Cloning, Expression And Purification Of PDF Gene Of Helicobacter Pylori And Studies On The Activity Of The Recombinant Protein

Helicobacter pylori (H. pylori), a gram-negative pathogenic bacterium, is strongly associated with peptic ulcer disease and gastric cancer. The infection rate of H. pylori in human population is very high, approaching 50% among people worldwide. The traditional therapeutic methods using antibiotics show some effects against H. pylori infection, but the bacteria is progressively developing resistance to these drugs. Thus, it is urgent and crucial to find novel drug targets for appropriate antimicrobial agent discovery against this pathogen.In bacteria, protein synthesis is initiated with N-formylmethionyl-tRNAi, resulting in N-terminal formylation of all nascent polypeptides. During elongation of polypeptide chain, peptide deformylase (PDF) catalyzes the removal of a formyl group from the N-terminal of peptides, while protein synthesis in mammalian cells does not involve N-formylation process and therefore has no need for deformylation. PDF enzyme is thought to be a unique and essential feature to prokaryotes. So it has been considered to be an attractive target for the discovery of novel antibiotics.To be a new target of therapeutic drugs, gene conservation of peptide deformylase (deformylase, def) from H. pylori needs to be verified. In this work, 14 strains were isolated from different regions of China. Bacteria DNA was extracted and used as template to amplify def gene by PCR. Def genes obtained were subsequently cloned into pGEM-T easy vector and recombinants were identified by enzyme digestion. Sequence alignment showed that this gene was extremely conserved in H. pylori. No variation in amino acid level was observed in three...