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Preparation method of egg yolk antibody injection for treating porcine respiratory syndrome

A yolk antibody and syndrome technology, applied in the direction of antibodies, antibacterial drugs, antiviral agents, etc., can solve the problems of unreported porcine respiratory syndrome, and achieve the effect of being suitable for large-scale promotion, quick effect, and improving production performance.

Inactive Publication Date: 2013-02-06
GUANGZHOU GLAM BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And so far, the use of egg yolk antibody treatment of porcine respiratory syndrome research has not yet been reported

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Example 1: Isolation and identification of PRRSV epidemic strains and virus TCID 50 determination

[0016] 1. Isolation and identification of popular strains of PRRSV

[0017] 1) Collect the serum of suspected PRRSV-infected pigs, inoculate MARC-145 cells, culture at 37°C, 5% CO2, and observe the cell lesions every day. If no cell lesions appear after 96 hours of culture, place the cell culture at -40°C for repeated freezing and thawing Centrifuge at 12,000 rpm for 10 minutes three times, and take the supernatant to inoculate MARC-145 cells to continue passage. Successive passages were carried out in this way. Cytopathic changes occurred 72 hours after the inoculation of the isolated strain when it was subcultured to the second generation.

[0018] 2) Design a pair of specific primers P1 and P2 according to the sequence of PRRSV registered in GENBANK (AY150564). Among them, P1: CCGACTGCTAGGGCTTCT, P2: TATCTGCCACCCAACACG amplified fragment size is 363bp. RNA was ext...

Embodiment 2

[0021] Preparation and immunization of embodiment 2 inactivated vaccine

[0022] 1. Bacteria toxin inactivation

[0023] Mix the bacterial venoms that measure the titer of good bacteria respectively, and the TCID of PRRSV in the vaccine prepared after mixing 50 not less than 10 8 / ml, TCID of PCV-2 50 not less than 10 6 / ml, the number of mycoplasma hyopneumoniae is not less than 10 10 / ml. After mixing, add 0.2% formaldehyde to inactivate at 37°C for 24 hours. It is ready for use after passing the inactivation test.

[0024] 2. Preparation of inactivated vaccine

[0025] Preparation of oil phase: 94 parts by volume of white oil for injection, add 6 parts by volume of Siben-80°C, after mixing, then add aluminum stearate (2 grams of aluminum stearate per 100 milliliters of mixed solution), and gradually stir until Until transparent, autoclave for later use.

[0026] Preparation of aqueous phase: Take 4 parts by volume of sterilized Tween at -80°C, add 96 parts by volum...

Embodiment 3

[0030] The preparation of embodiment 3 egg yolk antibody and titer determination

[0031] 1. Preparation of egg yolk antibody

[0032] Collect the eggs 14 days after the third immunization, use 75% ethanol to sterilize the eggshells, collect the yolks under aseptic conditions, and use an equal amount of phosphate buffered saline (0.1M PBS, PH7.2) to place in a tissue homogenizer, Homogenize at 5000 rpm for 1 minute, add lactic acid until the pH is 4.0, stir overnight at 2-8°C, freeze at -80°C and thaw, repeat freezing once, then centrifuge at 8000 rpm for 10 minutes, collect the supernatant, and follow the Add saturated ammonium sulfate at a ratio of 1:1, stir at 2-8°C for 2 to disappear, then stand overnight at 2-8°C, collect the precipitate by centrifugation at 8000 rpm for 10 minutes, add 0.1M PBS to dissolve and remove ammonium sulfate.

[0033] 2. Titer determination of PRRSV antibody

[0034] 1) Cell preparation Take well-grown MARK-145 cells, digest them with trypsin ...

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Abstract

The present invention relates to a preparation method of an egg yolk antibody injection for treating porcine respiratory syndrome, which comprises the following steps: (1), isolating the current prevalent PRRSV (porcine reproductive and respiratory syndrome virus), PCV-2 (porcine circovirus virus) and mycoplasma hyopneumoniae, and culturing the PRRSV with the MARC-145 cells, the PCV-2 with the PK15 cells and the mycoplasma hyopneumoniae with the mycoplasma culture medium respectively to prepare the triple inactivated vaccine after measuring the titer of virus and mycoplasma; (2), immunizing the nonimmune laying hen with the prepared triple inactivated vaccine for PRRSV / PCV2 and mycoplasma hyopneumoniae; (3), collecting the egg yolk in the aseptic conditions; (4), measuring the valence of neutralizing antibody in the PRRSV and PCV-2 through microneutralization experiment and measuring the valence of antibody of in the mycoplasma hyopneumoniae through ELISA (enzyme-linked immune sorbent assay); and (5), performing sterility test for the egg yolk after valence measurement and performing sterile packaging after testing qualified. The preparation method has the benefits of effectively treating pigs which are independently infected or mixedly infected with PRRSV, PCV2 and mycoplasma hyopneumoniae diseases.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a preparation method of yolk antibody for treating porcine respiratory diseases. Background technique [0002] Porcine Respiratory Disease Syndrome (PRDC) is a disease characterized by reduced growth rate, reduced feed utilization, loss of appetite, coughing, and dyspnea. Although the overall mortality rate is not high, it can seriously affect the economic benefits of pig farms. The main pathogens that cause porcine respiratory syndrome clinically are Mycoplasma hyopneumoniae, reproductive and respiratory syndrome virus (PRRSV), type 2 circovirus (PCV-2) and swine influenza virus, as well as other bacterial pathogens secondary to infection, Pathogens such as Pasteurella, Streptococcus and Actinobacillus pleuropneumoniae. Pigs at all stages after weaning can be sick, that is, from 2 weeks after weaning, respiratory disease syndrome may occur unt...

Claims

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Application Information

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IPC IPC(8): A61K39/395A61P31/04A61P31/14A61P31/16A61P31/20
Inventor 李帅伟庞程
Owner GUANGZHOU GLAM BIOTECH
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