Penyaringan Domain Adp-Ribosilasi Gen Toksin Daripada Burkholderia pseudomallei Virulen

The active part of bacterial toxin enzymes from Burkholderia pseudomallei, Pseudomonas aeruginosa and difteria is ADP-ribosilation domain. This particular domain is conserved among all three microorganisms. The ADP-ribosilation domain from B. pseudomallei was PCR amplified using B. pseudomallei virulent genome as a template and primers that were generated based on Pseudomonas aeruginosa ADP-ribosilation sequences. The amplification product was purified and used as a probe (HPCR2) to screen the DNA inserts from B. pseudomallei that have been cloned into expression vector pSport-I. The objective of this study was to screen eight positive clones obtained from early screening using immunoblot and antitoxin generated from rabbit. We also screened three clones that produced negative results during immunoblot screening. The results showed that only one clone (L31) from eight positive clones carried the ADP-ribosilation domain. Manual DNA sequencing of L31 using two primers was able to generate 450bp. Further analysis showed that only one polypeptide from six posibilities was able to be translated without existence of any stop codon.