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DNA loaded Brucella ghost composite vaccine

A technology of Brucella and Brucella nucleic acid, applied in the field of Brucella slough compound vaccine, can solve the problem that the protective immune response is not high enough, the gene silence is not expressed, and the protective antigen immunogenicity is not good, etc. question

Inactive Publication Date: 2018-10-23
INNER MONGOLIA HUAXI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, there is no DNA vaccine that can replace the existing dead vaccine and attenuated vaccine. In addition to the safety problem of DNA vaccine, the main reason is that the protective immune response induced by it is not high enough
The reason may be that the selected protective antigen immunogenicity is not good or that although the constructed Brucella DNA vaccine can express the target antigen in eukaryotic cells, its expression level in animals is low or a certain During the time period, there was a situation of gene silence and non-expression

Method used

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  • DNA loaded Brucella ghost composite vaccine
  • DNA loaded Brucella ghost composite vaccine
  • DNA loaded Brucella ghost composite vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Embodiment 1, the preparation of the brucella slough composite vaccine loaded with DNA

[0074] 1. Construction of temperature-controlled cleavage type suicide plasmid PUSacB-ΔwboA-TLC

[0075] 1. Synthesis of primers

[0076] According to the upstream and downstream homology arm nucleotide sequences of the wboA gene (genebank number: HQ845203.1) of the whole genome of Brucella disclosed by GenBank, primers wboA-N-F, primers wboA-N-R, primers wboA-C-F and Primers wboA-C-R.

[0077] The nucleotide sequences of each primer are as follows:

[0078] Primer wboA-N-F: 5'-CGC GAGCTC CTGGCGTCAGCAATCAGAG-3' (the underline is the recognition site of restriction endonuclease SacⅠ);

[0079] Primer wboA-N-R: 5'-CGC GGATCC GTGCAACGACCTCAACTTCC-3' (the underline is the recognition site of restriction endonuclease BamHI);

[0080] Primer wboA-C-F: 5'-CGC GTC GAC ACGCCATCGAACCTTTATCTG-3' (the underline is the recognition site of restriction endonuclease SalI);

[0081] Prime...

Embodiment 2

[0163] The application of the brucella slough composite vaccine loaded with DNA prepared by embodiment 2, embodiment 1

[0164] 1. Safety test

[0165] 1. Take 40 healthy female BALB / C mice aged 6-8 weeks and weighing 18-20 g, and randomly divide them into 4 groups, 10 mice in each group.

[0166] 2. After completing step 1, the four groups of mice were treated as follows:

[0167] Group 1 (S2 group): each mouse was inoculated with Brucella live vaccine S2 by intraperitoneal injection; the inoculation dose was 5×10 8 CFU / piece;

[0168] The 2nd group (RBG (PVAX1-BLP) group): every mouse is prepared by intraperitoneal injection the brucella crassa slough complex vaccine of loading DNA prepared in embodiment 1; Inoculation dose is 5 * 10 9 CFU / piece;

[0169] The 3rd group (FKB group): each mouse is inoculated with Brucella formaldehyde inactivated vaccine by intraperitoneal injection; Inoculation dose is 5 * 10 9 CFU / piece;

[0170] Group 4 (negative control group): each ...

Embodiment 3

[0193] Embodiment 3, the preparation of the smooth type brucella slough compound vaccine loaded with DNA

[0194] 1. Construction of temperature-controlled cleavage suicide plasmid PUSacB-Δbp26-TLC

[0195] 1. Synthesis of primers

[0196] According to the nucleotide sequence of the upstream and downstream homology arms of the bp26 gene (genebank number: AY166769) published by GenBank, primers bp26-N-F, primer bp26-N-R, primer bp26-C-F and primer bp26 were designed and synthesized -C-R. The nucleotide sequences of each primer are as follows:

[0197] Primer bp26-N-F: 5'-CGC GAGCTC CGTGTTGTGCGCCTGAAGCGCAAT-3' (the underline is the recognition site of restriction endonuclease SacⅠ);

[0198] Primer bp26-N-R: 5'-CGC GGATCC GACGAGCATGATTGTGGAAAATGA-3' (the underline is the recognition site of restriction endonuclease BamHI);

[0199] Primer bp26-C-F: 5'-CGC GTC GAC CTTGGCCGTGTGGTGGAAATCAG-3' (the underline is the recognition site of the restriction endonuclease SalI);

...

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Abstract

The invention discloses a DNA loaded Brucella ghost composite vaccine. The preparation method comprises following steps: introducing a suicide plasmid that contains a nucleic acid molecule encoding atemperature sensitive regulatory protein cI857, a nucleic acid molecule encoding a bacteriophage splitting protein E, and a nucleic acid molecule encoding a bacterial nuclease protein A into Brucella;utilizing a homologous recombination technology to obtain recombinant Brucella; culturing the recombinant Brucella to obtain a bacterial solution, processing the bacterial solution at a high temperature, collecting bacterial cells, and adding target DNA to obtain the DNA loaded Brucella ghost composite vaccine. The composite vaccine has following advantages: (1) the vaccine has the characteristics of bacterial ghost, compared with a conventional killed vaccine or an attenuated live vaccine, the side effect of the composite vaccine is small, the safety is high, and the protection effect is good; and (2) the bacterial ghost is a safe and effective carrier for delivering DNA vaccines, can introduce nucleic acid vaccines into antigen presenting cells, and performs high efficient expression. The composite vaccine has an important meaning for controlling the epidemic spreading of brucellosis and has a wide application range.

Description

technical field [0001] The invention belongs to the field of genetic engineering drugs, in particular to a DNA-loaded Brucella slough complex vaccine. Background technique [0002] Brucellosis (brucellosis) is a zoonotic infectious disease characterized by abortion and fever caused by Brucella. animal life and health. At present, the attenuated live vaccines used for the prevention and control of brucellosis mainly include bovine type 19 attenuated strain (S19), sheep type Rev.1 attenuated strain, sheep type 5 attenuated strain (M5) and porcine type 2 attenuated strain Strains (S2), the first two are from foreign countries, and the latter two are independently developed in China. These attenuated live vaccines have played an important role in the prevention and control of brucellosis, but there are still some fatal flaws in the immune prevention process of brucellosis, which mainly manifests in two aspects: First, the currently used attenuated live vaccines are highly toxi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74A61K39/10A61P31/04C12R1/01
CPCA61K39/098A61K2039/53A61P31/04C07K14/005C12N9/22C12N2795/00022
Inventor 温丽娟王林叶布和刘裕李敏高强
Owner INNER MONGOLIA HUAXI BIOTECH
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