Purification and characterization of the haemocoel insecticide Tp40 from Xenorhabdus nematophila

Abstract:

[Objective] Xenorhabdus nematophila is an insect pathogen bacterium symbiotically associated with entomopathogenic nematode. The bacteria produce a number of toxins to overcome immune response of insect hosts and kill their hosts. We purified a novel haemocoel insecticidal protein from X. nematophila HB310, cloned and analysed gene sequence of this novel protein. [Methods] We isolated and purified the insecticidal protein by methods of salting out and native-PAGE from the intracellular proteins of X. nematophila HB310. We tested the virulence of the protein by direct injection into fifth-instar Galleria mellonella larvae. The protein was identified by western blotting. The gene of insecticidal protein was cloned by PCR and analyzed in GenBank. [Results] We purified a novel haemocoel insecticidal protein that was named as Tp40. The injectable hemocoelic potency (LD50) of Tp40 was 68.54ng/larva against fifth-instar G. mellonella larvae. The SDS-PAGE spectrum of Tp40 only showed a single ~ 42kDa band. Western blotting with an antibody that was highly specific to the known Txp40 indicated that Tp40 was homologous to the known Txp40 and only existed inside cells. The nucleotide sequences of tp40 gene have been deposited in GenBank (accession number GenBank: EU095326). The size of the open reading frame of tp40 was 1107bp, encoding a peptide of 368 amino acid residues, with a theoretical molecular weight 41.5 kDa and an isoelectricpoint (IP) 8.66. The Tp40 shared 85%-99% homology of nucleotide sequences and 70%-99% amino acids with those of 13 group strains. [Conclusion] Tp40 is high injectable virulent for G. mellonella larvae and its gene/protein sequence is highly conserved, which play a key role during the bacterium-nematode killing host insects process.

Key words:Xenorhabdus nematophila; haemocoel toxin; Galleria mellonella; gene cloning; sequence analysis