Cloning and Expression of the Heterogenic Vacuolating Cytotoxin From an Iranian Helicobacter pylori Strain

Document Type: Research Paper

Authors

Biotechnology Research Center, Pasteur Institute of Iran and National Biotechnology Network, Tehran, I.R.Iran.

Abstract

Several reports indicate that the nonconserved genes of Helicobacter pylori (H. pylori) in particular its cytotoxin
are widely heterogeneous among various geographic locations and this is manifested at the protein
level ranging from 5-15% which demands access to locally deduced protein antigens for inclusion into
diagnostic kits and/or inclusion as a vaccine component for the target population. We have previously
demonstrated such variations via PCR-RFLP analysis between Iranian and western H. pylori strains. vacA
gene from a selected strain of the most prevalent RFLP category among Iranian strains, was partially
sequenced which revealed 8.3% dissimilarity with reference strains at protein level. This drastic difference
prompted us to subclone the vacA coding region into an expression vector to produce the recombinant protein.
Full sequencing of the coding region demonstrated 8-9% amino acid difference with American and
German reference strains. Recombinant protein expression yielded 4% of the total E. coli proteins.
Histidine tag allowed for purification of the recombinant VacA using immobilized metal affinity chromatography
(IMAC). Identity of the recombinant protein was repeatedly confirmed by Western blot analysis using
patient serum, rabbit hyper immune serum as well as anti-His monoclonal antibody.

Keywords