Construction and application of A19 Brucella VjbR deletion mutant strain

A technology of brucella and mutant strains, applied in the field of brucella vaccine research, can solve the problems of difficulty in distinguishing naturally infected immunized animals with vaccines, inability to use pregnant animals and humans, etc.

Pending Publication Date: 2019-06-07
SHENYANG AGRI UNIV
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  • Claims
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Problems solved by technology

However, the existing Brucella bovine A19 vaccine still has strong virulence, can not be used for pregnant animals and humans, has no specific missing markers, and various serum reactions and na

Method used

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  • Construction and application of A19 Brucella VjbR deletion mutant strain
  • Construction and application of A19 Brucella VjbR deletion mutant strain
  • Construction and application of A19 Brucella VjbR deletion mutant strain

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Experimental program
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Embodiment 1A19

[0056] The construction method of embodiment 1.A19 brucella VjbR gene deletion mutant strain, comprises the following steps:

[0057] Step 1 PCR primer design

[0058] According to the whole genome sequence of the A19 Brucella strain, two pairs of specific PCR primers VjbRKan-N-F / VjbRKan-N-R and VjbRKan-C-F / VjbRKan-C-R were designed on the upstream and downstream of the VjbR gene, and the forward primers VjbRKan-N-F and The 5'-end of the reverse primer VjbRKan-C-R of the downstream fragment adds the recognition sites and protective bases of restriction endonuclease BamH I and Sal I respectively, and the reverse primer VjbRKan-N-R of the upstream fragment and the downstream fragment The reverse complementary sequences of the upstream and downstream primers of the kana resistance gene were added to the 5'-ends of the forward primers VjbRKan-C-F, respectively.

[0059]According to the pK19mobsacB vector sequence, a pair of specific primers VjbRKan-F / VjbRKan-R were designed on bo...

Embodiment 2

[0092] The virulence and immune protection of embodiment 2.A19 Brucella strain VjbR gene deletion mutant

[0093] 1. Toxicity determination of mutant strains

[0094] Take TSA (tryptose soy agar) medium (tryptone 17.0g, soytone 3.0g, glucose 2.5g, NaCl 5.0g, K 2 HP0 4 2.5g, agar 20.0g, distilled water 1000mL) A19 brucella strain grown on and the mutant strain colony of VjbR gene deletion that step 1 obtains, make 1×10 with PBS respectively 5CFU bacterial suspension, and then 75 female BALB / c mice of 6-8 weeks were randomly divided into three groups, and each mouse was injected with 0.2mLA19 Brucella strain and VjbR gene mutant strain bacterial solution intraperitoneally, respectively. Each mouse in the group was injected with 0.2 mL of normal saline as a blank control. On the 8th, 15th, 28th, 42nd and 60th days after infection, 5 mice from each group were sacrificed, the spleen was removed under aseptic conditions, weighed, and then 5 mL of PBS homogenate was added, and 10...

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Abstract

The present invention relates to a construction and an application of a A19 Brucella VjbR deletion mutant strain and belongs to the field of Brucella vaccine research. By designing PCR primers for upstream and downstream regions of VjbR and kana resistance genes, the upstream and downstream two length homologous nucleotide fragments and kana resistance genes of a target gene are amplified and directionally connected together by a PCR amplification method to obtain target gene-deficient type homologous nucleotide fragments, the obtained target gene-deficient type homologous nucleotide fragmentsare linked to cloning vectors to obtain the target gene-deficient type homologous nucleotide fragment-containing recombinant vectors, the constructed target gene-deficient type homologous nucleotidefragment-containing recombinant vectors are transformed into Brucella A19, and positive clones are screened to obtain a target gene-deficient Brucella A19 attenuated vaccine mutant strain. The vaccinemutant strain is expected to develop into a labeled attenuated vaccine and play positive promotion roles in controlling spreading of brucellosis and healthy development of national economy.

Description

technical field [0001] The invention relates to the technical field of Brucella vaccine research, in particular to the construction and application of an A19 Brucella VjbR deletion mutant. Background technique [0002] Brucellosis, also known as Mediterranean relaxation fever, Malta fever, wave fever or undulating fever, is a zoonotic systemic infectious disease caused by Brucella, which can infect animals, such as cattle, sheep, pigs, and dogs. and animals such as camels and deer. Its clinical features are long-term fever, hyperhidrosis, arthralgia, and hepatosplenomegaly. The chronic phase of the disease may cause damage to multiple organs and systems. [0003] Brucella is a Gram-negative non-motile bacterium, non-capsulated (smooth type with microcapsule), positive for catalase and oxidase, absolutely aerobic, capable of reducing nitrate, intracellular Parasitic, can survive in many kinds of domestic animals. There are six species of Brucella: Brucella malta (Brucella...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/90C12N15/65C12N15/11G01N33/569A61K39/10A61P31/04C12R1/01
Inventor 陈泽良刘宝山韩小虎隋昀原张欢刘金玲沈国顺
Owner SHENYANG AGRI UNIV
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