Preparing and immunological efficiency analysis of pasteurella multocida ghost vaccine

A technology of Pasteurella and multocida, which is applied in the field of genetic engineering, can solve the problems of unsatisfactory immune effect, failure to provide strong protection, strong virulence of attenuated live bacteria vaccine, etc., and achieve high protection effect

Inactive Publication Date: 2018-09-28
HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the unsatisfactory immune effect of traditional inactivated vaccines, they cannot provide strong protection, and the attenuated live bacteria vaccines have the risk of returning to strong virulence. Therefore, the development of a safe and effective vaccine is of great significance for the control of pasteurellosis in animals.

Method used

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  • Preparing and immunological efficiency analysis of pasteurella multocida ghost vaccine
  • Preparing and immunological efficiency analysis of pasteurella multocida ghost vaccine
  • Preparing and immunological efficiency analysis of pasteurella multocida ghost vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1: Construction method of cleavage plasmid pPBA1100-E

[0082] 1. Using the pHH43 plasmid as a template to amplify the cleavage cassette

[0083] According to the sequencing results of the pHH43 plasmid, primers were designed to amplify the cleavage cassette, that is, the temperature control element and the cleavage gene E.

[0084] Upstream primer CI857-UP: CC GAGCTC TCA GCC AAA CGT CTC TTC AGG,

[0085] Downstream primer CI857-DN: CG GGATC C TCA CTC CTT CCG CAC GTA ATT

[0086] The primers were synthesized by Invitrogen (Shanghai) Co., and SacI and BamHI restriction endonuclease sites were introduced at the 5' ends of the upstream and downstream primers, respectively. Using the pHH43 plasmid as a template, use the designed primers to amplify the cleavage cassette. The PCR reaction system is shown in Table 1-1:

[0087] Table 1-1 PCR reaction system

[0088] DNA polymerase buffer

[0089] The operating parameters of PCR were: 1) pre-denaturat...

Embodiment 2

[0135] Embodiment two: the preparation method of Pasteurella multocida bacterium shadow

[0136] 1. Preparation of Pasteurella multocida electroporation competent cells

[0137] (1) Dilute the lyophilized Pasteurella multocida serotype B:2 strain with sterilized physiological saline or culture solution, inoculate it on the LB agar plate medium with an inoculation loop, and cultivate it at 37° C. for 12 hours.

[0138] (2) Pick a single colony and inoculate it in 5 mL of LB liquid medium, place it in a shaker at 37° C., 220 rpm, and shake for 12 hours.

[0139] (3) Inoculate 5 mL of the overnight culture into 200 mL of LB liquid medium, and culture with shaking at 37°C until OD 600nm The absorbance value is around 0.4.

[0140] (4) Pre-cool the bacterial solution in an ice bath for 20-30 minutes, then divide the bacterial solution into four pre-cooled 50 mL centrifuge tubes, centrifuge at 4°C and 4000 rpm for 10 minutes, and discard the supernatant under sterile conditions. ...

Embodiment 3

[0160] Embodiment three: the preparation method of Pasteurella multocida ghost vaccine

[0161] (1) Preparation of Pasteurella multocida Inactivated Vaccine

[0162] Centrifuge the cultured Pasteurella multocida bacteria liquid at 6000g for 10 minutes, discard the supernatant, and resuspend the bacteria with PBS solution to a culture concentration of 2×10 9 CFU / mL, then add 0.3% formaldehyde by volume, and inactivate overnight at 37°C. Use a tee to fully emulsify and mix the inactivated bacterial solution with an equal volume of ISA 206 adjuvant.

[0163] (2) Preparation of Pasteurella multocida ghost vaccine

[0164] According to the test results of Examples 2, 3 and 4, the induction time and initial induction concentration were selected for induction when the lysis rate was the highest, and the induced bacterial liquid was centrifuged to collect bacterial precipitates. Then resuspend the bacteria with sterile PBS, and dilute half of the bacterial pellet with sterile PBS s...

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Abstract

The invention discloses a preparing and immunological efficiency analysis method of a pasteurella multocida ghost vaccine. The preparing method comprises the steps that a pasteurella multocida ghost is prepared, the induction time and the initial induced concentration are induced when the ghost splitting rate is maximum, an induced pasteurella multocida solution is centrifuged, and pasteurella multocida body sediments are collected; then the pasteurella multocida ghost vaccine and an inactivated vaccine are prepared through an aseptic PBS re-suspended pasteurella multocida body; and finally, the prepared pasteurella multocida ghost vaccine is applied to conduct immunization on a mouse. The result shows that the prepared pasteurella multocida ghost vaccine can induce the mouse to produce high-level humoral immunity and cellular immunity, and high protective power can be provided for the mouse against attack of pasteurella multocida. The preparing and immunological efficiency analysis method of the pasteurella multocida ghost vaccine lays a foundation for further study on animal pasteurella ghost vaccines.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the preparation of a Pasteurella multocida ghost vaccine and its immune efficacy analysis method. Background technique [0002] Pasteurellosis in animals is an acute infectious disease caused by Pasteurella infection. At present, the prevention of the disease mainly depends on vaccination, and its vaccine mainly includes inactivated bacterial vaccine, attenuated live bacterial vaccine and subunit vaccine. Due to the unsatisfactory immune effect of traditional inactivated vaccines, they cannot provide strong protection, and the attenuated live bacteria vaccines have the risk of returning to strong virulence. Therefore, the development of a safe and effective vaccine is of great significance for the control of pasteurellosis in animals. [0003] Bacterial ghost technology is a new type of vaccine technology emerging in recent years, which is suitable for most Gram-negative bacte...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/102A61P31/04C12N15/70G01N33/569G01N33/68
CPCA61K39/102A61P31/04C12N15/70G01N33/56911G01N33/6854
Inventor 张晓轩翟军军倪宏波闻晓波周玉龙
Owner HEILONGJIANG BAYI AGRICULTURAL UNIVERSITY
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