Paper Information

Journal:   IRANIAN JOURNAL OF BIOTECHNOLOGY   April 2004 , Volume 2 , Number 2; Page(s) 123 To 131.
 
Paper: 

CLONING AND EXPRESSION OF THE HETEROGENIC VACUOLATING CYTOTOXIN FROM AN IRANIAN HELICOBACTER PYLORI STRAIN

 
 
Author(s):  TALEBKHAN Y.*, MAHBOUDI F., SARRAMI R., BARKHORDARI F., AMANI M., MOHAMMADI M.*
 
* 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.
 
Keyword(s): HELICOBACTER PYLORI, ESCHERICHIA COLI, RECOMBINANT, CYTOTOXIN, VACA, HETEROGENEITY, CLONING, EXPRESSION, PURIFICATION
 
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