Purification And Action Target Of Txp40Protein Toxin From Xenorhabdus Nematophila HB310

Txp40protein is a kind of insecticidal toxin with hemocoel injecting activity which is found fromthe Xenorhabdus nematophila. The txp40genes encoding this protein is widely exists in many speciesof symbiotic bacteria of entomopathogenic nematodes and shows very strong conservatism. Theresearch objectives are to reveal the insecticidal mechanism and the role of Txp40toxin in pathogenicprocess of symbiotic bacteria, and explore the development and application potential. This experimentchooses X. nematophila HB310strain as material. Txp40protein was gotten through non-denaturing gelelectrophoresis (native-PAGE) and prokaryotic expression of txp40gene, the action targets of Txp40protein in Galleria mellonella were searched by Co-Immunoprecipitation assay. The results of theexperiments are as follow:1. The total intracellular proteins were obtained by the salting out method of85%saturatedammonium sulfate from X. nematophila HB310cells, Txp40protein was isolated and purified with8%native-PAGE from the total intracellular proteins. SDS-PAGE analysis indicated that purified Txp40protein was monomeric protein with about40kDa molecular weight. The purified protein also showedinsecticidal activity to5th larvae of Galleria mellonella by hemocoel injection. The dot blothybridization of purified protein with Txp40antibody showed positive reaction. These results imply thatthe purified protein is Txp40toxin.2. In order to get plenty of Txp40toxin, txp40gene was cloned from X. nematophila HB310andconstructed to recombinant expression vector pET28a-txp40. The recombinant plasmid pET28a-txp40was transformed into Escherichia coli BL21(txp40) and expressed by IPTG induction. The analysis ofSDS-PAGE showed that the txp40gene was successfully expressed in E. coli. The expressed Txp40product had molecular weights of about42kDa. Western blotting analysis showed that the recombinantTxp40protein could bind specifically to Txp40antibodies. Bioassay results also showed that therecombinant Txp40protein had haemocoel injection activity against G. mellonella larvae.3. In order to understand the insecticidal mechanism of Txp40protein, The action targets of Txp40toxin in the larvae of G. mellonella were detected with immune Co-immunoprecipitation. The fiveproteins were captured from total proteins of larvae of G. mellonella.. The cytochrome P450andcysteine synthase in the five proteins were identified as potential action targets by MALDI-TOF-MS.