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Riemerella anatipestifer bacterial ghost vaccine adopting chitosan oligosaccharide as adjuvant

A technology of Riemerella anatipestifer and chitosan oligosaccharides, applied in the direction of bacteria, antibacterial drugs, bacterial antigen components, etc., can solve the problems of secondary infection, inability to provide mucosal immune barrier against infection, inability to produce local immunity, etc. To achieve the effect of preventing viral infection and comprehensive immune protection

Inactive Publication Date: 2015-10-14
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since Riemeria anatipestifer has basically lost its ability to colonize the respiratory tract after being prepared as a bacterium, it cannot produce local immunity and can only immunize animals by injection, so there are still problems in the following aspects: ① Immunization by injection, giving Animals cause great immune stress, often causing paramyxovirus and Escherichia coli to take advantage of the weak point, leading to severe secondary infection
② Inactivated vaccines can only induce humoral immunity, no or only slight local mucosal immunity, and cannot provide a reliable mucosal immune barrier against infection

Method used

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  • Riemerella anatipestifer bacterial ghost vaccine adopting chitosan oligosaccharide as adjuvant
  • Riemerella anatipestifer bacterial ghost vaccine adopting chitosan oligosaccharide as adjuvant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Implementation Example 1 Preparation of Riemerella anatipestifer bacterial shadow

[0018] 1.1 Preparation of protoplasts: After activating the RA2 strain numbered WF1, cultivate to OD 600 When the value is 0.7, place the bacterial liquid in an ice water bath for 30 minutes, take 10 mL of the bacterial liquid, centrifuge at 4°C, 5000 rpm for 10 minutes, discard the supernatant, wash the bacterial mud with 10 mL of ice-water bath sterilized water, centrifuge again, and repeat the washing three times. Suspend with ice-water bath hypertonic buffer, add preheated lysozyme solution to 5mg / mL, 37°C water bath for 30min, then add preheated EDTA solution to 0.01mol / L, 37°C water bath for 20min. During the action, take the treated bacterial solution on a glass slide every 5 minutes, stain with red, and observe the formation of protoplasts under an oil microscope. When the formation rate of protoplasts reaches 95% or more, centrifuge at 5000 rpm for 20 minutes, discard the supernat...

Embodiment 2

[0022] Implementation example 2 Vaccine immune protection test

[0023] In order to verify the clinical protective effect of the fungus vaccine prepared by the present invention on ducks, commercial meat ducks from Cherry Valley were selected as experimental animals. The fungus vaccine was vaccinated by adding different adjuvants through different immunization routes. At the same time, traditional formaldehyde was used. Control test of oil adjuvant vaccine of homologous strain prepared by inactivation method. The immune protection effect was evaluated by the comparison test of antibody level detection and challenge protection rate.

[0024] 1. Materials and methods

[0025] 1.1 Test materials

[0026] Fungus shadow + chitosan adjuvant: prepared according to the method described in Example 1.4.

[0027] Bacteria shadow + oil adjuvant: mix the prepared RA2 strain (WF2) with mineral oil adjuvant in a ratio of 1:1 to make the bacterial count reach 5×10 10 cfμ / mL.

[0028] Formaldehyde-ina...

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Abstract

The purpose of the present invention is to provide a riemerella anatipestifer bacterial ghost vaccine loaded with polyinosinic acid-polycytidylic acid having interferon inducing activity and added with chitosan oligosaccharide as an adjuvant. According to the technical scheme, a temperature control expression vector is constructed and is electrotransformed into riemerella anatipestifer protoplast, positive clones are screened and then are subjected to bacterial amplification culture at a temperature of 28 DEG C, lysis gene expression is induced at a temperature of 42 DEG C, polylysine is added to continuously act when the OD600 is no longer be reduced so as to completely lyze the live bacteria, centrifugation is performed to separate the bacteria, the dried bacteria and a 6 mg / mL Poly I:C solution according to an equal ratio so as to make the Poly I:C be embedded into the bacterial ghosts, a 2% chitosan oligosaccharide aqueous solution is added, stirring is performed to form a uniform mixture, sub-packaging is performed into penicillin bottles, and freeze-drying is performed so as to obtain the novel riemerella anatipestifer bacterial ghost vaccine. According to the present invention, the prepared riemerella anatipestifer bacterial ghost vaccine is immunized through drinking water, such that the local immunity on the respiratory tract mucous membrane and the digestive tract mucous membrane can be irritated, the humoral immunity can be stimulated, and the protection effect on the human body is not different from the injection immunity.

Description

technical field [0001] The invention belongs to the field of veterinary vaccine production, and relates to a method for preparing a bacterial vaccine for ducks, in particular to a method for preparing a Riemeria anatipestifer ghost vaccine with chitosan as an adjuvant. Background technique [0002] Riemerella anatipestifer ( Riemerella Anatipestifer , RA) mainly affects ducklings aged 2-7 weeks, characterized by neurological symptoms, fibrinous pericarditis, perihepatitis and air sacculitis, and is one of the main bacterial infectious diseases that endanger duck production. The disease can be infected through respiratory tract, digestive tract and skin wounds, and often forms mixed infections with pathogens such as Escherichia coli and paramyxovirus, causing a large number of deaths of ducks. [0003] Although Riemerella anatipestifer is sensitive to some antibiotics, it is difficult to completely remove it from the body; at the same time, due to the irrational use of antib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/02A61K39/39C12N1/21C12N15/74A61P31/04
Inventor 刘文华刘宗柱任慧英王晓雷张灿
Owner QINGDAO AGRI UNIV
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