中华蜜蜂<i>AcerOr1</i>基因昆虫表达载体pIB/V5-His的构建及功能分析

郭丽娜; 赵慧婷; 任有蛇; 徐兵; 姜玉锁

doi:10.19303/j.issn.1008-0384.2020.04.001

中华蜜蜂AcerOr1基因昆虫表达载体pIB/V5-His的构建及功能分析

Construction and Functions of Expression Vector pIB/V5-His of AcerOr1 in Apis cerana

摘要

摘要:
目的构建能在Sf9昆虫细胞中稳定高效表达中华蜜蜂气味受体AcerOr1的重组表达载体，并对其功能进行分析。
方法将目的基因AcerOr1和昆虫表达载体pIB/V5-His载体用Bam HI和EcoRI做双酶切，通过T₄ DNA连接酶构建成含目的基因AcerOr1的表达载体pIB/V5-AcerOr1。将重组表达载体用脂质体转染的方法转染至Sf9细胞中，利用免疫荧光和Western blot检测AcerOr1蛋白的亚细胞定位和表达情况；利用全波长多功能酶标仪检测该受体对4种花香物质刺激后细胞内Ca²⁺浓度的变化。
结果成功构建重组表达载体pIB/V5-AcerOr1并建立稳定转染细胞系；Western blot结果证明重组表达载体能在昆虫细胞Sf9中表达；免疫荧光显示重组表达载体在Sf9细胞膜上表达，与预测结果一致。转染pIB/V5-AcerOr1的细胞受花香物质月桂酸（Lauric acid）、亚麻酸（Linolenic acid）、α-松油醇（α-Terpineol）、十一酸（Undecanoic acid）刺激时，均能引起细胞内Ca²⁺浓度升高。
结论构建的重组表达载体pIB/V5-AcerOr1能在Sf9细胞中稳定表达，并具有对气味分子的识别功能。

Abstract:
Objective To construct and determine the functions of a recombinant expression vector which could stably and efficiently express the olfactory receptor AcerOr1 in Sf9 cells of Apis cerana.
Method AcerOr1 was amplified and using T4 DNA ligase ligated into the insect expression vector pIB/V5-His treated by BamHI and EcoRI. The recombinant DNA was liposome-transfected onto Sf9. Subsequently, AcerOr1 expression was determined by Western blot, and protein subcellular localization by immunofluorescence. A full-wavelength multifunctional microplate reader was employed to determine the intracellular Ca²⁺ concentration before and after the recombinant receptor was stimulated separately with lauric acid, linolenic acid, α–terpineol, and undecanoic acid.
Result The recombinant pIB/V5-AcerOr1 was successfully constructed with a stable, transfected vector expression. The recombinant expression vector was determined to be approximately 50 kDa and localized as predicted in the Sf9 cell membrane. The intracellular Ca²⁺ concentrations in the Sf9 cells transfected with pIB/V5-AcerOr1 increased correspondingly upon the stimulations by the floral substances.
Conclusion The recombinant pIB/V5-AcerOr1 was successfully constructed with a stable expression in the Sf9 cell of the bees with the olfactory function verified by exposing the cells to aromatic chemicals.

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