Cloning, Expression Hemolysin Gene From Xenorhabdus Stockiae And Its Biological Activities

Xenorhabdus sp. which form mutualistic symbioses with the entomopathogenic nematodes (EPN) Steinernema and colonize the intestine of the nematodes, are the Gram-negative bacteria of the family of Enterobacteriaceae. These bacteria show multiple bioactivities such as broad-spectrum and high efficient insecticidal activities, as well as antimicrobial and antitumor. Becasuse reversible phenotypic changes (which called phase variants) may occur when they are cultured in vitro, these bacteria are divided into primary form (phase I) and secondary form (phase II) based on different phenotypes. The protein XhlA of Xenorhabdus nematophila is a cell-surface associated haemolysin that can lyse the two major prevalent types of insect immune cells plasmatocytes and granulocytes, and XhlA is required for full virulence towards Manduca sexta larvae. Xenorhabdus Stockiae was isolated from Hunan Province by our laboratory, which was rarely reported all over the world, and its natural products owned strong insecticidal, antimicrobial and anti-tumor activities.In this study, primers were designed according to the encoding gene of Xenorhabdus Stockiae hemolysin, this gene xhlA was amplified from the genome of Xenorhabdus Stockiae HN_xs01by PCR technique, and was inserted on the expression vector pET28a, then the final expression vector pET28a-xhlA was transformed into Escherichia Coli Rosetta(DE3) by addition of IPTG for XhlA expression. SDS-PAGE showed that the recombinant protein was approximately155kDa, this protein was further identified by LC-MS. After optimizing of induction initiation time, induction time, IPTG concentration and inducement temperature, those results showed that the optimal induction condition was fermented2h at37℃,0.1mmol/L IPTQ inducted4h at37℃. Under this condition, recombinant hemolysin could efficiently express. Experiment of haemolysis and phospholipase confirmed that the ultrasonication supernatant protein from engineered bacteria after inducted could lyse rabbit erythrocytes, also could hydrolyze lecithin, those showed recombinant hemolysin expressed by engineered bacteria had the haemolysis and phospholipase activity. Then cell activity test showed that the recombinant hemolysin had weak effect on murine breast tumor cells4T1, but no effect on human hepatoma cells Hep-3B and murine melanoma cells B16.The complete genome sequence of Xenorhabdus Stockiae HN_xs01which isolated from the cadavers of Galleria mellonella was analyzed, and amplified the hemolysin encoding gene successfully. Then expressed active hemolytic protein in E. coli, found the optimal induction condition, established hemolysin expression system. This study provided preliminary research foundation for purifying hemolysin protein of Xenorhabdus Stockiae, and laid a solid foundation for further study on the mechanism of mutualism and pathogenesis in Xenorhabdus, also for the application of hemolysin.