Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis

The food grade Lactococcus lactis is a potential vehicle for protein delivery via the oral route. This study used lactococcal strains as models for producing the wzm gene that codes for the porin protein involved in transport of Vibrio cholerae lipopolysaccharide O-antigen. The 750 bp gene fragment...

Full description

Saved in:

Bibliographic Details
Main Author:	Zamri, Hana Farizah
Format:	Thesis
Language:	English English
Published:	2011
Subjects:	Lactococcus lactis
Online Access:	http://psasir.upm.edu.my/id/eprint/21548/1/FPSK%28m%29_2011_10R.pdf
Tags:	Add Tag No Tags, Be the first to tag this record!

id	my-upm-ir.21548
record_format	uketd_dc
spelling	my-upm-ir.215482022-01-26T04:45:42Z Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis 2011-07 Zamri, Hana Farizah The food grade Lactococcus lactis is a potential vehicle for protein delivery via the oral route. This study used lactococcal strains as models for producing the wzm gene that codes for the porin protein involved in transport of Vibrio cholerae lipopolysaccharide O-antigen. The 750 bp gene fragment was PCR-amplified from Vibrio cholerae O1 clinical isolates and cloned into the L. lactis nisin-controlled gene expression vector pNZ8048. The constructs were electrotransformed into L. lactis NZ9000 host strains where transcription of the gene on the RNA level was confirmed by reverse transcriptase PCR. Sequence comparison and multiple alignment of the translated cDNA nucleotides with that of known proteins reveal the presence of conserved structural domains of the ABC-2 membrane superfamily integral membrane protein component. Due to its hydrophobic nature, whole cell L. lactis protein extract was subjected to solubilisation with the detergents sodium dodecyl sulfate (SDS) and Triton X-100 before being separated on SDS-PAGE and analysed on Western blot. In the case of the current study, solubilisation using SDS was found to be more efficient when compared to Triton X-100 in retrieving the expressed ≈ 34 kDa wzm porin as observed upon western blot analysis. ELISA readings showed that oral administration of recombinant L. lactis into New Zealand White rabbits elicited a statistically significant increase of both IgG and IgA levels (P < 0·05) when compared to the control group given only the preparation buffer. Challenge study with virulent V. cholerae O1 strains via the oral route evoked watery diarrhoea in rabbits given only the buffer throughout the immunization period, but fecal passing of both the recombinant and non-recombinant L. lactis groups were normal. This indicates a positive effect of the Lactococcal cells itself, probably towards the intestinal microbiota, in protecting against the adverse effects of V. cholerae and in evading diarrhoea. The diarrhoea lasted approximately two days in the control group, while the others were observed to be diarrhoea-free until the end of the study. Bioinformatics and molecular methods have enabled prediction and detection of the wzm protein product which, as shown in this study, possesses the potential to elicit antibody production and enhance immunity. Administration of the L. lactis bacterium through the oral route was shown to increase mucosal immunity and assist in conferring protection against the diarrhoeal-causing disease cholera. These results provide more insight into the relatively unknown product of V. cholerae wzm, while providing a potential alternative for health improvement against cholera that could be further developed for a safer, convenient, and effective method in protection and prevention of this disease. Lactococcus lactis 2011-07 Thesis http://psasir.upm.edu.my/id/eprint/21548/ http://psasir.upm.edu.my/id/eprint/21548/1/FPSK%28m%29_2011_10R.pdf application/pdf en public masters Universiti Putra Malaysia Lactococcus lactis Faculty of Medicine and Health Sciences English
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English English
topic	Lactococcus lactis
spellingShingle	Lactococcus lactis Zamri, Hana Farizah Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis
description	The food grade Lactococcus lactis is a potential vehicle for protein delivery via the oral route. This study used lactococcal strains as models for producing the wzm gene that codes for the porin protein involved in transport of Vibrio cholerae lipopolysaccharide O-antigen. The 750 bp gene fragment was PCR-amplified from Vibrio cholerae O1 clinical isolates and cloned into the L. lactis nisin-controlled gene expression vector pNZ8048. The constructs were electrotransformed into L. lactis NZ9000 host strains where transcription of the gene on the RNA level was confirmed by reverse transcriptase PCR. Sequence comparison and multiple alignment of the translated cDNA nucleotides with that of known proteins reveal the presence of conserved structural domains of the ABC-2 membrane superfamily integral membrane protein component. Due to its hydrophobic nature, whole cell L. lactis protein extract was subjected to solubilisation with the detergents sodium dodecyl sulfate (SDS) and Triton X-100 before being separated on SDS-PAGE and analysed on Western blot. In the case of the current study, solubilisation using SDS was found to be more efficient when compared to Triton X-100 in retrieving the expressed ≈ 34 kDa wzm porin as observed upon western blot analysis. ELISA readings showed that oral administration of recombinant L. lactis into New Zealand White rabbits elicited a statistically significant increase of both IgG and IgA levels (P < 0·05) when compared to the control group given only the preparation buffer. Challenge study with virulent V. cholerae O1 strains via the oral route evoked watery diarrhoea in rabbits given only the buffer throughout the immunization period, but fecal passing of both the recombinant and non-recombinant L. lactis groups were normal. This indicates a positive effect of the Lactococcal cells itself, probably towards the intestinal microbiota, in protecting against the adverse effects of V. cholerae and in evading diarrhoea. The diarrhoea lasted approximately two days in the control group, while the others were observed to be diarrhoea-free until the end of the study. Bioinformatics and molecular methods have enabled prediction and detection of the wzm protein product which, as shown in this study, possesses the potential to elicit antibody production and enhance immunity. Administration of the L. lactis bacterium through the oral route was shown to increase mucosal immunity and assist in conferring protection against the diarrhoeal-causing disease cholera. These results provide more insight into the relatively unknown product of V. cholerae wzm, while providing a potential alternative for health improvement against cholera that could be further developed for a safer, convenient, and effective method in protection and prevention of this disease.
format	Thesis
qualification_level	Master's degree
author	Zamri, Hana Farizah
author_facet	Zamri, Hana Farizah
author_sort	Zamri, Hana Farizah
title	Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis
title_short	Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis
title_full	Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis
title_fullStr	Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis
title_full_unstemmed	Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis
title_sort	cloning and immunological characterization of recombinant vibrio cholerae o-antigen transport protein expressed in lactococcus lactis
granting_institution	Universiti Putra Malaysia
granting_department	Faculty of Medicine and Health Sciences
publishDate	2011
url	http://psasir.upm.edu.my/id/eprint/21548/1/FPSK%28m%29_2011_10R.pdf
_version_	1747811497012101120

Cloning and Immunological Characterization of Recombinant Vibrio Cholerae O-Antigen Transport Protein Expressed in Lactococcus Lactis

Similar Items