Yolk antibody oral preparation for treating piglet PED (porcine epidemic diarrhea) and preparation method thereof

A technology of epidemic diarrhea and egg yolk antibody, which is applied in the field of preparation of veterinary biological products and oral preparations of egg yolk antibody, can solve the problems of no good medicines for treating piglet epidemic diarrhea, and reduce the incidence of diarrhea, reduce the incidence, Significant effect of technological progress

Inactive Publication Date: 2016-03-23
SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the above-mentioned technical problems in the prior art, the present invention provides a method for preparing an oral yolk antibody preparation for treating piglet epidemic diarrhea. The preparation method solves the technical problem that there is no good medicine for treating piglet epidemic diarrhea in the prior art

Method used

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  • Yolk antibody oral preparation for treating piglet PED (porcine epidemic diarrhea) and preparation method thereof
  • Yolk antibody oral preparation for treating piglet PED (porcine epidemic diarrhea) and preparation method thereof
  • Yolk antibody oral preparation for treating piglet PED (porcine epidemic diarrhea) and preparation method thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Expression and purification of embodiment 1PEDVCOE protein

[0022] 1. Expression of COE protein

[0023] 1) Collect the intestinal tract of suspected PEDV-infected pigs, put an appropriate amount into a mortar, and add an appropriate amount of PBS for grinding. Centrifuge the grinding liquid at 3000 rpm for 10 minutes, take 100 μl of supernatant, and use the RNA extraction kit to extract RNA. A pair of primers were designed using Primer Premier 5.0 software for the amplification of the COE gene. Upstream primer M1: 5'-CG GGATCC ACTTCTTTTGTTACTTTGCCATC-3'; downstream primer M2: 5'-CG GAATTC AACACCTTCAAGTGGTTTAGG-3'. BamHI and EcoRI restriction enzyme sites were respectively designed at the 5' end and 3' end of the cloning primer, and the amplification size was 430bp (as shown in SEQ ID NO: 3). The amino acid sequence is (as shown in SEQ ID NO: 4)

[0024] 2) After gel recovery and purification of the PCR product, digestion with restriction endonucleases, the reco...

Embodiment 2

[0030] Preparation and immunization of embodiment 2 immunogen

[0031] 1. Preparation of immunogen;

[0032] Take Freund's adjuvant and COE protein, mix according to the volume ratio of 1:1, start the magnetic stirring bar to rotate and stir until the water-in-oil form is formed. Freund's incomplete adjuvant was used for the first immunization, and Freund's complete adjuvant was used for booster immunization.

[0033] 2. Immunity;

[0034] For 5-month-old Hailan brown layer hens, the first immunization dose is 1ml / feather, and three booster immunizations are carried out at 2 weeks, 4 weeks, and 8 weeks after the first immunization, and the immunization dose is 2ml / feather.

Embodiment 3

[0035] The preparation of embodiment 3 egg yolk antibody and titer determination

[0036] 1. Preparation of Egg Yolk Antibody

[0037] Collect the eggs 7 days after the third immunization, disinfect the surface of the eggs with 75% alcohol, collect the egg yolks under sterile conditions, pipette 200 μl of egg yolks, add 1 ml of lipid solvent (0.06% k-carrageenan, 0.18% low-fat pectin) , 10 µM CaCl 2 ), shake and mix, and place at 4°C for 2h. Centrifuge at 11000g at 4°C for 20min, separate the supernatant from the precipitate, discard the precipitate, add 0.5 times the volume of 60% ammonium sulfate solution to the supernatant, and place at 4°C for 1h. Centrifuge at 11000g at 4°C for 20min, discard the supernatant, resuspend with 1 volume of PBS, add 0.5 volume of 60% ammonium sulfate solution, centrifuge at 11000g, 4°C for 20min, discard the supernatant, and wash the pellet with 1x PBS Resuspend and store at 4°C for later use.

[0038] 2. Determination of PEDV egg yolk ant...

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Abstract

The invention provides a yolk antibody oral preparation for treating piglet PED (porcine epidemic diarrhea). The yolk antibody oral preparation is characterized by containing a yolk antibody extracted from egg yolks collected after nonimmune laying hens are immunized with PEDV (porcine epidemic diarrhea virus) epidemic strain protective antigen gene COE protein. The invention further provides a preparation method of the oral preparation, comprising the steps: taking a current epidemic strain as a basis, performing prokaryotic expression and purification on PEDV protective antigen gene COE, so as to obtain target protein; preparing immunogen with freund's adjuvant, and then inoculating the nonimmune laying hens; collecting eggs after immunity, and extracting a yolk antibody from yolk; and preparing the safe and efficient yolk antibody oral preparation from the yolk antibody. The yolk antibody oral preparation disclosed by the invention can be used for effectively treating diseased piglets with PED, and meanwhile, the yolk antibody oral preparation can be used for preventing un-infected piglets from getting infected in the same group. The oral preparation is simple in preparation technique, and is suitable for bulk production and clinical use.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a veterinary biological product, in particular to a preparation method of an egg yolk antibody oral preparation for treating piglet epidemic diarrhea. Background technique [0002] Porcine epidemic diarrhea (PED) is an acute and highly contagious infectious disease of pigs caused by porcine epidemic diarrhea virus (PEDV). . Piglets infected with PEDV often die from dehydration due to acute enteritis and fatal watery diarrhea. The mortality rate of piglets within 20 days of age can reach more than 95%, which brings huge economic losses to the pig industry. [0003] Vaccination is one of the main ways to prevent the occurrence of diseases. However, in this domestic PED epidemic, this method has not effectively curbed the occurrence and prevalence of PED. As a protein with strong immune function, yolk antibody has been reported to be used in the prevention and treatment of certain inte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/42A61K45/06A61P31/14C07K16/10C07K16/02A61K35/741
CPCC07K16/10A61K35/741A61K39/42A61K45/06A61K2039/505A61K2039/542C07K2317/11A61K2300/00
Inventor 宁昆刘佩红周锦萍王建张维谊杨德全齐新永葛菲菲李鑫
Owner SHANGHAI ANIMAL EPIDEMIC PREVENTION & CONTROL CENT
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