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Preparation method of bacterial ghost of O24 type duck pathogenic escherichia coli

A technology of Escherichia coli and pathogenicity, which is applied in the field of preparation of O24 duck pathogenic Escherichia coli slough, can solve the problems of high cost, affect animal health, complicated steps and the like, achieve simplified preparation steps and ensure no viable bacteria The effect of existence, simple preparation process

Inactive Publication Date: 2019-11-05
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The preparation process is simple and does not require the use of inducers, formaldehyde and freeze-drying equipment, which reduces costs and fills the gap in the field of O24 E. coli slough preparation in the industry. It solves the need to add inducer IPTG to other carriers, which has high costs and cumbersome steps. Technical problems of residual formaldehyde in formaldehyde-inactivated slough affecting animal health

Method used

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  • Preparation method of bacterial ghost of O24 type duck pathogenic escherichia coli
  • Preparation method of bacterial ghost of O24 type duck pathogenic escherichia coli
  • Preparation method of bacterial ghost of O24 type duck pathogenic escherichia coli

Examples

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Effect test

Embodiment 1

[0027] The preparation of embodiment 1 O24 type duck pathogenic Escherichia coli slough

[0028] The specific preparation steps of O24 type duck pathogenic Escherichia coli slough are:

[0029](1) Isolation and identification of pathogenic Escherichia coli in ducks: aseptically collect blood, liver and other disease materials of dead ducks in a waterfowl farm in Jiangsu Province, streak on MacConkey agar medium, and place the agar plate upside down at 37°C Cultivate overnight in a constant temperature incubator, take out the next day to observe the colony morphology, pick typical colonies on the MacConkey plate for Gram staining and microscopic examination, and transfer the bacteria that are Gram-negative in the microscopic examination to glucose and maltose , lactose, sucrose, mannitol, indole, MR, citrate, VP, hydrogen sulfide and other bacterial biochemical trace identification tubes and three sugar iron agar medium for biochemical identification. Referring to the primer s...

Embodiment 2

[0054] Embodiment 2 transmission electron microscope detection

[0055] The Escherichia coli pBV221-E / D and pBV221 / D bacterium liquid samples induced to express 2.5h in Example 1 were stained respectively by negative staining method. Add a drop of Escherichia coli bacterial solution to the copper grid, let it stand for 10 minutes, absorb the bacterial solution with filter paper, then add a drop of 2% phosphotungstic acid dye solution to the copper grid, absorb the dye solution after dyeing for 1 minute, and place it in an infrared drying lamp Under treatment for 10 minutes. The cell structure was observed with a philips transmission electron microscope (Tecnai 12).

[0056] The results of transmission electron microscopy Figure 5 As shown, the pBV221 / D control bacterium presents a complete bacterial structure ( Figure 5 -A), high electron density and uniform distribution. However, the electron density of induced pBV221-E / D cells decreased and the distribution was uneven ...

Embodiment 3

[0057] Example 3 Detection of immunogenicity of O24 duck pathogenic E. coli slough

[0058] Carry out immunogenicity detection to the O24 type duck pathogenic Escherichia coli slough prepared in Example 1. Forty 7-day-old Cherry Valley ducks with similar body weight were selected and randomly divided into two groups, 20 in each group, in the slough immune group and the PBS control group. Inoculate 200 μL of bacterial slough vaccine (1 × 10 9 CFU / bird), subcutaneously inoculate an equal volume (200 μL) of sterile PBS solution in the neck of Cherry Valley ducks in the PBS control group; two weeks later, the same dose was immunized again, and blood was collected two weeks after the first and second immunizations, respectively , to separate the serum. Whole bacterial antigen of duck pathogenic Escherichia coli (1×10 9 CFU / mL) was coated on the microtiter plate, the serum sample was diluted 500 times, and the serum antibody titer was detected by indirect ELISA.

[0059] The det...

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Abstract

The invention discloses a preparation method of a bacterial ghost of O24 type duck pathogenic escherichia coli. The preparation method comprises the steps of strain isolation, serotype identification,bacteriolysis plasmid construction, bacterial ghost preparation and inactivation treatment, wherein bacteriolysis plasmid construction is conducted on pathogenic escherichia coli strain with serotypebeing O24 by using a pBV221 carrier in bacteriolysis plasmid construction; and beta-propiolactone is adopted in inactivation treatment. The preparation process is simple, an inductive agent, formaldehyde and freeze-drying equipment are not needed in use, the cost is lowered, the bland of the field of O24 type duck pathogenic escherichia coli in the industry is filled, and the technical problems that an inductive agent IPTG is needed, the cost is high, the steps are tedious, and animal health is influenced by the residual formaldehyde in a formaldehyde inactivated ghost are solved.

Description

technical field [0001] The invention relates to the field of sloughs, in particular to a method for preparing O24 duck pathogenic Escherichia coli sloughs. Background technique [0002] Sludge is the shell of Gram-negative bacteria made by cleavage gene. It is a bacterial empty shell that does not contain cellular contents such as nucleic acid and cytoplasm. This bacterial empty shell maintains the complete inner and outer membrane structure of the original bacteria, including membrane Various antigenic components and some adhesive properties on the surface can therefore stimulate the body to produce good humoral, cellular and mucosal immunity, and are considered as a new type of candidate vaccine. In the 1980s, Austrian scholar W. Lubitz and others first developed this unique inactivated vaccine: after Gram-negative bacteria express the cleavage protein E of phage, Gram-negative bacteria under the action of the cleavage protein and osmotic pressure A transmembrane channel ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/70C12R1/19
CPCC07K14/00C12N15/70
Inventor 袁橙郭长明张步彩郝福星蔡丙严唐炳红刘剑华唐晨晨
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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