杆状病毒LEF-11蛋白自身相互作用关键区域的鉴定

doi:10.3864/j.issn.0578-1752.2017.20.017

摘要/Abstract

摘要： 【目的】家蚕核型多角体病毒（Bombyx mori nucleopolyhedrovirus，BmNPV）是蚕业上最常见且危害最为严重的病原微生物之一，其晚期表达因子（late expression factor，lef）LEF-11对病毒的增殖具有重要作用，相关研究证明LEF-11蛋白能够发生自身相互作用形成寡聚体，本研究通过逐步截短探究LEF-11蛋白自身相互作用的关键区域。【方法】利用双分子荧光互补（bimolecular fluorescence complementation）技术和荧光定位（fluorescent co-location）技术，首先根据BmNVP lef-11及各个截短片段序列和荧光蛋白序列设计引物，通过PCR技术扩增回收得到所有片段以及荧光标签片段，并且通过限制性内切酶处理回收后，将荧光标签片段分别连接到昆虫细胞表达载体pIZ/V5-His上，构建pIZ-DsRed和pIZ-EGFP质粒，然后用相同方法构建LEF-11全长和逐步截短的LEF-11红色荧光融合蛋白载体，分别与pIZ-LEF11-GFP共同转染到家蚕BmN-SWU1细胞中，更换普通培养基48 h后固定细胞并制片，最后在荧光共聚焦显微镜下观察荧光融合蛋白表达及定位情况，鉴定LEF-11蛋白自身相互作用的关键区域；荧光双分子互补试验相关载体的构建步骤与荧光融合表达载体构建方法一致，转染处理及荧光观察方法也相同。【结果】杆状病毒LEF-11蛋白2—61和62—112端均能与LEF-11蛋白全长发生相互作用，分别共定位于细胞质和细胞核；逐步截短过程中，LEF-11蛋白N-端2—61、12—61、22—61、32—61、42—61和2—51均能与LEF-11蛋白全长共同定位于细胞质中，但其52—61、2—41未发生共定位；C-端截短片段能够与LEF-11蛋白全长发生共定位的有62—112、72—112、82—112、72—101和72—91，但92—112和72—81区域未发现共定位现象；双分子荧光互补试验结果验证LEF-11蛋白N-端片段与LEF-11蛋白全长相互作用的有2—61、12—61、22—61、32—61、42—61、2—51，而2—41、52—61截短突变体则不能，与N-端片段荧光共定位结果保持一致。【结论】鉴定到了杆状病毒LEF-11蛋白的42-51和82-91区域为其自身相互作用关键区域，可用于LEF-11相关功能研究。

关键词: 家蚕, BmNPV, LEF-11, 双分子荧光互补, 自身相互作用

Abstract: 【Objective】Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the most common and most serious pathogenic microorganisms in sericulture, and LEF-11, one of the late expression factors of baculovirus, has been verified essential for virus proliferation. Related research proves that LEF-11 has the ability to form oligomers by self-interaction. the objective of this study is to explore the key areas of LEF-11 self-interaction via gradually truncated. 【Method】Bimolecular fluorescence complementation (BiFC) and fluorescent co-localization were utilized in this study. Firstly, the primers were designed based on the sequence of BmNPV lef-11 each truncated fragment as well as fluorescent protein sequence, all of the fragments were treated with restriction endonuclease after PCR. Then, pIZ/V5-His, the insect cell expression vector, was used to construct fluorescence fusion protein vector. Next, each truncated fragment fluorescence fusion protein vector was transfected together with pIZ-LEF11-GFP in BmN-SWU1, respectively. Cells were fixed after 48 h of the replacement of ordinary medium. Finally, the expression and localization of fluorescent fusion protein were observed under fluorescence confocal microscopy. As for the BiFC essay, the construction of vectors and the way of post processing were the same with fluorescence fusion protein.【Result】Both the 2-61 and 62-112 of the baculovirus LEF-11 protein could interact with the full length of the LEF-11 protein, and co-located in cytoplasm and nucleus, respectively. In the process of gradual truncation, LEF-11 protein N-terminal 2-61, 12-61, 22-61, 32-61, 42-61 and 2-51 could co-located with the full length of LEF-11 protein in cytoplasm, but N-terminal 52-61 and 2-41 did not co-locate. LEF-11 protein C-terminal 62-112, 72-112, 82-112, 72-101 and 72-91 could co-located with the full length of LEF-11 protein, but C-terminal 92-112 and 72-81 did not co-locate. The result of BiFC essay indicated that LEF-11 protein N-terminal 2-61, 12-61, 22-61, 32-61, 42-61 and 2-51 could interact with the full length of LEF-11 protein, but the 2-41 and 52-61 truncated fragments did not interact with the full length of LEF-11 protein. It was compatible with the result of N-terminal fragments fluorescence localization.【Conclusion】The 42-51 and 82-91 truncated fragments were identified as the key areas for the baculovirus LEF-11 protein self-interaction and could be used in LEF-11 protein related functional studies.

Key words: Bombyx mori, BmNPV, LEF-11, bimolecular fluorescence complementation, self-interaction

蒋亚明，董战旗，陈婷婷，胡楠，董非凡，黄亮，唐良彤，潘敏慧. 杆状病毒LEF-11蛋白自身相互作用关键区域的鉴定[J]. 中国农业科学, 2017, 50(20): 4028-4035.

JIANG YaMing, DONG ZhanQi, CHEN TingTing, HU Nan, DONG FeiFan, HUANG Liang, TANG LiangTong, PAN MinHui. Identification the key areas of Bombyx mori Nucleopolyhedrovirus LEF-11 self-interaction[J]. Scientia Agricultura Sinica, 2017, 50(20): 4028-4035.

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