A Study Of The Antigenicity Of Salmonella Enterica Subspecies Enterica Serovar Typhi 50 Kda Recombinant Proteins
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Date
2018-12
Authors
Teh, Boon Aun
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
S. Typhi 50 kDa outer-membrane protein (OMP) was reported to be antigenic and
has been used to develop the commercially successful Typhidot® kit. However, the
specificity of the kit varies from 37.5 to 98.8%. This might be due to contamination with
other proteins during the 50 kDa OMP preparation and it could have affected the specificity
of the 50 kDa OMP. The objective of this study was to investigate the identities of the
individual proteins contained in the 50 kDa protein complex to pinpoint the
immunodominant protein(s) responsible for its antigenicity, and thus improve the sensitivity
and specificity of the Typhidot® test. 2D-gel electrophoresis and LC-MS/MS were used to
identify the proteins in the complex. From the pool of identified proteins, TolC, GlpK, FliC
and SucB were identified as potential antigenic proteins in the 50 kDa OMP complex and
recombinant proteins were produced in E. coli. Due to the insoluble nature of the proteins,
purification was carried out using native and denaturing conditions to improve the yield of
soluble recombinant proteins. The ELISA results showed that recombinant GlpK (rGlpK)
and recombinant FliC (rFliC) against pooled typhoid sera have the highest absorbance
reading, while recombinant TolC (rTolC) and recombinant SucB (rSucB) showed low
absorbance reading. The ELISA results also showed that the antibodies reacted stronger with
the rGlpK and rFliC proteins when there were purified under native condition. This
suggested the presence of linear and conformational epitopes on these proteins. However
these two proteins were found to be unsuitable as biomarkers due to non-specific binding
with pooled normal serum and rFliC protein cross-reacted with antibodies in sera of subjects
infected with other Salmonella and non-Salmonella serovars.
Description
Keywords
Antigenicity , Salmonella Enterica