ANEPⅢ毒蛋白的表达调控及其抗虫效果的生物测定  

ANEPⅢ Gene Expression Regulation and Anti-insect Bioassay

作　　者：王占斌[1] 李勍[1] 严善春[1] 冯连荣[1] WANG Zhanbin;LI Qing;YAN Shanchun;FENG Lianrong(College of Forestry, Northeast Forestry University, Harbin 150040, China)

机构地区：[1]东北林业大学林学院,哈尔滨150040

出　　处：《中国生物防治学报》2018年第3期480-487,共8页Chinese Journal of Biological Control

基　　金：国家林业局"948"项目(2014-4-02)

摘　　要：蝎体内产生一系列毒素蛋白,ANEPⅢ是其中一种。试验构建重组表达载体pPIC9K-ANEPⅢ,将质粒pNJUTRXA-ANEPⅢ转化大肠杆菌BL21,通过IPTG诱导目标蛋白ANEPⅢ,并用Tricine-SDS-PAGE电泳进行定性分析,检测得到8000 kD目的蛋白。通过电转化的方法将pPIC9K-ANEPⅢ转入毕赤酵母宿主菌GS115中,筛选得到一株耐受2.0 mg/mL G418表型为His^+Mut^+的菌株。经0.5%的甲醇诱导,取不同诱导时间的表达上清液进行Tricine-SDS-PAGE电泳分析,确认7800 kD毒性目的蛋白已经获得分泌表达,在诱导72 h时表达量最高。对ANEPⅢ基因的原核与真核表达产物进行提取、初步纯化后进行了抗虫试验。结果表明,原核表达产物没有显示出生物活性,真核表达产物具有较好的生物学活性。浓度为1 mg/mL的真核表达产物,喂食舞毒蛾2龄幼虫5 d后,校正死亡率达37.9%,幼虫食叶量下降50%。黄粉虫的生物测定结果表明,喂食1 mg/mL的真核表达产物5 d后,黄粉虫幼虫的校正死亡率为20%。A series of toxin protein are created in Scorpion＇s body, anti-neuroexcitation peptide Ⅲ is one of them. The recombinant expression vector pPIC9K-ANEPⅢ was constructed, the plasmid pNJUTRXA-ANEPⅢ was transformed into BL21. The target protein was induced by IPTG, and the expressed protein was evaluated by Tricine-SDS-PAGE. The result indicated that the size of the fusion protein was about 8000 kD. The recombinant plasmid pPIC9K-ANEPⅢ was transformed into Pichia pastoris GS 115 by electro transformation, one of His＋Mut＋ strain which can survive on the 2.0 mg/mL G418 plate for expression was screened out. After inducing by 0.5% methanol, the expressed protein has been evaluated by Tricine-SDS-PAGE, the size of the secreted protein was about 7800 kD, and the highest expression level appeared at 72 h. Prokaryotic and eukaryotic expression product of ANEPⅢ gene was extracted, the insect resistance test was carried out after preliminary purification. The result showed that the prokaryotic expression product did not show biological activity, but the eukaryotic expression product had better biological activity. 2nd instar larvae of Lymantria dispar were feeded by eukaryotic expression product, which concentration was 1 mg/mL, the corrected mortality was 37.9% after 5 days, and the larval feeding capacity descended to 50%. The same experiment performed on larvae of Tenebrio molitor by eukaryotic expression product, its concentration was 1 mg·ml-1, the corrected mortality of larvae was 20.0% after 5 days.

关 键 词：抗神经兴奋肽 载体构建 蛋白表达 生物测定 

分 类 号：S476[农业科学—农业昆虫与害虫防治]