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

Abstract: 【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-DH-EGFP 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.

Key words: Lymantria dispar, diapause hormone, transactivator of transcription (TAT), protein transduction domain (PTD), fusion protein, transmembrane transport