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Rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine and preparation method thereof

A technology of rabbit hemorrhagic virus and baculovirus vector, applied in the field of immunization, can solve the problems of large individual differences in animals, the preparation process of Pasteurella multocida is not stable enough, the spread of virulent and other problems

Active Publication Date: 2018-11-30
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are major defects in this vaccine, which are mainly manifested in that the individual animals used to prepare rabbit haemorrhagic disease virus are quite different, which is not conducive to the large-scale production of vaccine antigens, and the quality control is difficult to carry out. The most important thing is that the vaccine preparation process is easy to cause strong toxicity. Diffusion, poor biosafety
In addition, the preparation process of Pasteurella multocida is not stable enough, the antigen immunogenicity varies greatly between batches, and the challenge protection rate in the efficacy test is low
The above defects have seriously affected the quality of vaccines and restricted the production efficiency of enterprises

Method used

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  • Rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine and preparation method thereof
  • Rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine and preparation method thereof
  • Rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1 recombinant rabbit hemorrhagic disease virus VP60 protein antigen cell culture tank suspension culture method

[0071] Cultivate the Sf9 cells in suspension culture at 27-28°C and 90-120r / min for 24-48 hours, continuously passage and expand, the passage ratio is 1:2-1:4, inoculate the cell culture tank, and first reach the culture tank In the process of cultivating cells in the cell culture tank, control the parameters of the cell culture tank within the normal range: temperature 27-28°C, pH value 6.0-6.2, dissolved oxygen (DO) 50% ~ 60%, stirring speed 50 ~ 70 r / min. After the cells in the cell culture tank reached a certain density (about 2×10 6 Individual / ml), inoculate the recombinant baculovirus at a ratio of 1% to carry out virus maintenance culture, and control the parameters such as temperature, pH value, DO, stirring speed, etc. in a suitable range. 4 to 5 days after inoculation, when the diseased cells reach more than 85%, the cell culture can b...

Embodiment 2

[0074] Example 2 Preparation of Rabbit Hemorrhagic Disease Virus Baculovirus Vector Cell Culture Inactivation Solution

[0075] Add formaldehyde solution with a final concentration of 0.2% to the recombinant rabbit hemorrhagic disease virus VP60 baculovirus cell culture prepared above, mix well, change the tank and inactivate at 37°C for 18-24 hours, stirring at a speed of 50-70r / min or inactivate at 37°C for 24 hours after changing the bottle, shake once every 2 to 3 hours for 3 minutes each time to obtain the inactivation solution of rabbit hemorrhagic virus baculovirus vector cell culture, and place the inactivation solution in 2 to 3 hours. Store at 8°C for no more than 6 months. In the inactivation test, take the inactivation solution of the rabbit hemorrhagic disease virus baculovirus carrier cell culture, inoculate well-grown Sf9 cells according to 1% of the total amount, culture at 27-28°C, and observe continuously for 5 days. There is no cytopathic change. Harvest th...

Embodiment 3

[0076] Embodiment 3 Pasteurella multocida fermentation culture method

[0077] 3 Preparation of Pasteurella multocida fermentation broth

[0078]3.1 Propagation and identification of primary seeds Dilute the freeze-dried bacteria of Pasteurella multocida C51-17 strain with sterilized physiological saline, and then streak and inoculate them on a Martin agar plate containing 4% serum and 0.1% lysed whole blood. Incubate at 37°C for 18-22 hours, select at least 5 typical colonies, inoculate several branches of Martin agar slant containing about 4% serum and 0.1% lysed whole blood, and culture at 37°C for 18-22 hours, as a primary seed. Store at 2-8°C and use for no more than 7 days.

[0079] 3.2 Propagation and identification of secondary seeds Take primary seeds and inoculate them in Martin broth containing 0.5% serum, place at 37°C, 200r / min, vibrate and cultivate for 14-18 hours, take samples for pure inspection, and use them as secondary seeds after passing the test. Store...

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Abstract

The invention relates to a rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine and a preparation method thereof, and belongs to the field of immune technology. Recombinant rabbit hemorrhagic disease virus VP60 baculovirus is inoculated into Sf9 insect cells and cultured at 27-28 DEG C. When cell lesion reaches 85% or more, a cell culture is harvested and inactivated, and the inactivated cell culture is used as a rabbit hemorrhagic disease virus antigen. Rabbit Pasteurella multocida capsular serotype A C51-17 strain is amplified and cultured, a bacterial solution is inactivated, and the inactivated bacterial solution is used as a Pasteurella multocida antigen. The rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine can be prepared by mixing the rabbit hemorrhagic disease virus antigen and the Pasteurella multocida antigen with adjuvants in proportion. The rabbit hemorrhagic disease virus baculovirus vector and pasteurella multocida bivalent inactivated vaccine has high safety, good immune effect and simple process, and can be used for preventing and controlling Rabbit Hemorrhagic Disease (Rabbit Plague) and Rabbit Pasteurella multocida.

Description

technical field [0001] The invention relates to a rabbit haemorrhagic disease virus baculovirus vector, pasteurellosis multocida dual inactivated vaccine and a preparation method thereof, belonging to the technical field of immunization. Background technique [0002] Rabbit hemorrhagic disease (RHD) is an acute, severe and highly contagious infectious disease caused by rabbit hemorrhagic disease virus (RHDV), commonly known as "rabbit plague". The disease is often fulminant, with extremely high morbidity and mortality, and the mortality rate is as high as about 90%. Rabbit Pasteurellosis (Pasteurellosis), also known as rabbit hemorrhagic septicemia (Hemorrhagic septicaemia, HS), is an acute, septic infectious disease caused by Pasteurella multocida (Pm) in rabbits. The disease can be divided into rhinitis type, pneumonia type, otitis media type and other disease types. The lesions of various disease types are inconsistent, but two or more disease types often occur in combin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/295A61K39/23A61K39/102A61P31/20A61P31/04C12N1/20C12N7/00C12R1/93C12R1/01
CPCA61K39/102A61K39/12A61P31/04A61P31/20C12N1/20C12N7/00A61K2039/70C12N2770/16051C12N2770/16063C12N2770/16034C12N2750/14334Y02A50/30
Inventor 范志宇王芳胡波魏后军宋艳华仇汝龙陈萌萌朱伟峰薛家宾
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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