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Neisseria gonorrhoeae-salmonella double-effect vaccine, and preparation method and application thereof

A technology of Neisseria gonorrhoeae and Salmonella, applied in the field of medical microbiology, achieves good safety, high loading efficiency, and high antibody level

Inactive Publication Date: 2017-03-15
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although Salmonella ghost immunity has achieved certain immune protection effects in animal models such as chickens, the immune protection effect still needs to be further improved (Nandre RM et al. Enhanced protective immune responses against Salmonella Enteritidis infection by Salmonella secreting an Escherichia coli heat-labile enterotoxin B subunitprotein Comparative Immunology, Microbiology and Infectious Diseases, 2013, 36:537-548)

Method used

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  • Neisseria gonorrhoeae-salmonella double-effect vaccine, and preparation method and application thereof
  • Neisseria gonorrhoeae-salmonella double-effect vaccine, and preparation method and application thereof
  • Neisseria gonorrhoeae-salmonella double-effect vaccine, and preparation method and application thereof

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preparation example Construction

[0033] 2. Preparation of Salmonella bacterial shadow

[0034] Insert the phage cleavage gene E into the temperature-controlled expression vector pBV220 to obtain the lytic plasmid p-E, and use p-E as a template. In order to prepare the plasmid pB-E that can induce expression in Salmonella, this part uses the PBBR-MCS2 cloning vector, The lysing cassette amplified by PCR was recombined into the above-mentioned cloning vector and transformed into Salmonella. The recombinant bacteria were inoculated in 5 ml of Kan-resistant LB liquid medium, and cultured with shaking at 200 rpm at 28°C for 8-10 hours. The next day, they were transferred to 2000ml Kan-resistant LB liquid medium at a ratio of 1:500, and cultured at 28°C with shaking at 200rpm. Bacteria were cultured to OD 600 When the value is about 0.3-0.4, rapidly raise the temperature to 42°C to induce expression to prepare Salmonella ghost SE-ghost. Bacterial solutions before and after induction were serially diluted and spr...

Embodiment 2

[0038] In the present embodiment 2, the Neisseria gonorrhoeae-Salmonella double-effect vaccine prepared in the embodiment 1 has been evaluated for immune efficacy, and its specific steps are as follows:

[0039] (1) Evaluation of immune efficacy as gonorrhea vaccine:

[0040] 1) Animal grouping and immunization:

[0041] Thirty 6-week-old SPF BALB / c mice were randomly divided into 3 groups: SE-ghost(PorB) group, pVAX1-PorB group and PBS control group. The mice were deprived of food and water 10 hours before immunization, and 100 μl of 10% sodium bicarbonate solution was orally administered to the mice half an hour before immunization, and water was given to the mice after oral administration for 3 to 4 hours. SE-ghost (PorB) group immunization dose was 1×10 9 CFU, pVAX1-PorB is 100 μg. Vaccine every two weeks, a total of 3 immunizations.

[0042] 2) Detection of humoral immune response level in immunized mice:

[0043] Two weeks after the third immunization, blood was col...

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Abstract

The invention provides a neisseria gonorrhoeae-salmonella double-effect vaccine, and a preparation method and an application thereof, wherein a salmonella ghost is used as a delivery vector for loading a neisseria gonorrhoeae PorB DNA vaccine. The preparation method mainly includes the following steps: 1) with a neisseria gonorrhoeae genome as a template, carrying out PCR amplification to obtain a porin (PorB) gene and constructing a PorB DNA vaccine; 2) constructing a temperature control expression vector pB-E, transforming into a salmonella competent state to obtain recombinant bacteria SE (pB-E), and carrying out temperature induction to prepare a salmonella ghost vaccine freeze-dried powder; and 3) incubating the neisseria gonorrhoeae PorB DNA vaccine with the salmonella ghost freeze-dried powder, to obtain the salmonella ghost-loaded neisseria gonorrhoeae PorB bacterial ghost vaccine SE ghost (PorB). The double-effect vaccine has good safety, can also induce a body to generate and increase specific humoral immune responses and mucosal immune responses on neisseria gonorrhoeae and salmonella, has relatively high antibody level, and is of great significance in prevention and treatment of gonorrhea and / or salmonella.

Description

technical field [0001] The invention relates to the field of medical microbiology, in particular to the preparation and application of a double-effect immunoenhancing vaccine based on Neisseria gonorrhoeae PorB gene vaccine-Salmonella ghost vaccine. Background technique [0002] Gonorrhea is an acute or chronic suppurative inflammation of the genitourinary system mucosa caused by Neisseria gonorrhoeae, ranking first among sexually transmitted diseases in my country. At present, about 106 million gonorrhea patients are newly added every year in the world, and the harm is very serious. With the continuous emergence of drug-resistant strains of Neisseria gonorrhoeae, antibiotic treatment is becoming more and more difficult. However, due to the lack of understanding of the mechanism by which Neisseria gonorrhoeae induces immune responses, there is still no effective gonorrhea vaccine available. The development of gonorrhea vaccine is still an important strategy for gonorrhea co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/095A61K39/112A61K47/46A61P31/04A61P13/00
CPCA61K39/0275A61K39/095A61K2039/523A61K2039/55594A61K2039/575Y02A50/30
Inventor 焦红梅杨慧贺丽赵丹李国才陈瑾
Owner YANGZHOU UNIV
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