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Cloning, expression and purification of Brucella abortus 544 recombinant protein L7L12-Truncated Omp2b

ghasemian, melina and Gheibi, Nematollah and bouzari, saeid and golshani, maryam (2016) Cloning, expression and purification of Brucella abortus 544 recombinant protein L7L12-Truncated Omp2b. Masters thesis, qazvin university of medical sciences.

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Abstract

Abstract: Background: Brucella spp. are gram negative intracellular rod shape bacteria infecting both human (Undulant fever or Malta fever) and a wide range of animals, thus bring about a considerable economical cost on governments. However, controlling of the disease depends on the prevention and eradication of the infected animals. Due to current attenuated vaccines’ limitations, such as pathogenic effect of S19 on both human and cattle or cross reaction with serological tests, extensive researches are done to find a proper vaccine. Therefore, in this study, we designed a recombinant fusion protein containing two previously demonstrated immunodominant proteins, L7/L12 (ribosomal protein) and truncated omp2b (outer membrane protein 2b), of Brucella abortus 544. These two immunogenic proteins are also conserved among B. melitensis and B. abortus genus (proved by bioinformatic studies). Material and method: To this aim Escherichia coli BL21 (DE3) was transformed with pET28a-L7/L12-Omp2b, and protein expression was then induced by IPTG for 4hours incubation. The expressed fusion protein was then purified by affinity chromatography using Ni-NTA resin. The fusion protein was confirmed by using western blot analysis using anti-his tag-rabbit antibody. Result: The Blast analysis and sequencing results confirmed the correct sequence of the fusion protein. It was successfully cloned and expressed in pET28a and western blot analysis confirmed the expression. Conclusion: The purified protein seems to be a good candidate for stimulating the cellular immune system (the main route of the immune system against intracellular pathogens) against Brucella abortus and melitensis however further studies are in progress.

Item Type: Thesis (Masters)
Subjects: R Medicine > RP Paramedical > RP105 Biotechnology
Divisions: University Thesis > Faculty of Paramedicals > Biotechnology
Depositing User: Paramedicals School Students
Date Deposited: 18 May 2016 08:47
Last Modified: 14 Jan 2017 08:25
URI: http://eprints.qums.ac.ir/id/eprint/3820

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