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Cloning and Expression of Some Virulent Proteins of Aeromonas Hydrophila

KrishiKosh

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Title Cloning and Expression of Some Virulent Proteins of Aeromonas Hydrophila
 
Creator Deepanjali, A.
 
Contributor Indrani Karunasagar
Pai, R.
Venugopal, M.N.
Shamsunder, B. A.
Maragal, M.M.
Krishna Bhatt, C.H.
 
Subject bacteria, proteins, biological phenomena, diseases, genes, irrigation, enzymes, electrophoresis, water, amino acids
 
Description Ph.D. Thesis
Aeromonas species are known to be of great importance both economically and medically. They form a complex group of ubiquitous Gram-negative, facultatively anaerobic, motile, water borne bacteria that are widely isolated from clinical, environmental and food samples and are considered opportunistic human pathogens and important pathogens of fish causing a variety of disease conditions. They sometimes cause serious damage in the aquaculture of freshwater fish. The epidemiological investigation of Aeromonas has been hampered by the lack of a rapid identification method, taxonomic complexicity, and the limitation of conventional methods to classify and discriminate different isolates. The pathogenesis of A. hydrophila infection is complex and multifactorial, with the involvement of a number of virulence factors. The major physiological functions attributed to the outer membrane proteins (OMPs) include maintenance of the structural integrity and morphology of the cell and porin activity, as well as a role in conjugation and bacteriophage binding. A role in bacterial virulence has been implicated for some of these proteins. OMPs are highly immunogenic bacterial components due to their exposed epitopes on the cell surface. OMPs from Aeromonas spp. have been identified as suitable targets for vaccine development in fish. Although several studies have shown that different vaccine formulations including subunit, whole killed and live attenuated vaccines may provide protection against A. hydrophila infections, there are currently no vaccines commercially available against these important pathogens. The present study was initiated with the aim of identifying useful vaccine candidate proteins of A. hydrophila that would serve to protect fish against infection by these bacteria. To achieve this, the genes coding for outer membrane proteins were cloned and expressed in E. coli. The immunogenicity of these recombinant proteins was investigated. The outcome of the investigation is summarized below:

1. A. hydrophila is autochthonous to the aquatic environment and ubiquitous in fresh water. Hence we could obtain several isolates of the organism from fish culture ponds, aquarium fish and from moribund and dead fish.
2. SDS-PAGE of whole-cell protein profiles reveals that all isolates were typable. The isolates of A. hydrophila had a differentprofile when compared with other species of Aeromonas. Slight variation in profiles within the isolates of A. hydrophila was also noticed.

3. A dendrogram of the cluster analysis based on protein profiles indicate that environmental and clinical isolates of A. hydrophila formed different clusters except for 3 environmental isolates namely Ah65, Ah41 and Ah71.

4. A. hydrophila is a highly heterogeneous group and whole cell protin profile of the organism cannot be used as an index for further characterization of isolates within this group.

5. OMP pattern of A. hydrophila was different from the pattern obtain from Aeromonas spp. The unique MP patterns can either be a feature of A. hydrophila or it could be natural selection due to the environment faced by these microorganisms or common clonal origin of these strains.

6. OMP profile indicates the presence of several proteins which may be OMPs and the immunogenicity of these OMPs need to be investigated.

7. Primers were designed to amplify genes coding for two OMPs, OMP48 of A. veronii and OMP W of A. hydrophila. Omp 48 gene could be amplified in all A. hydrophila isolates and also in A. jandaei, A. caviae, A. sobria and A. veronii. Similarly OMP W could be detected in all isolates of A. hydrophila and in A. veronii, A. sobria, A. schubertii, A. trota and A. jandaei.

8. Omp48 and OmpW were cloned and expressed in a bacterial expression system.
9. The cloned envelope protein genes were sequenced and the nucleotide sequences were compared with GenBank sequences using bioinformatic tools. The gene OMP48 showed 97% similarity with OMP 48 gene of A. veronii and OMPW showed 82% similarity with OMPW of A. hydrophila (ATCC 7966).

10. The recombinant proteins were purified by affinity chromatography and antisera were raised in rabbits and fish. The specificity and the titer of polyclonal antibodies were determined by Western blotting and plate ELISA. These recombinant proteins proved to posses’ good immunogenicity.
 
Date 2016-07-04T15:18:40Z
2016-07-04T15:18:40Z
2010-05-20
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/68427
 
Language en
 
Format application/pdf
 
Publisher Karnataka Veterinary, Animal and Fisheries Sciences University, Bidar