斜纹夜蛾蜕皮激素受体与超气门蛋白的原核表达及活性检测

双酰基肼类杀虫剂模拟天然蜕皮激素作用影响幼虫蜕皮。昆虫蜕皮激素受体的高度敏感性和专一性要求必须建立新的杀虫活性检测技术， 以适应快速准确和大批量筛选的要求。本研究采用RT-PCR技术， 获取斜纹夜蛾Spodoptera litura蜕皮激素受体（EcR）与超气门蛋白（USP）功能域目的基因， 构建EcR、 USP功能区基因原核表达载体（pEHISEGFPTEV-EcR_cde和pEHISEGFPTEV-USP_cde）。载体经诱导表达和蛋白纯化， 获得EcR和USP功能区纯化蛋白。在蛋白浓度l mg/mL， ³H-PonA终浓度8 nmol/L的条件下， 采用放射性配基受体结合分析测定了4种药剂（虫酰肼、 呋喃虫酰肼、 抑食肼和灭幼脲）不同浓度下的放射性比活的变化。结果显示： 随着药剂浓度的逐渐增大， 前3种药剂的放射性比活都有不同程度的降低， 其中虫酰肼的放射性比活降低程度最大， 其次是呋喃虫酰肼和抑食肼， 灭幼脲的放射性比活基本无变化。这些结果表明相同条件下虫酰肼比呋喃虫酰肼和抑食肼有更高的杀虫活力， 本研究的方法可对双酰基肼类杀虫剂或者先导化合物进行初步筛选。

Prokaryotic expression and activity determination of ecdysone receptor and ultraspiracle protein from Spodoptera litura (Lepidoptera: Noctuidae)

Nonsteroidal ecdysteroid agonists act in a similar fashion with ecdysone to affect the insect pest larval molting. The ecdysone receptor is so sensitive and specific that the new screening assay technique must be set up to detect the activity of insecticidal lead compounds. In this study, the functional domains of the ecdysone receptor and ultraspiracle protein from Spodoptera litura were cloned by RT-PCR. These two fragments were inserted into expression vectors and named as pEHISEGFPTEV-EcR_cde and pEHISEGFPTEV-USP_cde, respectively, and the fusion proteins were purified after induction. With l mg/mL fusion protein and 8 nmol/L ³H-PonA, the radioligand receptor binding assays were carried out to detect the radioactivity changes in the count per minute (CPM) in treatments with four insecticides (tebufenozide, fufenozide, dibenzoylhydrazide and chlorbenzuron) at different concentrations. The results showed that when the concentrations of tested compounds gradually increased, the CPM of the first three insecticides was reduced in different degrees. Tebufenozide had the highest degree of radioactivity reduction, followed by fufenozide and dibenzoylhydrazide, while the radioactivity of chlorbenzuron had almost no change. This study indicated that among the tested four insecticides, tebufenozide has the highest insecticidal activity under the same conditions, and the method can be used to preliminarily screen out the insecticidal activity of nonsteroidal ecdysteroid agonists or lead compounds.