Characteristics Of The Insoluble Complex Formed By AcMNPV Late Expression Factor Lef-10

Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) is the type speciesof nucleopolyhedrovirus which belongs to baculoviridae. Gene expression in AcMNPV canbe divided into four stages (immediate early gene expression，early gene expression，late geneexpression and immediate late gene expression). In recent years, nineteen genes have beenidentified as late expression factor. Nine genes are involved in viral DNA replication, and tengenes directly affect late transcription. However, the researches related to lef-10have notalmost been reported.AcMNPV lef-10expression vectors constructed (pTriEx-lef-10, pTriEx-lef-10-1-52,pTriEx-lef-10-27-78, pTriEx-lef-10-1-65, pTriEx-lef-10-GFP) were transformed into BL21(DE3) cells for protein expression induced by IPTG. Besides, pTriEx-lef-10and linear bacmidwere co-transfected into sf9cells for protein expression. The result of LEF-10-GFP，LEF-10，LEF-10-1-52，LEF-10-27-78and LEF-10-1-65analysed by Western blot revealedthat C-terminal and N-terminal truncation and C-terminal fusing of GFP could prevent orpartly prevent LEF-10from forming high polymer compound, but C-terminal truncatingthirteen amino acids could not prevent high polymer compound from being formed. Wespeculate that the formation of LEF-10high polymer compound may be related to thirteenamino acids between52and65amino acids. SDS-PAGE and Western blot analysis of purifiedLEF-10protein expressed by E.coli BL21(DE3) processed with phenol–chloroform–isoamylalcohol revealed that the high molecular complex could partly disassembled byphenol–chloroform–isoamyl alcohol, and the high molecular complex contains some proteinsfrom E.coli BL21. LEF-10protein molecular weights were estimated between520kDa and3250kDa using SDD-AGE. By transmission electron microscopy (TEM), LEF-10proteincomplexes expressed by prokaryotic and eukaryotic expression systems were observed invarious sizes as sphere or near sphere.The results suggested that LEF-10protein could form high polymer compound in bothprokaryotic and eukaryotic expression systems. As the high polymer compound could not becompletely opened by strong denaturants, we speculated that covalent bonds could beinvolved in the forming of the complexes. Characteristics of the LEF-10high polymer compound were investigated in order to reveal the mechanism of the forming of highmolecular weight compound in different aspects in this study.