Cry1Ah蛋白活性片段在溶液中的单体和寡聚体杀虫活性分析

Toxicity analysis of activated Cry1Ah of monomeric and oligomeric forms in the solution

[目的]分析NaCl对寡聚化的影响,以及寡聚化对Cry1Ah杀虫活性的影响。[方法]利用Superdex-75分子筛,将Cry1Ah活性片段的寡聚体和单体进行分离,通过戊二醛交联试验分析寡聚化程度,结合生物活性测定,分析NaCl对寡聚化的影响以及寡聚化对Cry1Ah毒力的影响。[结果]在纯化的组分峰Ⅰ中寡聚体占总蛋白的63.35%。NaCl对寡聚化有促进作用,而寡聚体又以多种形式存在,并且表现出不同的生物活性。寡聚体峰Ⅰ、寡聚体峰Ⅱ、单体峰Ⅲ对棉铃虫的LC50分别为47.192、3.329、3.977μg/mL。[结论]NaCl对寡聚化有促进作用,而且,寡聚体的毒力比Cry1Ah活性片段的毒力有不同程度的降低,最多下降49.6倍。推测这种体外形成的寡聚体不同于昆虫体内形成的寡聚体,引起插膜穿孔能力下降,导致毒力不同。

[Objective] Our aim is to analyze the effect of sodium chloride on oligomerization, and the toxicity of oligomeric units. [Method] To analyze the effect of sodium chloride on oligomerization, and toxicity of oligomeric units, toxin protein was purified by chromatography with Superdex75 to separate the oligomeric units and monomer unit. Oligomerization reaction was detected by glutaraldehyde crosslinking, and bioassay of all the oligomeric units was performed against the larvae of Helicoverpa armigera. [Result] Oligomeric units accounted for 63.35% of total Cry1Ah toxin in Peak 1. Sodium chloride could promote oligomerization. The 50% lethal concentrations （LC50） of oligomeric units （Peak 1 and Peak 2） and monomer unit （Peak 3） against H. armigera were 47.192 μg/mL, 3.329 μg/mL and 3.977 μg/mL, respectively. [Conclusion] Sodium chloride could promote oligomerization; the toxicity of oligomeric units declined compared with Cry1Ah polypeptide： Peak1 was 49.6 fold lower than that of activated Cry1Ah polypeptide. We deduced that the oligomerization was different in structure between vivo and in vitro reactions, and thereby poreforming ability and insecticidal activity were decreased, respectively.