TAT-Cloan-DH-EGFP融合蛋白的原核表达及其在舞毒蛾幼虫体内的跨膜转导  

Prokaryotic expression of TAT-Cloan-DH-EGFP fusion protein and its transduction in larvae of the gypsy moth,Lymantria dispar(Lepidoptera:Lymantriidae) by oral administration

作　　者：周洲[1] 李永丽[1] 源春彦 曲良建[2] 

机构地区：[1]河南科技大学林学院,河南洛阳471003 [2]中国林业科学研究院森林生态环境与保护研究所,北京100091

出　　处：《昆虫学报》2014年第12期1361-1367,共7页Acta Entomologica Sinica

基　　金：国家自然科学基金项目(U1204324);国家高技术研究发展计划("863"计划)项目(2012AA101503)

摘　　要：【目的】为了探究滞育激素经昆虫取食的生物效应,需设计一种能够加速跨膜运输的融合蛋白,克服经昆虫消化道的降解作用。【方法】本研究以分月扇舟蛾Clostera anastomosis滞育激素基因Cloan-DH和TAT穿膜肽编码序列为基础,构建TAT-Cloan-DH融合基因;然后以增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)为标签,构建在p ET-22b载体中。在0.2 mmol/L IPTG诱导下,在大肠杆菌Escherichia coli BL21 Rosetta(DE3)中表达出TAT-CloanDH-EGFP融合蛋白。再分别将表达有TAT-Cloan-DH-EGFP和EGFP融合蛋白的大肠杆菌BL21菌体拌入人工饲料,经舞毒蛾Lymantria dispar幼虫持续取食0,8,24,48,72和90 h后,随机挑选试虫进行组织固定并制作石蜡切片,选取中肠所在体段切片进行组织荧光分析。【结果】37℃0.2 mmol/L IPTG诱导条件下,表达的TAT-Cloan-DH-EGFP融合蛋白的分子量为61 k D,主要以包涵体形式表达,融合蛋白约占细胞总蛋白含量的18.1%;随着试虫取食含TAT-Cloan-DHEGFP融合蛋白饲料的增加,虫体组织的绿色荧光强度逐渐增加,取食72 h后,组织荧光强度达到最高。【结论】实验结果说明融合蛋白TAT-Cloan-DH-EGFP可以通过消化道横向跨膜运输,到达虫体其他组织,且该过程与蛋白的摄入量成比例。【Aim】In order to explore the biological effects of diapause hormone by insect feeding,a fusion protein is designed to accelerate its transmembrane transport and overcome the degradation in insect digestive tract.【Methods】The diapause hormone from Clostera anastomosis L.（Cloan-DH） was over-expressed as a fusion protein with a transactivator of transcription（TAT） protein of HIV-1 and an enhanced green fluorescent protein（EGFP）.Escherichia coli BL21 cells expressing TAT-Cloan-DHEGFP and EGFP were added to artificial diets respectively to rear the larvae of the gypsy moth,Lymantria dispar.Paraffin-embedded sections of the abdominal segments containing the midgut were collected from larvae fed with the artificial diets for 0,8,24,48,72 and 90 h,respectively.Then cellular transduction was evaluated by observing thin sections of gastric tissues to detect intracellular EGFP.【Results】After induction with 0.2 mmol/L IPTG at 37℃,the TAT-Cloan-DH-EGFP fusion protein was over-expressed and reached up to 18.1% of total cellular proteins mainly in inclusion bodies.Strong green-fluorescent signals of EGFP were detected in the inner layer of the midgut,muscle and cuticle sections of larvae fed with TAT-Cloan-DH-EGFP.It was found that the fluorescence intensity of EGFP increased with the quantity of the fusion protein uptake by the larvae.At 72 h after feeding the artificial diets containing TAT-Cloan-DH-EGFP,the fluorescence intensity of EGFP was up to the highest.【Conclusion 】 The results indicate that TAT-Cloan-DH-EGFP can transfer across intestine and enter other tissues by oral applications,and the quantity of transferred protein increases in proportion to the intake by the larvae.

关 键 词：舞毒蛾 滞育激素 TAT穿膜肽 蛋白转导域 融合蛋白 跨膜运输 

分 类 号：Q966[生物学—昆虫学]