Riemerella anatipestifer pre-bacterial ghost vaccine preparation method

A Riemerella anatipestifer and vaccine technology, applied in the field of poultry breeding disease prevention and control, can solve the problems of large immune stress, weakened immunogenicity, time-consuming and labor-intensive immunization operations, etc., achieve high immune protection rate and avoid stress , the effect of convenient application

Active Publication Date: 2015-10-14
QINGDAO AGRI UNIV
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AI Technical Summary

Problems solved by technology

However, there are still some problems in the following aspects: ①The traditional vaccine preparation technology changes the antigen structure through the inactivation process of chemical drugs such as formaldehyde, which weakens the immunogenicity and reduces the immune protection rate
②The vaccines reported so far are all immunized by injection, and the immune stress is large. Moreover, under the condition of large-scale breeding, the immunization operation is time-consuming and laborious
③ Inactivated vaccines can only induce humoral immunity, no or only slight cellular immunity and local mucosal immunity, and cannot provide a reliable mucosal immune barrier against infection

Method used

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  • Riemerella anatipestifer pre-bacterial ghost vaccine preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Implementation Example 1: Preparation of Riemerella anatipestifer precursor shadows

[0020] 1.1 Toxicity verification of clinical isolates The virulence of a strain of Riemerella anatipestifer (coded as LY1) isolated from clinical laying ducks and identified as type 2 by serotype was verified. The strain does not produce gelatinase, and the inoculated ducklings have not shown pathogenicity for three consecutive generations, but they can produce more than 90% protection when challenged with the same type of strong virus, indicating that the strain is a non-pathogenic or relatively pathogenic strain. Strains with low and good immunogenicity can be used as vaccine candidate strains.

[0021] Preparation of protoplasts After the LY1 strain was activated, cultured to OD 600 When it is 0.7, place the bacterial solution in an ice-water bath for 30 minutes, take 10 mL of the bacterial solution, and centrifuge at 5000 r / min for 10 minutes at 4 °C, discard the supernatant, ...

Embodiment 2

[0026] Implementation Example 2: Vaccine Immunization Protection Test

[0027] In order to verify the clinical protective effect of the mycobacteria vaccine prepared by genetic engineering means on ducks, Cherry Valley commercial meat ducks were selected as experimental animals, and the homologous strain oil adjuvant vaccine prepared by the method of formaldehyde inactivation was used to carry out the duckling under the same conditions. Duck immune protection test.

[0028] 1. Materials and methods

[0029] 1.1 Test material

[0030] Proghost vaccine: prepared according to the method described in Example 1.5.

[0031] Inactivated vaccine with oil adjuvant: formaldehyde-inactivated LY1 strain and mineral oil adjuvant were mixed at a ratio of 1:1 to make the bacterial content reach 10 10 cfu / mL.

[0032] Experimental animals: 1-day-old Cherry Valley commercial meat duck.

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Abstract

The present invention relates to a method for conversion preparation of a riemerella anatipestifer pre-bacterial ghost vaccine by using phage PhiX174 E gene and staphylococcal nuclease A gene, and an application method. According to the technical scheme, the phage PhiX174 E gene and the staphylococcal nuclease A gene are coupled to form the series coupling gene through the flexible coupling arm (Gly4Ser), a temperature control expression vector pBV-E-SN is further constructed, the pBV-E-SN is transformed into riemerella anatipestifer protoplast through electric shock conversion, the positive clones are screened and are subjected to bacterial amplification culture at a temperature of 28 DEG C, the bacteria are separated, a protection agent is added, and sub-packaging and freeze-drying are performed. According to the present invention, the prepared live bacterial 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, and the humoral immunity can be stimulated.

Description

technical field [0001] The invention belongs to the field of poultry breeding epidemic prevention and control, and relates to a preparation method of a bacterial vaccine, in particular to a method for preparing a pre-ghost vaccine of Riemeria anatipestifer by utilizing the E gene of phage PhiX174 and the staphylococcal nuclease A gene transformation methods and applications. Background technique [0002] Duck infectious serositis, caused by Riemerella anatipestifer ( Riemerella Anatipestifer , RA) is an acute or chronic contagious disease of ducks, geese, turkeys and other poultry and wild birds, mainly affecting ducklings aged 2-7 weeks, with neurological symptoms and fibrinous pericarditis, perihepatitis and air sacculitis as a feature. [0003] At present, Riemeria anatipestifer infection is harmful to the breeding industry. In addition to directly causing animal morbidity and death, the indirect economic loss caused by the reduction of feed conversion rate and growth r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/55C12N15/34C12N1/21A61K39/02A61P31/04C12R1/01
Inventor 刘文华刘宗柱任慧英韩先杰邹玲
Owner QINGDAO AGRI UNIV
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