Molecular Analysis of the EMM Gene of Group a Streptococcus Strain Di323
The epidemiological studies and characterization of group A streptococci (GAS) are mainly based on serological M and T typing, but although T typing is useful it is not M specific. In addition, it is difficult to prepare the M antisera and the increasing number of new M types makes them non-typea...
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| Main Author: | |
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| Format: | Thesis |
| Language: | English English |
| Published: |
2001
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/9320/1/FSAS_2001_53.pdf |
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| Summary: | The epidemiological studies and characterization of group A streptococci (GAS)
are mainly based on serological M and T typing, but although T typing is useful it
is not M specific. In addition, it is difficult to prepare the M antisera and the
increasing number of new M types makes them non-typeable with the available
reference sera. The M protein is a major virulence factor of GAS which is encoded
by the emm gene. The 5' ends of this gene are highly heterogenous and encode for
specificity of the M serotypes used for M typing. Therefore sequencing of the 5'
ends of the emm gene is the choice alternative to the serological typing in the
characterization of GAS when M antisera are not available.
The Malaysian GAS strain, D 1323 shows unique serotype specificity based on the
homology searches of the 5' end emm gene sequence. The emm gene of D1323
was amplified using 'all M' primers and cloned into pCR®2.1-TOPO® vector for
its sequence determination as well as into pTrcHis2-TOPO® vector for its
expression. Plasmids of positive clones in pCR®2.1-TOPO® were sequenced and the positive clones in pTrcHis2-TOPO® were analysed for protein expression by
SDS-PAGE and Western immunoblotting.
The complete deduced sequence of the emm gene ofD1323 was shown to contain
an open reading frame of 1416 nucleotides which encodes for 429 amino acid
residues of the mature M protein. There are three copies of C repeats in the
sequence. The cleavage site of a signal peptide was predicted to be located at amino
acid residue 42. Conserved regions of the C-terminus which are shared among
various M serotypes and that of the leader peptide were also determined based on
multiple sequence alignment. The M protein ofD1323 was predicted as M Class I
protein based on the alignment of the C-terminus and phylogenetic analysis. The
fusion M protein was successfully expressed in the Escherichia coli system and its
size was determined. |
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