中华蜜蜂AcerOr2基因昆虫表达载体pIB/V5-His的构建

doi:10.3969/j.issn.1004-1524.2020.06.07

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (6): 994-999.DOI: 10.3969/j.issn.1004-1524.2020.06.07

中华蜜蜂AcerOr2基因昆虫表达载体pIB/V5-His的构建

郭丽娜¹, 赵慧婷², 任有蛇¹, 徐兵³, 姜玉锁^1,*

1.山西农业大学动物科技学院,山西太谷 030801;
2.山西农业大学生命科学院,山西太谷 030801;
3.山西农业大学工学院,山西太谷 030801

收稿日期:2019-12-05 出版日期:2020-06-25 发布日期:2020-06-24
通讯作者: *姜玉锁,E-mail:jiangyusuo-001@163.com
作者简介:郭丽娜(1987—),女,河南安阳人,博士,讲师,主要从事蜜蜂分子生物学研究。E-mail: linaguo@126.com
基金资助:
山西省“1331工程”高校科技创新(J241942004); 山西农业大学校青年科技创新(J141902180); 优秀博士来晋奖励(K271899063)

Construction of insect expression vector pIB/V5-His of AcerOr2 in Apis cerana cerana

GUO Lina¹, ZHAO Huiting², REN Youshe¹, XU Bing³, JIANG Yusuo^1,*

1. College of Animal Science and Veterinary Medicine, Shanxi Agricultural University, Taigu 030801, China;
2. College of Life Science, Shanxi Agricultural University, Taigu 030801, China;
3. College of Engineering, Shanxi Agricultural University, Taigu 030801, China

Received:2019-12-05 Online:2020-06-25 Published:2020-06-24

摘要/Abstract

摘要： 为构建能稳定高效表达中华蜜蜂气味受体AcerOr2的重组表达载体,并在Sf9昆虫细胞中表达,将目的基因AcerOr2和昆虫表达载体pIB/V5-His用BamHⅠ和EcoRⅠ做双酶切,通过T4 DNA连接酶构建成含目的基因AcerOr2的表达载体pIB/V5-His-AcerOr2。将重组DNA用脂质体转染的方法转染至Sf9细胞中,利用Western blot和免疫荧光检测AcerOr2蛋白的表达和亚细胞定位情况。结果表明,成功构建重组表达载体pIB/V5-His-AcerOr2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,融合蛋白相对分子质量为56 ku左右;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。说明构建的重组表达载体pIB/V5-His-AcerOr2能在Sf9细胞中稳定表达,为进一步研究中华蜜蜂气味受体AcerOr2的功能奠定了基础。

关键词: 中华蜜蜂, 气味受体AcerOr2, 载体构建, 亚细胞定位

Abstract: In order to construct a recombinant expression vector which can stably and efficiently express olfactory receptor AcerOr2 in Apis cerana cerana, and then express in the Sf9 insect cell, AcerOr2 was amplified and the insect expression vector pIB/V5-His were treated with BamHⅠ and EcoRⅠ, and AcerOr2 were ligated into pIB/V5-His vector using T4 DNA ligasein this experiment. The recombinant DNA was transfected into Sf9 cells by liposome transfection, and the expression and subcellular localization of AcerOr2 protein were detected by Western blot and immunofluorescence. The results showed that the recombinant expression vector pIB/V5-His-AcerOr2 was successfully constructed and a stable transfected vector was expressed. Western blot results showed that the recombinant expression vector could be expressed in the insect cell Sf9, the fusion protein was about 56 ku. Immunofluorescence showed that the recombinant expression vector was localized in the Sf9 cell membrane, which was consistent with the predicted results. The results showed that the construction of recombinant expression vector pIB/V5-His-AcerOr2 could stably express in Sf9 cell, which will lay a foundation for the further study on the function of AcerOr2 in Apis cerana cerana.

Key words: Apis cerana cerana, olfactory receptor AcerOr2, vector construction, subcellular localization

中图分类号:

S891

郭丽娜, 赵慧婷, 任有蛇, 徐兵, 姜玉锁. 中华蜜蜂AcerOr2基因昆虫表达载体pIB/V5-His的构建[J]. 浙江农业学报, 2020, 32(6): 994-999.

GUO Lina, ZHAO Huiting, REN Youshe, XU Bing, JIANG Yusuo. Construction of insect expression vector pIB/V5-His of AcerOr2 in Apis cerana cerana[J]. , 2020, 32(6): 994-999.

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中华蜜蜂AcerOr2基因昆虫表达载体pIB/V5-His的构建

Construction of insect expression vector pIB/V5-His of AcerOr2 in Apis cerana cerana

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