Sf9细胞存在Dm0-like核纤层蛋白的证据

Evidence for the existence of Dm0-like Lamin in Sf9 cells

为了确定Sf9细胞是否存在核纤层(lamina)及其性状,该文首先用已知的昆虫的核纤层蛋白(Lamin)的基因序列在Spodobase数据库搜索Sf9细胞的同源序列,并将推导的氨基酸序列与其他物种的同源蛋白进行比对。再利用抗果蝇Lamin Dm0抗体ADL67通过免疫印迹法(Western blotting)对Sf9细胞的蛋白裂解物进行检测,并通过免疫荧光技术(immunofluore-scence)对Sf9细胞进行染色。在Spodobase数据库搜索到1条Sf9细胞的Dm0-like lamin EST序列,同源比对显示它与其他物种的Lamin存在一定的同源性,尤其与家蚕、果蝇的同源性相对较高。免疫印迹结果表明Sf9细胞裂解物中存在大小约为70ku的蛋白,免疫荧光检测表明Sf9细胞核周呈现阳性反应,这些特征与已知的其他物种的核纤层的性状相似。结果表明,Sf9细胞可能存在Dm0-like核纤层蛋白,可作为探讨杆状病毒核衣壳穿过核纤层的机制之依据.

Summary The nuclear membrane of mammalian cells was composed of inner nuclear membrane,outer nuclear membrane and perinuclear space.The lamina was localized under the nucleoplasm face of inner nuclear membrane.It has been known that the lamina was distributed in the nucleus of mammalian cells,insect cells and plant cells.Lamina plays important roles in the celluar life cycle,including DNA replication,transcription,chromatin organization as well as nuclear assembly.Moreover,lamina is an obstacle for the egress of some viruses,like herpes simplex virus（HSV）.Sf9 cells were mostly used for the infection of Autographa californica multiple nucleopolyhedrovirus（AcMNPV）.However,there was no comprehensive study to observe whether Sf9 cells also have the lamina.So,it is difficult to know how AcMNPV pass through the lamina of Sf9 cells and then arrive at the inner nuclear membrane for nuclear egress of capsids.To determine the nucleotide sequence of lamingene in Sf9 cells,we searched the Spodobase database with theknown insect lamingenes.The identity of the nucleotide and amino acid sequences of the homologous lamins was respectively analyzed.To analyze the molecular mass of Lamin of Sf9 cells,the monolayer cells were harvested and the whole-cell protein extracts were separated by SDS-polyacrylamide gel electrophoresis（SDS-PAGE）and the protein size of Lamin was detected using Drosophila Dm0 monoclonal antibody ADL67 by Western blotting.To observe the shape and distribution of lamina of Sf9 cells,the living cells were immunostained with ADL67 by immunofluorescence assay.From the Spodobase database,aDm0-like lamin EST of Sf9 cells was obtained.The nucleotide and amino acid homology comparison indicated that this sequence showed some identities to the lamins of other species,especially Bombyx mori.Western blotting assay showed that the protein size of Dm0-like Lamin of Sf9 cells was approximately70 ku.Immunofluorescence staining indicated that the observed lamina of Sf9 cells was localized evenly along with the nuclear membrane.These results indicate that the Dm0-like Lamin may exist in Sf9 cells.How the capsids of budded virus of AcMNPV pass through the lamina will depend on the structure and cellular distribution of lamina.If the distribution of lamina in the inner nuclear membrane is discontinuous,the viral capsids will cross the blind spots to arrive at the inner membrane directly.Also,We cannot exclude the possibility that the lattice of lamina of Sf9 cell is enough wide and the layers of lamina are thin,and the capsids will pass through the lattice without difficulties.On the contrary,if the distribution of lamina is compact and the layers of lamina are thick,the baculovirus may disrupt the lamina for capsids egress.This study may lay the foundation for the exploration of the mechanism of baculovirus capsids transport across the lamina.