Characterization Of STX Phage Endolysin Of Escherichia Coli O157

Escherichia coli O157 is the most popular serotype of enterohemorrhagic Escherichia coli (EHEC). It can cause Diarrhea and Hemorrhagic colitis in human, and some serious complications such as hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) and encephalitis, which could result in high mortality. First found in U.S in 1982, E.coli O157 infection sporadically or endemically happened in the world. Because of the trend of epidemic outbreak, high morbidity and mortality, and deterioration after antibiotic treatment, it became a global public health concern. To prevent and control O157 infection is the very urgent issue recently. Lysins, a protein enzyme encoded by bacteriophage, can exert lysis activity on bacteria. With the advantages of high effectiveness, synergize with antibiotics and has no drug resistance, lysins were thought to be a promising anti-bacteria drug in future application. Endolysin of Escherichia coli O157 Stx phage, was obtained by recombinant expression, and the anti-bacterial activity have been characterized in this research.Using Signal3.0P, NCBI CDD, SWISS-MODEL, and MEMSAT3 to predict the constructure and function of endolysin, there are signal peptide and enzymatic active spot within. To analyze antibacterial activity of endolysin, amplify two fragments of endolysin gene by PCR, and targeted fragments was respectively cloned into expression Escherichia coli vector pET28a, recombinant vector pET28a-LysEC1 with signal peptide and pET28a-LysEC2 without signal peptide were transformed into BL21. To investigate the active lysis effect, recombinant BL21/pET28a-LysEC1 with signal peptide and truncated recombinant BL21/pET28a-LysEC1 without signal peptide were expressed. By plate lysis and turbidity decrease test, it deduced that LysEC1 with signal peptide could lyse E.coli Min27 and MC1061, while LsyEC2 without signal peptide which doesn't has signal could not lyse these strains. Recombinant BL21/pET28a-LysEC1 was induced by IPTG and analyzed by SDS-PAGE, and result shows the expression product was about 23KD. After purification with His-trap, the recombinant LysEC1 protein was obtained and the concentration is about 1mg/ml.Lysis tests in vitro showed LysEC1 can exert effective lysis activity on different kinds of E.coli. Using turbidity decrease test to detect enzymatic unite of LysEC1, the enzyme specific activity is 800 unit/mg. The lysis spectrum of LysEC1 was analyzed, not only E. coli O157 strain Min27 and E. coli MC1061 could be lysed, but also other bacteria including E. coli O46 and E. coli O115 could be lysed too. However, there is no other bacteria apart from E. coli could be affected by LysEC1.