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Application of protective antigen Omp25c to preparation of brucellosis vaccine

A brucella, protective technology, applied in the application field of preparing brucella vaccine, can solve the problem of lack of immune protection and the like, and achieve the effect of improving immune protection effect

Inactive Publication Date: 2011-05-04
INST OF PLA FOR DISEASE CONTROL & PREVENTION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But not all immunogenic proteins are immune protective, such as Omp31, Bp26 and other immunogenic proteins are not immune protective

Method used

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  • Application of protective antigen Omp25c to preparation of brucellosis vaccine
  • Application of protective antigen Omp25c to preparation of brucellosis vaccine
  • Application of protective antigen Omp25c to preparation of brucellosis vaccine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Embodiment 1, expression and immunogenicity analysis of Omp25c protein

[0022] 1. Expression of Omp25c protein

[0023] 1. Design of expression primers

[0024] B. melitensis 16M has completed the whole genome sequencing. According to the 16M sequence and annotation information included in GenBank (GenBank No.: AE008918), the signal peptide sequence of the gene was analyzed with SignalP software and the corresponding signal peptide sequence was removed, and the protein antigen was analyzed with DNAStar software After properties and hydrophobicity, Primer Premier 5 (PREMIERBiosoft International, Palo Alto) was used to design specific expression primers for Omp25c protein: BMEI1829-E-F: 3'-ACGTGGATCCGACGCCGTCATTGAACAGG-5' and BMEI1829-E-R: 3'-AGCTAAGCTTGAACTTGTAAGCGACACCG-5'. According to the multicloning restriction site information of the expression vector pET32a, a BamH I restriction site was added to the 5' end of the upstream primer, and a Hind III restriction site...

Embodiment 2

[0031] Embodiment 2, the construction of omp25c mutant strain

[0032] 1. Primer design

[0033] According to the ORF of the omp25c gene (BMEI1829) and the sequences on both sides, design primers for amplifying the N-terminal and C-terminal homology arms. The N-terminal homology arm amplification primers are: BMEI1829-N-F and BMEI1829-N-R, 5' end enzyme The cleavage sites are KpnI and Xho I, respectively; the C-terminal homology arm amplification primers are: BMEI1829-C-F and BMEI1829-C-R, and the 5'-end restriction sites are Sal I and Hind III, respectively. In addition, mutant identification primers (Puc19K-F and BMEI1829-I-R) were designed on the inside of the kana gene and outside the homology arm of the target gene to determine the correct replacement of the omp25c gene. The designed primers and their sequences are listed in Table 1.

[0034] Table 1 Primer Sequence

[0035]

[0036]

[0037] 2. Construction of omp25c mutation cassette and mutation vector

[003...

Embodiment 3

[0041] Embodiment 3, animal experiments

[0042] 1. Experimental animals and animal immunity

[0043] 6-8 weeks old Balb / c mice (Animal Center, Academy of Military Medical Sciences) were selected and randomly divided into 4 groups, 30 mice in each group. The first group is the live attenuated M5 vaccine (China National Institute for the Control of Pharmaceutical and Biological Products); the second group is the mutant strain M5Δomp25c immunization group, and the immune dose is 2×10 5 CFU / rat; the third group was the normal saline control group, and the immunization dose was 200 μL / rat. Intraperitoneal injection was taken, and the frequency of immunization was 1 time.

[0044] 2. Detection of the survival ability of the omp25c deletion mutant strain in the spleen of mice

[0045] The mice were immunized with the vaccine strain M5 and the omp25c deletion mutant strain M5Δomp25c respectively, and the spleens of the mice were aseptically removed on the 7th, 14th and 28th days a...

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Abstract

The invention discloses protective antigen Omp25c protein of brucellosis and application thereof to preparation of a brucellosis vaccine. The test result shows that compared with a wild type vaccine strain, an omp25c mutant strain has reduced toxicity, so an omp25c gene plays a role in the toxicity of the vaccine strain. The immune level and the immune protection effect of body fluid and cells induced by an omp25c deletion strain are reduced, so the omp25c promotes the immune response and the immune protection effect of the vaccine strain. Accordingly, the omp25c protein is an important protective antigen of the brucellosis and can enhance the vaccine strain-induced immune response and the immune protection effect. The brucellosis vaccine is prepared from the omp25c protein serving as an active ingredient, so the immune protection effect of the vaccine can be improved.

Description

technical field [0001] The invention relates to a new application of the protein, in particular to the application of the Omp25c protein, an important immunoprotective antigen of the Brucella vaccine strain M5, in the preparation of the Brucella vaccine. Background technique [0002] Brucellosis (brucellosis, referred to as brucellosis) is a kind of zoonotic disease caused by Brucella. It is an important zoonotic disease and is widely prevalent in the world. Brucella can infect humans, a variety of domestic animals and wild animals. The main symptoms of patients are chronic arthritis and nerve damage, and the main symptoms of sick animals are abortion and infertility (Shang Deqiu. Progress in Brucellosis Research. Chinese Journal of Endemic Disease Control 2004, 19:204-212; Corbel, M.J. Brucellosis: an overview. Emerg Infect Dis 1997, 3:213-21.). Brucellosis not only causes huge economic losses to my country's animal husbandry production, but also seriously threatens people...

Claims

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Application Information

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IPC IPC(8): A61K39/10A61K47/42A61P39/04
Inventor 王玉飞陈泽良曲勍钟志军徐杰汪舟佳杜昕颖孙岩松黄留玉
Owner INST OF PLA FOR DISEASE CONTROL & PREVENTION
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