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Pseudomonas aeruginosa gene and DNA vaccine thereof

A Pseudomonas aeruginosa, DNA vaccine technology, applied in the field of molecular biology and infection immunity, can solve the problem of low immune efficacy of DNA vaccines

Active Publication Date: 2016-06-29
THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The research on Pa vaccine has a history of more than half a century. The research on Pa vaccine has roughly gone through several stages of inactivated and attenuated live vaccine → conjugate vaccine → genetic engineering vaccine. As a new type of genetic engineering vaccine, So far, the DNA vaccine of Pseudomonas aeruginosa is still in the stage of animal experiment research. Although some progress has been made, it has been found in the research that DNA vaccines generally have the disadvantage of low immune efficacy. (theoutermembraneproteinF, OprF) still has such problem as the DNA vaccine of antigen
Therefore, there is no DNA vaccine that can really effectively prevent and treat Pa infection so far.

Method used

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  • Pseudomonas aeruginosa gene and DNA vaccine thereof
  • Pseudomonas aeruginosa gene and DNA vaccine thereof
  • Pseudomonas aeruginosa gene and DNA vaccine thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1 pVAX1-OprF-VP22 construction method

[0022] Construction of Pseudomonas aeruginosa DNA vaccine pVAX1-OprF-VP22 by overlapping PCR

[0023] 1. PCR amplification of the OprF gene: According to the instructions of the bacterial genome extraction kit of Beijing Tiangen Biological Co., Ltd., the Pseudomonas aeruginosa genome was extracted from Pseudomonas aeruginosa PAO1, and then the open reading frame 1~ of the OprF gene sequence was amplified by primers. 1050bp (GenBank: NC_002516.2). P3: 5′-CGC GGATCC ACCATGAAACTGAAGAACAC-3'), where the BamHI restriction site is underlined; P7: 5'-CTGAAGCCAAGATGACCTCTCGCCG-3'. PCR parameters: pre-denaturation temperature and time 94°C 3min; denaturation temperature and time 94°C 30sec, annealing (refolding) temperature and time 55°C 30sec, extension temperature and time 72°C 1min, a total of 30 cycles; stop after 72°C 5min reaction.

[0024] 2. PCR amplification of the VP22 gene: pcDNA3-VP22 was used as a template, and...

Embodiment 2

[0026] Example 2 Immunization of mice with pVAX1-OprF-VP22 DNA vaccine and preparation of mouse antiserum

[0027] 1. Mice immunized with DNA vaccine: SPF level 6-8 week old healthy female BALB / c mice were randomly divided into PBS immunization group (negative control), pVAX1-OprF, pVAX1-OprF-VP22 groups, 8 mice in each group . The mice in the pVAX1-OprF and pVAX1-OprF-VP22 groups were injected intramuscularly with 20ug of the corresponding plasmid, and the negative control group was injected with PBS. The mice were immunized three times with an interval of 2 weeks.

[0028] 2. Preparation of mouse serum: The mice were killed 1 week after the last immunization, and the venous blood was collected by enucleation. The collected blood samples were placed at 37°C for 1 hour and then fully coagulated overnight at 4°C. Centrifuged at 4,000rpm for 10 minutes at 4°C to collect serum , Store at -70°C after aliquoting.

[0029] 3. Preparation of mouse T lymphocytes: the mice were killed ...

Embodiment 3

[0030] Example 3 pVAX1-OprF-VP22 enhances the humoral immunity of OprFDNA vaccine

[0031] 1. ELISA indirect method determination:

[0032] 1) Antigen coating: Dilute the purified OprF with 0.05M pH9.6 carbonate buffer at an optimal dilution (1ug / ml), add it to a polystyrene microplastic plate, 100ul per well, 4 ℃ coated overnight (16h-24h).

[0033] 2) Washing: Shake off the liquid in it, pat dry on absorbent paper, add washing solution, 300ul / well, shake, place at room temperature for 5min, and wash 3 times in total.

[0034] 3) Blocking: blocking solution 300ul / well. Incubate at 37°C for 2 hours, wash 3 times after blocking (same as before).

[0035] 4) Primary antibody incubation: Add serially diluted serum to be tested (starting at 1:500), add 100ul to each well (double wells), set blank control wells and negative controls at the same time, and incubate at 37°C for 1h.

[0036] 5) Wash 3 times (same as before).

[0037] 6) Secondary antibody incubation: dilute the se...

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Abstract

The invention discloses a pseudomonas aeruginosa OprF-VP22 gene, the application of the gene for making vaccines for controlling pseudomonas aeruginosa infection, and a pseudomonas aeruginosa DNA vaccine containing the gene.

Description

technical field [0001] The invention belongs to the fields of molecular biology and infection immunity, and in particular relates to a Pseudomonas aeruginosa OprF-VP22 gene and a DNA vaccine containing the gene. Background technique [0002] Pseudomonas aeruginosa (Pseudomonas aeruginosa, species Pa), also known as Pseudomonas aeruginosa, Pseudomonas genus, is a kind of obligate aerobic Gram-negative bacteria, widely distributed in nature, soil, water, air, normal people The skin, respiratory tract and intestinal tract of the human body all have this bacterium. The bacterium is an opportunistic pathogenic bacterium, and the body is susceptible to infection under certain conditions, such as reduced resistance, severe burns, metabolic diseases, blood diseases, malignant tumors, and postoperative or certain treatments. Pseudomonas aeruginosa is an important pathogenic bacterium of nosocomial infection. According to the national monitoring data in my country: from 1994 to 2006,...

Claims

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Application Information

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IPC IPC(8): C12N15/62A61K48/00A61K39/104A61P31/04C12R1/385
CPCA61K39/104A61K48/00A61K2039/53C07K14/21C07K2319/10
Inventor 余娴赵春景
Owner THE SECOND AFFILIATED HOSPITAL OF CHONGQING MEDICAL UNIV
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