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嗜线虫致病杆菌Xenorhabdus nematophila在侵入到寄主昆虫血腔后能够成功地逃避或抑制寄主昆虫的免疫反应并快速杀死昆虫。为深入了解嗜线虫致病杆菌的杀虫机理,明确关键的致病因子,作者应用盐析和制备型非变性凝胶电泳等方法,从嗜线虫致病杆菌HB310菌株的细胞内分离纯化了一种新的杀虫蛋白——Tp40,该蛋白对大蜡螟Galleria mellonella具有高血腔注射活性,对大蜡螟5龄幼虫的LD50为68.54ng/头。本文检测了该毒素对大蜡螟幼虫的致病特性,注射Tp40毒素后,大蜡螟幼虫表现出兴奋和痉挛等症状,当以不低于(70±0.02)ng/头的剂量注射Tp40,大蜡螟幼虫均在20min内死亡,但试虫的体色、血淋巴的颜色以及血细胞的形态没有发生明显的变化。对大蜡螟体内酶活性的测定结果显示,在注射LD50剂量的Tp40蛋白后,试虫体内羧酸酯酶和乙酰胆碱酯酶活力都明显的高于对照(P<0.05),而酚氧化酶活力显著低于对照(P<0.05)。对大蜡螟幼虫中肠的组织病理学研究显示:这种42kDa蛋白能够破坏试虫的中肠组织,导致其肠壁细胞出现排列紊乱、脱落和围食膜消失。据此推测,Tp40与嗜线虫致病杆菌对寄主昆虫的免疫抑制有关,寄主中肠组织可能是其作用靶标之一。
Xenorhabdus nematophila successfully invades or inhibits the host insect’s immune response and rapidly kills the insect after it invades the bloodstream of the host insect. In order to further understand the mechanism of insecticidal action of the nematode-borne Bacillus and clarify the key causative agents, the authors isolated and purified the intracellular of Neisseria gonorrhoeae HB310 strain using methods such as salting-out and preparative non-denaturing gel electrophoresis The new insecticidal protein, Tp40, has high intracoccular injection activity against Galleria mellonella and LD50 of 68.54ng / g for 5th instar larvae of wax worm. In this paper, the pathogenicity of the toxin to C. larvae was tested. Toxoplasma gondii larvae showed signs of excitement and spasm after Tp40 toxin injection. When Tp40 was injected into the larvae at a dose of not less than (70 ± 0.02) ng / Larvae borer larvae died within 20min, but the body color of hemolymph, hemolymph color and blood cell morphology did not change significantly. The results showed that the activities of carboxylesterase and acetylcholinesterase were significantly higher than those of the control (P <0.05) after the injection of LD50 dose of Tp40 protein, while the activity of phenoloxidase Significantly lower than the control (P <0.05). Histopathology of the midgut of larvae showed that the 42kDa protein could destroy the midgut tissues of the test insects, resulting in disordered arrangement of the cells in the intestinal wall, loss of the peritoneal membrane, and disappearance of the peritrophic membrane. Therefore, it is speculated that Tp40 is associated with the immunosuppression of host insects by nematode-induced pathogenic bacilli, and the host midgut tissue may be one of its targets.