Recombinant bacteria obtained from Brucella abortus 104-M vaccine strain with Omp25 gene knockout and application

A Brucella and recombinant bacteria technology, applied in the biological field, can solve problems such as abortion, natural infection of antibodies, and vaccine toxicity

Active Publication Date: 2017-10-20
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, the attenuated strain of Brucella vaccine is the most effective vaccine to control Brucella in practical application, but most vaccines show certain toxicity, and when used before pregnancy, it will cause abortion, and the antibodies produced by immunity are difficult to be compared with natural infection the difference

Method used

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  • Recombinant bacteria obtained from Brucella abortus 104-M vaccine strain with Omp25 gene knockout and application
  • Recombinant bacteria obtained from Brucella abortus 104-M vaccine strain with Omp25 gene knockout and application
  • Recombinant bacteria obtained from Brucella abortus 104-M vaccine strain with Omp25 gene knockout and application

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Experimental program
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Effect test

Embodiment 1

[0076] Embodiment 1, the Omp25 gene of knockout Brucella vaccine strain 104M

[0077] In this study, the cloning vector pMD19-T Vector (hereinafter referred to as T vector) was used as the vector to construct the insertional inactivation mutant strain of Brucella. On this basis, the pMD19-T plasmid with the kana resistance gene was constructed, and the deletion mutant strain of Brucella was constructed by replacing the resistance gene. This method avoids the influence of the vector and only needs one time Mutant strains can be obtained through resistance screening. The method of fusion PCR is used to fuse the upstream and downstream homology arms of the gene to be deleted with the kanamycin resistance gene to construct a targeting fragment, wherein the resistance gene is located between the upstream and downstream homology arms of the gene. Then, the targeting fragment is connected to the T vector to construct a mutant vector, and then the deletion mutant strain of Brucella i...

Embodiment 2

[0115] Embodiment 2, the influence of Omp25 gene deletion strain △ Omp25 on bacterial virulence and immunogenicity

[0116] 1. Immunization program

[0117] 1. Determination of immune dose

[0118] To select an appropriate infection dose, 1×10 5 -1×10 8 CFU / mL of 104M bacterial solution was used to inoculate the mice by intraperitoneal inoculation, and the mice were sacrificed at different time points (4-8 days) after infection, the spleen was isolated, and the average spleen weight and the average number of bacteria contained in the spleen per gram were calculated (spleen index =spleen weight (mg) / mouse body weight (g); number of spleen bacteria in grams=total bacterial count in mouse spleen / spleen weight).

[0119] After the mice were immunized with the 104M strain and infected, the mice were observed and sacrificed 4-8 days later, the spleen was isolated, and counted by smearing (see Figure 8 ). No bacteria were isolated from the spleens of mice in the negative contro...

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Abstract

The invention discloses recombinant bacteria obtained from brucella 104M with Omp25 gene knockout for a vaccine strain and an application. The recombinant bacteria are obtained for reducing and / or inhibiting protein activity of Omp25 in brucella 104M. For the recombinant bacteria, reduction and / or inhibition of protein activity of Omp25 in brucella 104M refer / refers to inhibition or silencing of expression of an Omp25 protein coding gene in brucella 104M. Experiments prove that the recombinant bacteria are obtained by knocking out virulence gene Omp25 from brucella 104M, and delta Omp25 as a brucella attenuated vaccine candidate for human is screened out by means of research in virulence and immunogenicity of the recombinant bacteria.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant bacterium for knocking out the Omp25 gene of a Brucella 104M vaccine strain and its application. Background technique [0002] Brucellosis is a chronic infectious disease caused by Brucella (BruceLLa) infection, and it is also a major zoonotic disease that endangers public health safety worldwide. Among domestic animals, cattle, sheep, and pigs occur most frequently, and can infect humans and other domestic animals. It is characterized by inflammation of reproductive organs and fetal membranes, causing abortion in female animals, infertility in male animals, and local lesions in various tissues. Cattle, sheep, pigs, dogs, etc. are also the main source of infection for human brucellosis. Human infection with brucellosis can cause fever, arthralgia, fatigue and weakness, and some patients turn into chronic patients that are difficult to cure. Brucellosis is widespread a...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74A61K39/10A61P31/04C12R1/01
CPCA61K39/098A61K2039/522C07K14/23C12N15/74
Inventor 李山虎王秉翔周建光王莹
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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