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Rabbit bordetella bronchiseptica subunit vaccine

A technology of Bordetella sanguinis and subunit vaccines, which can be applied in vaccines, veterinary vaccines, antibacterial drugs, etc., can solve the problems of bacterial toxin residues and immunization rabbits

Active Publication Date: 2017-05-10
YEBIO BIOENG OF QINGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inactivated Bordetella bronchiseptica rabbit vaccine is prone to the problem of bacterial toxin residues, which often leads to the disease of immune rabbits. Therefore, there is an urgent need for a safe and effective vaccine that can prevent and control the disease, so as to make up for the existing technology. lack of

Method used

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  • Rabbit bordetella bronchiseptica subunit vaccine
  • Rabbit bordetella bronchiseptica subunit vaccine
  • Rabbit bordetella bronchiseptica subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Isolation and cultivation of Bordetella bronchiseptica in rabbits

[0015] A batch of 1.5-3.0 kg rabbits were purchased from a rabbit farm in Weifang, Shandong Province for the test of swine fever spleen leaching vaccine. The disease was characterized by acute onset, rapid death, mouth and nose bleeding, lung bleeding, and congestion. According to the incidence and death of rabbits Clinical manifestations, autopsy lesions, preliminarily suspected infection of Pasteurella rabbit or Bordetella bronchiseptica rabbit, so the lung tissue was taken to isolate and culture the bacteria, and the slide agglutination test was carried out for verification; Pasteurella (Pm) gene or Bordetella (Bb) gene was amplified by PCR, cloned, and sequenced. It was confirmed that the batch of rabbits died of infection caused by Bordetella bronchiseptica.

[0016] 1.1 Incidence situation A batch of 1.5-3.0kg rabbits was purchased from Weifang, Shandong Province for the temperature mea...

Embodiment 2

[0030] The purification of the protein of embodiment 2 rabbit Bordetella bronchiseptica (QDBb01 strain)

[0031] The isolated, identified and preserved Bordetella bronchiseptica (QDBb01 strain) was cultured in 3% TSB, and the bacteria were collected at 4000r / min, and the bacteria were resuspended with PH7.2 Tris-Hcl, and the resuspended bacteria were ultrasonically broken. Work for 1s, the interval time is 3s, the whole time is 12min, a total of 180 times. 4000r / min low-speed centrifugation to remove unbroken bacterial cells and heavier bacterial intracellular organelles, take the supernatant at 100000r / min, ultracentrifuge for 20min, and remove the supernatant. Dissolve the precipitate containing outer membrane protein with Tris-Hcl buffer containing 2% Triton X-100, incubate at room temperature for 1 hour to remove inner membrane protein, then perform ultracentrifugation at 100,000 r / min for 30 minutes, resuspend the precipitate with buffer, and use The semi-permeable membr...

Embodiment 3

[0032] Embodiment 3: Rabbit Bordetella bronchiseptica (QDBb01 strain) subunit vaccine preparation

[0033]Purified Bordetella bronchiseptica protein is carried out in absolute quantification by spectrophotometry according to the following steps: (1) add 0.15ml of PBS for protein measurement and 2.85ml of Coomassie Brilliant Blue staining solution for protein quantification in a cuvette As a contrast, carry out zero adjustment; (2) add 0.14ml of PBS for the protein to be tested, add 0.01ml of the protein solution to be tested and 2.85ml of Coomassie brilliant blue staining solution for protein quantification, measure the absorbance value y in the cuvette to be 0.204, pass The formula y=0.152x+0.068 gives the protein concentration x=0.83 mg / ml, which is 895 μg / ml. The above reagents for protein measurement were purchased from TIANGEN Company. Dilute the purified protein solution to 150ug / ml, mix the vaccine with aluminum hydroxide gel adjuvant at a volume ratio of 1:4, and prepa...

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Abstract

The invention provides a rabbit bordetella bronchiseptica subunit vaccine, particularly relates to rabbit bordetella bronchiseptica (QDBb01 strain with the preservation number of CCTCC M 2016607) identified through isolated culture, Bb gene specific primer PCR amplification and sequencing. Rabbit bordetella bronchiseptica OMP (osteoblast milk protein) is extracted, antigenic protein with the protein molecular weight larger than 40 kDa is purified, then the protein concentration is measured with a Bradford protein content measuring method and diluted to be 150 ug / ml, the subunit vaccine ( about 30 ug / ml for finished products) is prepared by emulsifying the protein and a aluminum hydroxide adjuvant in a volume ratio being 1:4, the vaccine is injected subcutaneously into a rabbit with the immunity weight being about 1.5 kg in the dosage of 1 ml for each rabbit, after 14 days, ear intravenous infusion challenge is performed with the rabbit bordetella bronchiseptica QDBb01 strain (LD50 being 4.8*105 CFU) according to 100 LD50, the rabbit is observed for 7 days after challenge, and the challenge protection rate of the rabbit is 10 / 10 through statistics.

Description

technical field [0001] The invention belongs to the technical field of veterinary biological products, and in particular relates to a rabbit subunit vaccine of Bordetella bronchiseptica. Background technique [0002] Bordetella bronchiseptica in rabbits occurs frequently in autumn, winter and early spring. It is mainly transmitted through the air and infected through the respiratory tract. Young rabbits have a high morbidity rate and there have been fatal cases. Adult rabbits are less affected. The contamination rate of this bacterium was 64.4% in group-raised rabbits, and 20% in free-range rabbits. Under natural conditions, this bacterium parasitizes in the upper respiratory tract of various mammals, often causing mutual infection of chronic respiratory diseases. Especially in sudden changes in weather, decreased resistance of rabbits, poor hygiene in the rabbit house, and dirty air, etc., the disease can all be caused. The incubation period of the disease is 7-10 days...

Claims

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Application Information

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IPC IPC(8): A61K39/02A61K39/39A61P31/04A61P11/02A61P11/00
CPCA61K39/0208A61K39/39A61K2039/521A61K2039/552A61K2039/55505
Inventor 张恒郭莉莉张会范根成杜元钊
Owner YEBIO BIOENG OF QINGDAO
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