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Monogene deletion rough-type Brucella and production method of Brucella vaccine

A technology for Brucella and vaccine production, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve problems such as potential biosafety risks limiting vaccine development, and achieve the effect of stabilizing genetic traits

Active Publication Date: 2014-08-27
CHINA INST OF VETERINARY DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although it has been reported that recombinant Brucella crassa was obtained by inserting a resistance gene, the potential biosafety hazards caused by the resistance gene limit the possibility of its development as a vaccine

Method used

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  • Monogene deletion rough-type Brucella and production method of Brucella vaccine
  • Monogene deletion rough-type Brucella and production method of Brucella vaccine
  • Monogene deletion rough-type Brucella and production method of Brucella vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Pancreatic Soy Broth (TSB, BD Company, USA) or other suitable culture medium is used as the culture medium for recombinant bacteria, and an appropriate amount of commonly used antifoaming agent is added according to the medium volume, and 1% to 2% of the medium volume is added after sterilization. For the recombinant Brucella r2308-ΔWboA strain seed liquid, gradually increase the ventilation at 36-37°C, and ferment and cultivate for 36 hours according to the conventional method. 1% to 2% of the base amount.

[0080] After the cultivation is completed, try to use trypan soy agar to test the purity according to the method stipulated in "Chinese Veterinary Pharmacopoeia", and it is pure.

[0081] At the same time, take samples and count live bacteria with TSA for reference during concentration.

[0082] Add 0.15% to 0.3% of the total amount of sodium carboxymethyl cellulose to the purely qualified bacterial liquid to precipitate the bacterial cells, absorb the supernatant...

Embodiment 2

[0087] Product testing

[0088] The relevant inspection methods involved in the present invention are carried out according to the method of "Chinese Veterinary Pharmacopoeia"

[0089] (1) Physical properties The vaccine should be loose and fast, and can dissolve rapidly within 1 minute after adding the diluent.

[0090] (2) Pure test The vaccines tested are all pure and free from other bacteria.

[0091] (3) Dilute the vaccine to contain 10 per 1ml 9 CFU live bacteria, subcutaneously inoculated 5 Kunming mice of 18-20 g in the groin, each 0.25 ml / only, observed for 6 days, and the results were all healthy and alive (Table 3).

[0092] Table 3 Safety inspection results of live brucellosis brucellosis vaccine (r2308-ΔWboA strain)

[0093]

[0094] -: healthy survival, no abnormality at the injection site; +: death or morbidity

[0095] (4) Counting of live bacteria Use TSA plates to count the live bacteria of the vaccine, and the number of live bacteria per serving shoul...

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Abstract

The invention relates to a monogene deletion rough-type Brucella and a production method of a Brucella vaccine. According to the recombinant strain, base sequences between 1-897bp of the WboA gene of a Brucella 2308 strain are deleted by virtue of a non-resistance gene screening technology so as to lose the conditions for the formation of O-chain in a smooth-type Brucella cell wall LPS (Lipopolysaccharide) structure, and thus the smooth-type Brucella is transformed into a rough -type Brucella. The safety of the rough-type recombinant strain is significantly improved, but a good immune effect on brucellosis is still maintained, the recombinant bovine Brucella is named Brucella r2308-delta WboA strain (CGMCC No.8885). The current situation that Brucella vaccine immune animals are hard to distinguish from wild-strain-infected animals is changed by a vaccine produced by the strain, and the safeties of existing vaccines are effectively improved.

Description

[0001] Technical field The present invention relates to a production method of single-gene deletion Brucella crassa and its vaccine, which is a live vaccine for preventing brucellosis of cattle, sheep and pigs, and belongs to the field of veterinary biological products. technical background [0002] Brucellosis (brucellosis) is a zoonotic disease characterized by abortion and fever caused by Brucella or Brucella, which seriously threatens the life and health of humans and various animals. This disease not only has serious harm to the reproduction and production performance of animals, but more importantly, people are often difficult to cure after being infected with Brucella, thus causing serious public health problems. Elimination of brucellosis has therefore been one of the most important goals of public health programs in countries where Brucella is endemic. At present, the main way to eliminate the disease in the world is to combine culling and immunization. In China, wher...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21A61K39/10A61P31/04C12Q1/68C12Q1/04C12R1/01
Inventor 丁家波王楠李慧娇王忠田王芳蒋卉顾进华宁宜宝张广川毛开荣戴志红李宁印春生
Owner CHINA INST OF VETERINARY DRUG CONTROL
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