Cry1AH蛋白活性片段在溶液中的单体和寡聚体杀虫活性分析

摘要

[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,通过戊二醛交联试脸分析寡聚化程度,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时寡聚化有促进作用,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. [Conclasion] 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 pore-forming ability and insecticidal activity were decreased, respectively.