Application of Brucella GroEL protein and method for recombinant expression of Brucella GroEL protein

A Brucella and protein technology, applied in the field of preparation of Brucella subunit vaccines, can solve problems such as non-immune protection

Inactive Publication Date: 2011-09-28
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Leclerq S and colleagues cloned the Brucella GroEL gene into a mammalian expression vector to construct a DNA vaccine, but the results showed that the DNA vaccine could only induce a Th1 immune response, but no

Method used

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  • Application of Brucella GroEL protein and method for recombinant expression of Brucella GroEL protein
  • Application of Brucella GroEL protein and method for recombinant expression of Brucella GroEL protein
  • Application of Brucella GroEL protein and method for recombinant expression of Brucella GroEL protein

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Embodiment 1

[0037] Embodiment 1, recombinant expression Brucella GroEL protein

[0038] In this example, the Gateway technology was used to express the Brucella protective antigen GroEL gene in Escherichia coli to obtain the Brucella GroEL protein. It should be understood that this embodiment is only used to clearly illustrate a way to obtain the Brucella GroEL protein, and the acquisition of the Brucella GroEL protein does not exclude the introduction of other documents (such as the document 3 mentioned in the background technology) prepared in the same manner and may also be obtained from possible commercial sources.

[0039] 1. PCR amplification of Brucella protective antigen GroEL gene

[0040] 1. Primer design

[0041] The Primer 5.0 software was used to design primers for amplifying the GroEL gene (BMEII1048) and the L7 / L12 gene (BMEI0748) as a positive control, and the corresponding bridging sequence was introduced at the 5' end of each pair of primers, and the upstream was 5'-AA...

Embodiment 2

[0057] Embodiment 2, Brucella GroEL protein immunogenicity and immunoprotective verification

[0058] Following experiment uses the Brucella GroEL protein that embodiment 1 obtains to carry out, but those skilled in the art all understands, and this experimental conclusion is equally applicable to the Brucella GroEL protein that other ways (as literature introduction or commercial purchase) obtain ( Any active Brucella GroEL protein).

[0059] Experiment 1: Mice immunization, splenocyte stimulation and IFN-γ determination

[0060] Since Brucella is an intracellular parasite, cell-mediated immune response plays an important role in the body's defense against the invasion of Brucella. In order to identify whether GroEL is a candidate protective protein of Brucella, the T cell reactivity of GroEL was first verified. Test method: 6-8 weeks old female BALB / C mice (purchased from the Experimental Animal Center of the Academy of Military Medical Sciences) were randomly divided into...

Embodiment 3

[0077] Embodiment 3: the preparation of brucella subunit vaccine

[0078] References (1: Estein SM, Fiorentino MA, Paolicchi FA, et al. The polymeric antigen BLSOmp31 confers protection against Brucella ovis infection in rams. Vaccine, 2009, 27: 6704-6711. 2: Estein SM, Cassataro J, Vizcaíno N, et al.The recombinant Omp31 from Brucella melitensis alone or associated with rough lipopolysaccharide induces protection against Brucella ovis infection in BALB / c mice.Microbe and Infect, 2003, 5:85-93.) method for preparing Brucella subunit vaccine . Among them, the Brucella GroEL protein is used as a protective antigen, and immune adjuvants can also be incorporated in the preparation, including Freund's adjuvant, microbial adjuvant and their products (commonly used microorganisms include mycobacteria, Corynebacterium pumilus, Bacillus pertussis And the extract lipopolysaccharide of left blue negative bacillus, the extract substance muramyl dipeptide from mycobacteria, etc.), polynuc...

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Abstract

The invention discloses an application of Brucella GroEL protein and a method for recombinant expression of Brucella GroEL protein, in particular an application of Brucella GroEL protein used as a protective antigen in preparation of Brucella subunit vaccines, and a process for recombinant expression of the Brucella protective antigen GroEL. The experiment proves that the Brucella GroEL protein can be combined with adjuvants for immunizing to induce generation of specific antibodies and excite cell immunity, and the Brucella GroEL protein has a better immune protection effect on Brucella and can be used for preparing Brucella subunit vaccines. The Brucella GroEL protein disclosed by the invention plays an important role in epidemic prevention of Brucella, and has wide application prospects.

Description

technical field [0001] The invention relates to a recombinant expression method of Brucella GroEL protein and its application as a protective antigen in the preparation of Brucella subunit vaccines. Background technique [0002] Brucellosis caused by Brucella (referred to as brucellosis) is a typical zoonotic infectious disease, which has caused huge economic losses to my country's animal husbandry production and seriously threatened the healthy life of the people. It is an important public health issues. Brucellosis is a chronic infectious disease, preventing the occurrence of the disease is an important measure to control the disease. At present, the prevention of brucellosis is mainly achieved through vaccination. Vaccines prepared from inactivated and attenuated Brucella pathogens have received effective immune responses and immune protection effects after immunization of animals, but there are strong allergic reactions after vaccination, it is difficult to differentiat...

Claims

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Application Information

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IPC IPC(8): A61K39/10C12N15/31C12N15/63C07K14/23A61P31/04
Inventor 王玉飞陈泽良黄留玉付思美徐杰袁静杜昕颖汪舟佳宋宏彬
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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