Method for preparing gram-negative original bacillus having cross-protection power for killing living vaccine

A Gram-negative bacteria, Gram-negative technology, applied in the field of preparation of inactivated vaccines of Gram-negative pathogenic bacteria, can solve the problems of complex outer membrane protein extraction process, high vaccine cost, and inability to be widely used, and achieve the goal of preparing The process is simple, promotes industrialization, and eliminates the effect of separation and purification process

Inactive Publication Date: 2007-09-19
徐海军
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The vaccine prepared by this process has a good cross-immune protection effect, but the extraction process of the outer membrane protein is complicated and requires the use of ultra-fast refrigerated centrifugation, which makes the cost of the prepared outer membrane protein vaccine too high and cannot be widely used in production

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Embodiment 1: The present invention is used in the preparation method of fowl cholera propolis inactivated vaccine and its immunoprotective experiment.

[0014] The virulent freeze-dried strain of Pasteurella multocida type A involved in this example was purchased from the China Veterinary Drug Control Institute, and was identified and preserved by the institute.

[0015] 1. Preparation of bacteria solution for strain rejuvenation and seedling preparation:

[0016] After the freeze-dried bacteria were dissolved in 1ml of sterile physiological saline, they were streaked and inoculated on the blood Martin agar medium, and cultured at 37°C for 20-22 hours. The grown bacteria were washed and rejuvenated by continuous passage twice in the chicken body. The liver disease material was taken and inoculated on the blood Martin agar plate by streaking, and cultured at 37°C for 20-22 hours. 4-6 typical colonies were streaked and inoculated on blood Martin agar slant, and cultured...

Embodiment 2

[0032] Embodiment two: the present invention is used for the preparation method of Escherichia coli inactivated vaccine and its immunoprotective experiment

[0033] The standard Escherichia coli CMCC44115 strain freeze-dried bacterial classification that this embodiment relates to (serotype is O 2 type), purchased from the China Veterinary Drug Control Institute, identified and preserved by the Institute.

[0034] 1. Rejuvenation of strains and preparation of bacterial solution for seedling production

[0035]After the freeze-dried bacteria were dissolved in 1ml sterile saline, they were streaked on the blood Martin agar medium and cultured at 37C for 20-22 hours. The grown bacteria were washed and rejuvenated by continuous passage twice in the chicken body. The liver disease material was taken and inoculated on the blood martin agar culture plate by streaking, and cultured at 37°C for 20-22 hours. 4-6 typical colonies were streaked and inoculated on blood Martin agar slant,...

Embodiment 3

[0045] Embodiment 3: The present invention is used in the preparation method of the R. anatipestifer vaccine and its immune protection experiment.

[0046] The freeze-dried strain of R. anatipestifer (serotype I) involved in this example was isolated from clinically dead ducks.

[0047] 1. Rejuvenation of strains and preparation of bacterial solution for seedling production

[0048] Freeze-dried strains were dissolved in 1ml sterile saline, inoculated in serum nutrient broth, and cultured at 37°C for 16-18 hours. Take the culture solution and inoculate it on a serum nutrient agar plate, and incubate at 37°C for 16-18 hours. The grown bacteria were washed with sterilized saline and used as seed solution. The seed liquid is inoculated into the broth medium containing heme serum used for making the vaccine in the proportion of 5% of the culture fluid volume, and 200umol / L 2,2'-bipyridyl has been added in the broth medium as an iron chelating agent, It can also be replaced by 5...

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Abstract

The invention discloses a preparation method for inactivated vaccine of gram negative pathogen with cross-protection. The said method comprises the following steps, a) the rejuvenated pathogen being cultured in vitro in solid medium or liquid medium adding iron chelating agent; b) bacteria cultured in solid medium being washed by aseptic vaccine diluent untill the final concentration of bacterial suspension achieves to the own trade standard; or the bacterial suspension with a concentration lower the own trade standard and the bacteria cultured in liquid medium being condensed to reserve the bacterial cell and deciduous outer-membrane protein, in order to make the final concentration of bacterial suspension achieve to the own trade standard; c)the obtained bacterial suspension from b)step, being inactived by 0.1-0.3% of formaldehyde by volume percent; d) adding adjuvant to make the said vaccine. The said method is easy to realise industrialization with simple tenique and low cost.

Description

technical field [0001] The invention relates to a preparation method of an inactivated vaccine of Gram-negative pathogenic bacteria. Background technique [0002] Many Gram-negative bacteria are important pathogens, such as certain serotypes of Escherichia coli, Salmonella, Pasteurella multocida, and R. anatipestifer. These pathogenic bacteria lead to high morbidity and mortality in poultry, and have brought great harm to the poultry farming industry and human health. Due to the complex antigenic composition and numerous serotypes of Gram-negative bacteria, inactivated vaccines prepared by in vitro culture can only protect against infection by homotype bacteria, but basically have no protective effect on infection by heterotype bacteria. This feature of Gram-negative bacteria has brought great difficulties to the immune prevention of corresponding poultry infectious diseases. [0003] Studies have shown that Gram-negative bacteria can express cross-protective factor (CPF) ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61K39/106A61K39/108A61K39/39A61P31/04
Inventor 徐海军
Owner 徐海军
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