嗜线虫致病杆菌血腔毒素Tp40的纯化和特性分析

Purification and characterization of the haemocoel insecticide Tp40 from Xenorhabdus nematophila

【目的】嗜线虫致病杆菌是一种昆虫病原线虫共生菌,它能够产生多种杀虫毒素。本研究旨在从嗜线虫致病杆菌Xenorhabdus nematophila HB310菌株的细胞内纯化新的杀虫蛋白毒素,并对其进行基因克隆和序列分析。【方法】应用盐析和制备型非变性凝胶电泳等方法纯化蛋白,再通过对5龄大蜡螟幼虫血腔注射进行活性筛选。对获得的目的蛋白与已知蛋白进行同源分析,克隆出该目的蛋白的基因序列,从而进行相应的基因和氨基酸序列分析。【结果】本研究纯化的Tp40蛋白对大蜡螟LD50为68.54ng/头,其SDS-PAGE电泳图谱只显示出一条分子量约为42kDa的多肽。Western印迹分析表明Tp40与已知的Txp40为同源蛋白,并且仅存在于细胞内。编码该蛋白的基因开放读码框全长1107bp(GenBank登录号:EU095326),编码368个氨基酸残基,预测分子量为41.5kDa,等电点为8.66,与GenBank中的其余13株昆虫病原线虫共生菌所包含的相似基因核苷酸序列及推导的氨基酸序列比较,同源性分别为85%～99%和70%～99%。【结论】Tp40蛋白具有很高的血腔杀虫活性,其基因序列具有较强的保守性,是昆虫病原线虫共生菌复合体杀虫过程中的一种关键因子。

[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. neraatophila 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. neraatophila HB310. We tested the virulence of the protein by direct injection into fifth-instar Galleria raellonella 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. raellonella 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. raellonella larvae and its gene/protein sequence is highly conserved, which play a key role during the bacterium-nematode killing host insects process.