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Campylobacter jejuni chitose nano DNA vaccine and preparation method and use thereof

A Campylobacter jejuni, chitosan nanotechnology, applied in recombinant DNA technology, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of low antibody titer, weak preventive effect, poor immune effect, etc. To achieve the effect of convenient operation and safe immune process

Inactive Publication Date: 2008-12-31
YANGZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0010] The purpose of the present invention is to overcome the existing traditional inoculation of Campylobacter jejuni inactivated bacteria or naked DNA vaccine as a preventive vaccine, the antibody titer produced is low, the immune effect is poor, immune evasion is easy to occur, and there are only weaker preventive effects and other defects, thereby Provide a chitosan nano-DNA vaccine that can effectively reduce the amount of Campylobacter jejuni carried in chickens

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  • Campylobacter jejuni chitose nano DNA vaccine and preparation method and use thereof
  • Campylobacter jejuni chitose nano DNA vaccine and preparation method and use thereof
  • Campylobacter jejuni chitose nano DNA vaccine and preparation method and use thereof

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Embodiment 1

[0032] Embodiment 1. Campylobacter jejuni DNA vaccine of the present invention--the construction of pCAGGS-flaA DNA vaccine

[0033] The construction steps of the pCAGGS-flaA DNA vaccine of the present invention are as follows:

[0034] ① Extraction of Campylobacter jejuni genome template

[0035] Take 0.5ml of the pure culture of Campylobacter jejuni, centrifuge at 8000rpm for 5min, discard the supernatant, add 100μl of ultrapure water, mix it evenly with a pipette, boil in water for 20min, take it out and put it on ice immediately, and centrifuge at 8000rpm for 5min. Take the supernatant and store it in a -20°C refrigerator for later use.

[0036] ② Design specific primers: According to the GenBank entry sequence (gi: 30407139), use DNAstar software to analyze, select the nucleotide sequence of Campylobacter jejuni flagellin gene flaA, and design synthetic primers according to the correct reading order. Restriction sites and protective bases are added. The upstream primer...

Embodiment 2

[0053] Example 2. Identification of transient expression of exogenous flaA gene in vitro by pCAGGS-flaA DNA vaccine

[0054] To establish a transient expression system for COS-7 cells in vitro, the eukaryotic expression plasmid pCAGGS-flaA constructed in Example 1 was prepared in small quantities with QIAfilter plasma midi kit, and transfected into COS-7 cells cultured in 24-well plates via liposomes In order to form DNA-liposome complex; 48h later, the expression of flaA gene was detected by indirect immunofluorescence test. The transfected cells were washed 3 times with PBS, dried naturally, fixed with -20°C pre-cooled anhydrous methanol for 5 min, the fixative was discarded, and washed 3 times with PBS. Add 800 μL 1:400 dilution of chicken anti-Campylobacter jejuni polyantiserum, incubate in 37°C incubator for 60 min, wash with PBS three times, add 800 μL 1:160 dilution of FITC-labeled rabbit anti-chicken fluorescent secondary antibody, 37°C incubator Incubate for 40 min, ...

Embodiment 3

[0055] Embodiment 3. Preparation of chitosan-plasmid nanoparticles

[0056] Chitosan (Chitosan, hereinafter referred to as CS) was purchased from Zhejiang Jinke Biochemical Co., Ltd., batch number D070831151, yellow powder, molecular weight 201Kda, deacetylation degree 87%, viscosity 20mpa.s. Weigh 200mg of chitosan and add 800μl of acetic acid, then add SW to 5mL, shake overnight at 37°C 180r / min to completely dissolve chitosan (purchased from Zhejiang Jinke Biochemical Co., Ltd.), add water to 480mL the next day and adjust with NaOH The pH is 5.5, and the final volume is 500mL, and vacuum filtration (0.22μm membrane) is sterilized to obtain liquid a.

[0057] Dissolve the constructed pCAGGS-flaADNA vaccine in sterile 5mmoL / L Na 2 SO4 solution to 300 μg / mL to obtain liquid b.

[0058] Heat equal amounts of liquid a and liquid b to 55°C for 10-15 minutes respectively, mix evenly at a ratio of 1:1, shake for 30 seconds to obtain chitosan-coated plasmid DNA immune microcapsule...

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Abstract

The invention relates to a campylobacter jejuni chitosan nanometer DNA vaccine, a preparation method and application thereof. The campylobacter jejuni chitosan nanometer DNA vaccine is a nano-microcapsule composed of a chitosan and a recombinant plasmid; the recombinant plasmid is a pCAGGS-flaA which consists of a campylobacter jejuni flagellin gene flaA and a eukaryotic expression vector; the flagellin gene flaA has a nucleotide sequence showed by an SEQ NO.1. The campylobacter jejuni chitosan nanometer DNA vaccine is made by dissolving the constructed recombinant plasmid in chitosan solution; the vaccine can also be used for preparing the drugs for preventing chicken campylobacter jejuni. Tests prove that the vaccine can stimulate to produce specific humoral and local mucosal immune response; after oral challenge, the cloaca bacteria exhausting rate shows a significant declining trend, and the number of the campylobacter jejuni in blood, small intestine, large intestine and caecum can be decreased.

Description

technical field [0001] The invention relates to the preparation and application of recombinant plasmids and nano-plasmid nanoparticles, in particular to a campylobacter jejuni chitosan nano DNA vaccine and the preparation and application of plasmid-chitosan nanoparticles. technical background [0002] Since the accidental discovery that plasmid DNA can be transformed and expressed in muscle cells in 1990, and it was speculated that the injection of DNA into the body may produce an immune response, a wave of DNA vaccine research has rapidly set off around the world. DNA vaccine can induce humoral immunity and cellular immunity in the body, and has the effectiveness of attenuated live vaccine and the safety of subunit vaccine. It is very easy to manipulate plasmid DNA to change the quality and quantity of immune response, and it is simple, economical, The advantages of easy storage and transportation are considered to be the third generation of vaccines. In May 1995, the worl...

Claims

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Application Information

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IPC IPC(8): A61K48/00A61K9/50A61K47/36A61P31/04C12N15/11C12N15/79C12N15/31
Inventor 焦新安黄金林尹衍新李求春张弓潘志明孙林陈祥殷月兰
Owner YANGZHOU UNIV
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