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

Abstract

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

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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Identification the key areas of Bombyx mori Nucleopolyhedrovirus LEF-11 self-interaction

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