HOMOLOGUES OF virB OPERON OF AGROBACTERIUM TUMEFACIENS IN HELICOBACTER PYLORI – CHARACTERISATION AND FUNCTIONAL EVALUATION
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Date
2004
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ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD
Abstract
Genetic and functional studies have indicated that the type IV secretion system
(TFSS) of Helicobacter pylori forms a secretion complex in the cell envelope that
protrudes towards the outside in order to inject CagA protein into gastric epithelial cells.
However, the proposed structural model is based on partial amino acid homology with
the components of the Agrobacterium tumefaciens. The molecular structures and
functional mechanisms of many of the proteins of the TFSS of H. pylori are not known.
Towards that end, the present study was aimed at cloning cagT (virB7 homologue), a
functional component of the TFSS.
The genomic DNA of H. pylori J99 was amplified using the primers designed
for the cagT gene. The purified product of cagT was subjected to double digestion with
the restriction enzymes, NcoI and XhoI. The expression vector, pRSET-A isolated by
alkaline lysis, was also digested with the same restriction enzymes. The digested
products of cagT and pRSET-A were ligated and transformed into competent E. coli
DH5 cells by giving a heat shock.
Out of the eleven colonies, which were screened for the presence of cagT by
PCR, five were found to be positive for the insert. Three of the above positive clones
were further subjected to restriction digestion with NcoI and XhoI for the confirmation
of the same. The recombinant plasmids were transformed into competent BL21(DE3)
cells for the expression studies.
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Keywords
HOMOLOGUES , OPERON, AGROBACTERIUM, TUMEFACIENS, HELICOBACTER , PYLORI – CHARACTERISATION, FUNCTIONAL, EVALUATION