropriate nutritional level of the diet; (3) feed during lactation; (4) feed Liquid feed and fermented feed; (5) Use food-attracting regulators; (6) Improve livestock and poultry breeding environment, etc. Using modern bioengineering technology and products to improve animal feed intake is a current r...

Claims

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Application Information

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IPC IPC(8): A23K1/16C07K16/44C07K16/02
Inventor 胡文锋张炜阳李岩吴同山胡斌
Owner 胡文锋
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