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Preparation method and application of pseudomonas aeruginosa outer membrane protein vaccine

A technology of Pseudomonas aeruginosa and outer membrane protein, applied in chemical instruments and methods, medical preparations containing active ingredients, antibacterial drugs, etc., can solve the problems that there is no Pseudomonas aeruginosa vaccine on the market, and achieve Effects against infection

Inactive Publication Date: 2017-11-28
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] No effective Pseudomonas aeruginosa vaccine is on the market to date, although a small number of vaccines have entered clinical trials

Method used

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  • Preparation method and application of pseudomonas aeruginosa outer membrane protein vaccine
  • Preparation method and application of pseudomonas aeruginosa outer membrane protein vaccine
  • Preparation method and application of pseudomonas aeruginosa outer membrane protein vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Expression, purification and coating of embodiment 1.His-OprH vaccine:

[0071] Using the genomic DNA of the PA14 strain as a template, the oprH gene fragment not containing the start codon was amplified by PCR. The above PCR product was digested and cloned into the plasmid pET28b to construct the protein expression plasmid pET28b-His-OprH. The protein expression plasmid stored in DH5α was extracted, and transformed into Escherichia coli BL21(DE3) by electroporation. The expression of His-OprH fusion protein was induced by 1 mMIPTG, the bacteria expressing the protein were collected by centrifugation and sonicated, and the His-OprH fusion protein was purified by Ni-NTA, and the purified protein was further purified by molecular sieve ( figure 1 ). The finally obtained protein was concentrated by ultracentrifugation, diluted 10 times in a coating solution containing 3% DHPC, and placed at 37° C. for 72 hours. The coated fluid is dialyzed, and the protein is concentrat...

Embodiment 2

[0072] The immune effect measurement of embodiment 2.His-OprH vaccine:

[0073] The coated His-OprH vaccine was mixed with 20mg / ml immune adjuvant Curdlan suspension 1:1, and the control sample was mixed with Curdlan by exchange buffer 1:1. After the mice were anesthetized with 7.5% anhydrous chloral, the mice were immunized through the nasal cavity, and 10 μl of the vaccine or control sample was dripped into each nostril, and each mouse was immunized with a total of 20 μl. A total of three immunizations were performed at intervals of one week, and follow-up experiments could be carried out three weeks after the last immunization was completed.

[0074] The strains required for infection were cultured to the logarithmic phase, the bacteria were washed twice with PBS, and then resuspended to the desired concentration. After the mice were anesthetized with 7.5% anhydrous chloral, the mice were infected through the nasal cavity, 10 μl was instilled into each nostril, and each mo...

Embodiment 3

[0075] Example 3. Detection of antibody levels against OprH in mouse serum after immunization:

[0076] Three weeks after the last immunization, the sera of the mice in the OprH immunized group and the control group were taken out for the experiment. The OprH protein dissolved in PBS at a concentration of 1 mg / ml was used to coat the microtiter plate at 4°C overnight, and the protein solution in each well was 100 μl. After the coating was completed, the coating solution was removed, and 200 μl of PBST solution containing 1% BSA was added for blocking, and incubated at 37° C. for 2 h. After blocking, add 100 μl of mouse serum after doubling dilution to each well, and incubate at 37°C for 1 hour. Each well was washed 3 times with PBST, then 100 μl of diluted HRP-crosslinked anti-mouse IgG antibody was added, and incubated at 37°C for 1 h. Wash again with PBST for 3 times, add 200 μl TMB horseradish peroxidase color development solution, and carry out color reaction in the dark...

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Abstract

The invention relates to a preparation method and application of a pseudomonas aeruginosa outer membrane protein vaccine. The preparation method comprises the following steps: constructing an Escherichia coli strain BL21(DE)3 / pET28b-His-OprH capable of expressing pseudomonas aeruginosa outer membrane proteins, inducing the strain to express a His-OprH fusion protein by using IPTG (Isopropyl-beta-d-Thiogalactoside), purifying by using Ni-NTA and a molecular sieve, and coating the fusion protein by using DHPC (1,2-Dihexanoyl-sn-glycero-3-Phosphocholine). The protein vaccine prepared by the method disclosed by the invention can be used for immunization of mice, and is capable of producing protective effects on two pseudomonas aeruginosa strains PA14 and PA103 of different serotypes and inducing the mice to produce an IgG antibody targeting at the pseudomonas aeruginosa OprH protein. The antibody is capable of mediating phagocytosis of bone marrow macrophage on the strain PA14.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of a novel Pseudomonas aeruginosa outer membrane protein vaccine. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa) belongs to Pseudomonas family, Pseudomonas genus, Gram-negative bacteria. It is widely distributed in nature (soil, water and air), plant surfaces, human skin, intestinal tract and respiratory tract. Pseudomonas aeruginosa can naturally resist a variety of antibiotics, and can form biofilms, making it about 1000 times more resistant to antibiotics. As a result, Pseudomonas aeruginosa infections remain difficult to clear despite increasing concentrations of antibiotics. [0003] Vaccines are an alternative strategy to combat Pseudomonas aeruginosa infection. Over the past few decades, various vaccines have been attempted in animal models of acute and chronic P. aeruginosa infection, including the O antigen of b...

Claims

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Application Information

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IPC IPC(8): A61K39/104C12N15/70C12N15/66C07K14/21C07K1/34A61P31/04A61P11/00
Inventor 吴卫辉金守光刘畅程志晖靳永新白芳陈菲夏斌潘晓磊
Owner NANKAI UNIV
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