Evaluate the immunogenicity of plasmid encoding pscC protein of Pseudomonas aeruginosa in rats
Pseudomonas aeruginosa is a non-sporulating Gram negative, aerobic bacillus universally distributed in natural environment such as soil and water, which causes serious lethal infections. Its main targets are immunocompromised patients with attenuated host defense function which include burn victims,...
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| Format: | Thesis |
| Language: | English English |
| Published: |
2015
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| Online Access: | https://eprints.ums.edu.my/id/eprint/38613/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/38613/2/FULLTEXT.pdf https://eprints.ums.edu.my/id/eprint/38613/ |
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| Summary: | Pseudomonas aeruginosa is a non-sporulating Gram negative, aerobic bacillus universally distributed in natural environment such as soil and water, which causes serious lethal infections. Its main targets are immunocompromised patients with attenuated host defense function which include burn victims, organ transplant and cancer patient's long term therapy involves the use of antibiotics. Currently, there is no commercially available vaccine that can confer immunity to P. aeruginosa; therefore this study was conducted to determine the antigenic potential of a protein associated with the cell surface, which could subsequently be translated into a DNA based vaccine. The complete genome of P. aeruginosa strain PA0l was screened to determine an outer membrane surface coat Type III secretion protein (pscC) which is an important virulence determinant of type III secretion system (T3SS). Specific primers used were designed based on P. aeruginosa PA0l genome sequence available at the GenBank (NP250407) and subsequently cloned into two different Escherichia coli expression vector pGS-21a and pET-22b. The recombinant protein was analyzed by MALDI TOF-TOF mass spectrometer followed by purification using size exclusion chromatography. Finally, pscC gene was cloned onto a mammalian expression vector for plasmid immunization. The pMC2. l-pscC recombinant plasmid was directly injected intramuscularly in laboratory Sprague Dawley rats. Recombinant pscC antigen induced a specific humoral immune response against the antigen which was validated by agglutination and ELISA tests. The results clearly demonstrated that anti-pscC antibody was elicited using the animal model. The antibody level increased in 3 weeks of post immunization of all experimental doses compared with control group. The surface virulence Type III secretion protein (pscC) which is encoded by the outer membrane of T3SS genes will lead to the development of commercial plasmid vaccines to induce protective immunity against virulent Pseudomonas infection. |
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