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Method for purifying haemophilus parasuis serotype 4 and serotype 5

A technology of Haemophilus suis and serum, applied in the direction of biochemical equipment and methods, methods based on microorganisms, bacteria, etc., can solve problems such as banning, and achieve the effects of simple process control, large processing capacity, and simple operation

Inactive Publication Date: 2019-07-30
JIANGSU NANNONG HI TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The use of antibiotics to prevent Haemophilus parasuis disease has played a certain role, but the drug resistance and food safety problems caused by the abuse of antibiotics will eventually ban the use of antibiotics to prevent Haemophilus parasuis disease

Method used

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  • Method for purifying haemophilus parasuis serotype 4 and serotype 5
  • Method for purifying haemophilus parasuis serotype 4 and serotype 5
  • Method for purifying haemophilus parasuis serotype 4 and serotype 5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Preparation of Haemophilus parasuis antigen serotype 4:

[0021] Add Haemophilus parasuis culture medium according to 70% of the volume of the fermenter, inactivate at 121°C for 30 minutes, and when the medium is naturally cooled to 37°C, add Haemophilus parasuis serum type 4 seeds according to 2% of the volume of the medium solution, while adding calf serum with a final concentration of 10% (V / V) and 0.001% (W / V) NAD, cultured at 37°C and 180r / m for 12h, and harvested the bacterial solution.

Embodiment 2

[0023] Purification of Haemophilus parasuis serotype 4:

[0024] The harvested Haemophilus parasuis serotype 4 was concentrated by ultrafiltration using a hollow fiber with a pore size of 0.1um, and the shear force was controlled at 2000sec -1 , the transmembrane pressure was 10psi, the antigen solution was concentrated to 1 / 6 of the original volume, and the permeate was discarded;

[0025] After the concentration is completed, use sterile PBS (pH7.2-7.4) for isokinetic rehydration according to the flow rate at the permeation end. The rehydration volume is 5 times the concentrated volume, which is equivalent to 5 times of diafiltration, and the shear force is controlled at 2000sec. -1 , the transmembrane pressure is 10psi, the permeate is discarded, and the retentate is retained;

[0026] Dilute the concentrated antigen solution with sterile PBS (pH7.2-7.4) to the volume before concentration, which is the purified Haemophilus parasuis antigen.

[0027] The total protein cont...

Embodiment 3

[0035] Purification of inactivated antigen of Haemophilus parasuis serotype 4

[0036] The harvested Haemophilus parasuis antigen solution was added with a final concentration of 0.3% (V / V) formaldehyde, and inactivated at 37°C for 24 hours.

[0037] The harvested Haemophilus parasuis serum type 4 inactivated antigen was concentrated by ultrafiltration using a hollow fiber with a pore size of 0.1um, and the shear force was controlled at 2000sec -1 , the transmembrane pressure was 10psi, the antigen solution was concentrated to 1 / 6 of the original volume, and the permeate was discarded;

[0038] After the concentration is completed, use sterile PBS (pH7.2-7.4) for isokinetic rehydration according to the flow rate at the permeation end. The rehydration volume is 5 times the concentrated volume, which is equivalent to 5 times of diafiltration, and the shear force is controlled at 2000sec. -1 , the transmembrane pressure is 10psi, the permeate is discarded, and the retentate is r...

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Abstract

The invention relates to the technical field of antigen material purification, in particular to a method for purifying haemophilus parasuis serotype 4 and serotype 5. The method comprises the steps that an antigen culture medium and an inactivated antigen culture serve as raw materials, and ultra-filtration concentration and washing filtration are conducted in sequence. According to the technicalscheme, the advantages that a hollow fiber membrane is low in shearing force and simple in process control are utilized, the operation conditions are optimized, the stability of an antigen and the removal rate of impurities are ensured by limiting the transmembrane pressure and shear rate. By applying the method for purifying the haemophilus parasuis serotype 4 and serotype 5, after six-time concentration, the removal rate of miscellaneous protein is 90% or above, the antigen recovery rate is 60% or above, and the content of serum and endotoxin is obviously lowered compared with that of an original antiserum. Meanwhile, due to the fact that the processing capacity is large, the processing speed is high, operation is easy and the process is stable, automatic or semi-automatic production canbe achieved.

Description

technical field [0001] The present invention relates to the technical field of purification of antigenic substances, and further relates to the purification technology of Haemophilus parasuis used in vaccines, in particular to a method for purifying Haemophilus parasuis, which is mainly applicable to Haemophilus parasuis serotypes 4 and 5 type. Background technique [0002] Haempohlius parasuis (HPS) is a Gram-negative bacterium that mainly affects piglets aged 4-8 weeks. The disease caused by this bacterium is called Haemophilus parasuis disease, which can cause fibrinous pleurisy, peritonitis, pericarditis, arthritis, and even meningitis and other symptoms in affected piglets. The incidence rate reaches 20%-30%, and the mortality rate is severe. Up to 50%. In recent years, with the scale and intensification of the pig industry, HPS has become one of the important bacterial diseases affecting the healthy development of the pig industry due to the changes and influences of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N1/02C12R1/21
CPCC12N1/20C12N1/02
Inventor 缪芬芳肖澄董彦鹏徐凤胡静雅郁伟军车巧林周琳耿晓眉谢洁
Owner JIANGSU NANNONG HI TECH
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