Dual-gene deletion rough type bovine brucellosis and production method for vaccine thereof
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A technology of Brucella bovis and Brucella, applied in the field of veterinary biological products, can solve the problems of roughness and the inability of Brucella to form LPS, etc.
Inactive Publication Date: 2014-09-10
CHINA INST OF VETERINARY DRUG CONTROL
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Problems solved by technology
Therefore, the deletion or destruction of the WboA gene will also cause Brucella to fail to form LPS containing the O-chain completion structure, thus showing a rough type
Method used
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Embodiment 1
[0111]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. Recombinant Brucella rS2 strain seed bacteria 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 amount.
[0112] 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.
[0113] At the same time, take samples and count live bacteria with TSA for reference during concentration.
[0114] Add 0.2% to 0.4% of the total amount of sodium carboxymethyl cellulose to the bacterial liquid that has passed the pure inspection to precipitate the bacterial cells, absorb the supe...
Embodiment 2
[0119] Product testing
[0120] The relevant inspection methods involved in the present invention are carried out according to the method of "Chinese Veterinary Pharmacopoeia"
[0121] (1) Physical properties The vaccine should be loose and fast, and can dissolve rapidly within 1 minute after adding the diluent.
[0122] (2) Purely tested vaccines are purely free of other bacteria.
[0123] (3) Safety inspection Dilute the vaccine to contain 1×10 per 1ml 9 CFU live bacteria, subcutaneously inoculate 5 Kunming mice of 18-20 g in the groin, each 0.25 ml / mouse, observe for 6 days, all are healthy and alive (Table 3).
[0124] Table 3 Safety inspection results of rough brucellosis live vaccine (2308-ΔWboA-ΔvjbR strain)
[0125]
[0126] -: Healthy survival, no abnormality at the injection site; +: Death or morbidity.
[0127] (4) Viable bacteria count Use TSA to count the viable bacteria to count the viable bacteria of the vaccine, and the content of viable bacteria in each...
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Abstract
The invention relates to dual-gene deletion rough type bovine brucellosis and a production method for a vaccine thereof. A recombinant bacterial strain deletes a WboA gene and a vjbR gene of a bovine brucellosis 2308 strain by virtue of a non-resistance gene screening technology. Due to deletion of two genes, on the one hand, a condition (cased by WboA gene deletion) for forming an O chain in a smooth type bovine brucellosis cell wall LPS (lipopolysaccharide) structure is lost, and colonial morphology is changed into a rough type from a smooth type; and on the other hand, due to deletion of vjbR gene, toxicity of recombinant bacteria and viability in a cell are descended, so that infection ability of bovine brucellosis on a target animal is lowered, and therefore, safety of the vaccine is further improved. By using the bacterial strain to produce the vaccine, a current situation that a bovine brucellosis vaccine immune animal and a wild strain-infected animal are difficult to distinguish is changed, and safety of the existing vaccine is effectively improved.
Description
[0001] Technical Field The present invention relates to a production method of double-gene deletion Brucella crassa and its vaccine. The vaccine 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 method to eliminate the disease worldwide is the combination of culling and immunization....
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