Sf9細胞存在Dm0-like核纖層蛋白的證據(jù)

衛(wèi)文強?, 季少平?*, 張銀燕

(河南大學醫(yī)學院,河南 開封475004)

摘要為了確定Sf9細胞是否存在核纖層(lamina)及其性狀,該文首先用已知的昆蟲的核纖層蛋白(Lamin)的基因序列在Spodobase數(shù)據(jù)庫搜索Sf9細胞的同源序列,并將推導的氨基酸序列與其他物種的同源蛋白進行比對。再利用抗果蠅Lamin Dm0抗體ADL67通過免疫印跡法(Western blotting)對Sf9細胞的蛋白裂解物進行檢測,并通過免疫熒光技術(immunofluore-scence)對Sf9細胞進行染色。在Spodobase數(shù)據(jù)庫搜索到1條Sf9細胞的Dm0-like lamin EST序列,同源比對顯示它與其他物種的Lamin存在一定的同源性,尤其與家蠶、果蠅的同源性相對較高。免疫印跡結果表明Sf9細胞裂解物中存在大小約為70 ku的蛋白,免疫熒光檢測表明Sf9細胞核周呈現(xiàn)陽性反應,這些特征與已知的其他物種的核纖層的性狀相似。結果表明,Sf9細胞可能存在Dm0-like核纖層蛋白,可作為探討桿狀病毒核衣殼穿過核纖層的機制之依據(jù).

關鍵詞Sf9細胞； 核纖層； 免疫熒光； 免疫印跡

中圖分類號Q 96文獻標志碼A

Evidence for the existence of Dm0-like Lamin in Sf9 cells. Journal of ZhejiangUniversity(Agric. & LifeSci.), 2015,41(3)：245-251

Wei Wenqiang?, Ji Shaoping?*, Zhang Yinyan (MedicalCollege,HenanUniversity,Kaifeng475004,Henan,China)

SummaryThe 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 ofAutographacalifornicamultiple 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 oflamingene in Sf9 cells, we searched the Spodobase database with the known insectlamingenes. The identity of the nucleotide and amino acid sequences of the homologouslaminswas 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 usingDrosophilaDm0 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-likelaminEST of Sf9 cells was obtained. The nucleotide and amino acid homology comparison indicated that this sequence showed some identities to thelaminsof other species, especiallyBombyxmori. Western blotting assay showed that the protein size of Dm0-like Lamin of Sf9 cells was approximately 70 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.

Key wordsSf9 cells; lamina; immunofluorescence; Western blotting

核纖層(lamina)是真核細胞核膜的組成部分,位于內核膜的核質面。核纖層由核纖層蛋白(Lamin)組成,在維持核膜的形態(tài)、保持染色質的有序性、基因表達、細胞分裂、核膜的解體和重建等過程中發(fā)揮重要作用[1-4]。人類lamin的突變與多種疾病有關,例如,早衰綜合征、核纖層蛋白病、右心室心律失常型心肌癥、新疆維吾爾族特發(fā)性擴張性心肌病等[5-10].lamin突變在乳腺癌、結腸癌的發(fā)生和發(fā)展中也可能起一定的作用[11-13]。Lamin性狀受蛋白激酶的調控。細胞或病毒編碼的蛋白激酶可磷酸化Lamin導致其溶解[14-15]；JIL-1激酶與果蠅Dm0蛋白相互作用,影響細胞核的形態(tài)與完整性[16]。對各種生物Lamin結構與功能的研究,豐富了人們對核纖層的認識,為人類lamin突變所致疾病的治療奠定了理論基礎[17-18]。目前已知Lamin在人、小鼠、果蠅等真核生物的細胞內均存在。

核纖層附著在內核膜上,核纖層纖維排列通常比較致密,病毒等大分子很難通過它。而病毒則通過某種機制打破核纖層,以促進子代病毒的出核。對單純皰疹病毒(herpes simplex virus,HSV)、巨細胞病毒(human cytomegalovirus,HCMV)、鼻咽癌病毒(epstein-barr virus,EBV)等動物細胞的DNA病毒研究表明,子代病毒粒子出核必須打破核纖層[19-23]。昆蟲細胞Sf9是來自草地貪夜蛾(Spodopterafrugiperda)蛹期卵巢組織的商業(yè)細胞系,是研究桿狀病毒模式株AcMNPV感染機制的常用細胞。研究表明,桿狀病毒感染后在細胞核內裝配后的核衣殼沿著內核膜排列,通過出芽的方式進入核周間隙[24-25]。通過麥胚凝集素-膠體金(WGA-gold)標記核膜技術發(fā)現(xiàn),桿狀病毒感染致核膜向核質內伸出形成突起,核衣殼則移向此突起,沿著突起上形成的小孔,移向細胞質[26]。這使我們想到Sf9細胞是否存在核纖層,以及桿狀病毒如何穿越核纖層這個有趣的問題。但對Sf9細胞核纖層的研究目前還比較少,限制了對該問題的理解。

本研究利用抗果蠅Lamin Dm0單抗ADL67通過免疫印跡法對Sf9細胞裂解物進行檢測;再利用抗體ADL67通過免疫熒光技術染色Sf9細胞并在熒光共聚焦顯微鏡下觀察。同時,用其他昆蟲的已知lamin序列在Spodobase[27]數(shù)據(jù)庫(專門存放來自草地貪夜蛾的EST序列數(shù)據(jù)庫)搜索Sf9細胞的lamin同源序列,并進行核苷酸和氨基酸序列比對。結果初步表明,Sf9細胞存在Dm0-like核纖層蛋白。

1材料與方法

1.1材料和試劑

1.1.1昆蟲細胞Sf9細胞.用含10%胎牛血清的Grace培養(yǎng)基置于28 ℃培養(yǎng).胎牛血清購自Gibco公司.Grace培養(yǎng)基購自Invitrogen公司.

1.1.2抗體ADL67抗體由美國石溪大學Paul A。Fisher教授饋贈.四甲基異硫氰酸羅丹明(tetraethyl rhodamine isothiocyanate,TRITC)標記的羊抗鼠IgG抗體購自北京中衫金橋生物技術公司。辣根過氧化物酶(horseradish peroxidase,HRP)標記的羊抗鼠抗體購自北京博奧森生物技術公司。

1.2方法

1.2.1序列比對用已知的lamin基因搜索Spodobase數(shù)據(jù)庫,通過Multalian在線軟件(http://multalin.toulouse.inra.fr/multalin/)將搜到的Sf9細胞Dm0-likelaminEST序列及推導出的氨基酸序列與其他物種的lamin進行比對,并用ClustalW2在線軟件(http://www.ebi.ac.uk/tools/msa/clustalw2/)分析它們的核苷酸和氨基酸的同源性.

1.2.2免疫印跡Sf9細胞(1×106/35-mm小皿)收獲后加入5×蛋白上樣緩沖液,煮樣10 min后上樣進行十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-polyacrylamide gel electrophoresis,SDS-PAGE),將蛋白條帶經濕轉法轉到PVDF膜上。5%脫脂奶粉室溫孵育1 h。以單抗ADL67(1∶100)4 ℃過夜孵育,用帶吐溫-20的Tris緩沖鹽溶液(tris-buffered saline with tween-20,TBST)洗3次,每次10 min。加入辣根過氧化物酶標記的羊抗鼠二抗(1∶2 500)室溫孵育1 h,TBST洗3次,每次10 min。同時,取表達Dm0的細菌裂解物(由Nico Stuurman教授饋贈的質粒pETDmLFL在BL21細胞中誘導表達果蠅Dm0蛋白)作為陽性對照。最后以化學發(fā)光法檢測Lamin.

1.2.3免疫熒光檢測Sf9細胞(1×106/35-mm小皿)以4%多聚甲醛室溫固定10 min,再用磷酸緩沖鹽溶液(phosphate buffer saline,PBS)洗3遍.然后,分別以0.15% Triton X-100處理10 min,100%純甲醇-20 ℃處理20 min,PBS洗滌3次,每次5 min.以1%牛血清白蛋白(albumin from bovine serum,BSA)37 ℃封閉1 h.加ADL67(1∶10)室溫過夜孵育,用帶吐溫-20的磷酸緩沖鹽溶液(phosphate buffer saline with tween-20,PBST)洗3次,每次5 min.加四甲基異硫氰酸羅丹明標記的羊抗鼠二抗(1∶100),室溫孵育1 h,PBST洗3次,每次5 min.加入Hoechst 33258,室溫孵育20 min.用PBST洗3次.將細胞置于熒光共聚焦顯微鏡下,觀察細胞核內核纖層的分布、形態(tài)特征.

2結果

2.1Sf9細胞Dm0-like Lamin的大小

為了確定Dm0-like Lamin的分子質量,取Sf9細胞總蛋白,經SDS-PAGE后轉移到PVDF膜上,然后用抗體ADL67通過免疫印跡進行檢測.結果顯示,在大約70 ku處有1條明顯的陽性條帶,其大小與果蠅的Dm0蛋白接近(圖1).

Dm:果蠅的Dm0蛋白；Sf9:Sf9細胞的Dm0-like Lamin;M:蛋白標志物. Dm: Dm0 protein of D. melanogaster; Sf9: Dm0-like Lamin of Sf9 cells; M: Protein marker. 圖1　免疫印跡檢測Sf9細胞Dm0-like Lamin Fig.1　Detection of Dm0-like Lamin of Sf9 cells by Western blotting

2.2Sf9細胞Dm0-like Lamin與其他物種同源蛋白序列比對

為了確定Sf9細胞是否存在lamin序列,我們用已知的lamin序列搜索Spodobase數(shù)據(jù)庫.結果搜到1條來自Sf9細胞的Dm0-likelamin同源序列(Sf9LR450003-5-1-C1112),再由其核苷酸序列推導出氨基酸序列.核苷酸序列比對結果顯示,Sf9Dm0-likelamin與其他物種的lamin核苷酸序列同源性達56%～80%,其中,與家蠶的同源性達80%,與果蠅的同源性達64%(圖2A),但與其他物種lamin同源性相對較低.氨基酸序列比對結果顯示,Sf9 Dm0-like Lamin與其他物種的同源蛋白同源性達16%～96%.該序列與家蠶的Lamin同源性最高(96%),其次是果蠅(59%),而與其他物種的同源性較低(圖2B).

其他物種包括：Bombyx:家蠶(GI:512922266);Drosophila:果蠅(GI:667674288)；Mus:小鼠(GI:15929760);Homo:人(GI:224901);Xenopus:非洲爪蟾(GI:156119432). Other species include Bombyx (Bombyx mori， GI: 512922266)， Drosophila (Drosophila melanogaster， GI: 667674288)， Mus (Mus musculus， GI: 15929760)， Homo (Homo sapiens， GI: 224901)， Xenopus (Xenopus laevis， GI: 156119432). 圖2　Sf9和其他物種Lamin的核苷酸(A)與推導的氨基酸(B)序列比對 Fig.2　Comparison of nucleotide (A) and deduced amino acid (B) sequences of Lamin among Sf9 cells and other species

2.3Sf9細胞Dm0-like Lamin的定位

為了觀察Sf9細胞是否存在核纖層及核纖層蛋白的細胞定位,使用抗體ADL67對Sf9細胞進行免疫熒光染色.同時,用Hoechst 33258對細胞核進行染色.熒光共聚焦顯微鏡觀察顯示在Sf9細胞的核外周分布一層環(huán)狀的蛋白,其形狀不規(guī)則,有的呈規(guī)則的圓形,有的則呈橢圓形,這與其他物種的核纖層形態(tài)特征相似(圖3).

圖3　免疫熒光檢測Dm0-like Lamin在Sf9細胞的定位 Fig.3　Localization of Dm0-like Lamin of Sf9 cells observed by immunofluorescence

3討論

本研究通過生物信息學分析、免疫印跡、免疫熒光等方法初步證明Sf9細胞存在Dm0-likelamin.這為下一步研究桿狀病毒核衣殼穿過核纖層的機制提供了依據(jù).

利用已知的昆蟲lamin序列,在Spodobase[27]數(shù)據(jù)庫進行序列同源性搜索,結果找到1條Sf9細胞的Dm0-likelaminEST序列.生物信息學分析表明,該序列及其翻譯后的氨基酸序列與其他物種的Lamin具有一定的同源性,尤其與家蠶的Lamin同源性最高.Nico Stuurman通過對Lamin內含子的進化分析認為,目前在昆蟲中僅知道果蠅存在2種Lamin蛋白,即Dm0和Lamin C,Lamin C為果蠅所獨有.而家蠶僅有1種Lamin蛋白.Sf9細胞也很可能只存在1種Lamin蛋白[28-29].所以,抗體ADL67檢測到的細胞蛋白可能是由搜到的這個Dm0-likelamin基因表達。隨后,根據(jù)這段EST序列設計特異性引物,利用從Sf9細胞提取的RNA進行反轉錄PCR(reverse transcription PCR,RT-PCR),擴增到預期大小的條帶(未展示),測序結果也符合預期.該段序列的發(fā)現(xiàn)為下一步克隆Sf9Dm0-likelamin基因全長并深入研究其蛋白結構與功能奠定了基礎.

雖然在Spodobase數(shù)據(jù)庫搜到1個Sf9Dm0-likelaminEST序列,但其完整的開放閱讀框(open reading frame,ORF)尚未獲得,故其蛋白大小也無法預測.盡管Sf9 Dm0-like Lamin與家蠶Lamin的同源性最高,但家蠶Lamin的抗體市面上尚沒有.而針對果蠅Dm0的單抗ADL67目前已經存在,而且Sf9 Dm0-like Lamin與果蠅的Dm0 Lamin的同源性也達到59%.所以,我們嘗試用該抗體對Sf9細胞的Dm0-like Lamin的性狀進行分析.利用ADL67對Sf9全細胞裂解物進行免疫印跡檢測,結果檢測到大小約為70 ku的蛋白條帶.果蠅的Dm0蛋白(NM_001258963)大小為71 ku.這說明Lamin蛋白大小在不同昆蟲間可能是相對保守的.根據(jù)氨基酸序列比對的結果可知,獲得的Sf9細胞的Dm0-likelaminEST翻譯后的氨基酸序列是Lamin N端的一部分,而ADL67的抗原識別表位對應果蠅的Dm0的C端的548～620位氨基酸,故目前無法對果蠅ADL67抗原表位與Sf9細胞Dm0-like Lamin的序列同源性進行比對.但ADL67能識別Sf9細胞的Dm0-like Lamin,說明ADL67識別表位在不同昆蟲Lamin間可能具有較高的保守性.

核纖層蛋白一般分布于真核細胞核膜的核質面,熒光顯微鏡下觀察呈環(huán)形.為了確定抗體ADL67檢測到的70 ku左右的蛋白在Sf9細胞內是否也定位于核膜,再次利用該抗體進行了免疫熒光實驗.結果顯示,在細胞核的周圍觀察到環(huán)狀的陽性熒光信號，與其他物種的細胞核纖層形態(tài)、分布相似.免疫熒光實驗直觀地說明Sf9細胞存在核纖層蛋白.

基于上述實驗結果,初步認為在Sf9細胞存在Dm0-like核纖層蛋白.這表明桿狀病毒BV的核衣殼出核應先通過核纖層再靠近內核膜.至于桿狀病毒是如何穿越核纖層則取決于核纖層的結構,推測有3種可能性：一是核纖層排列致密且層數(shù)較多,核衣殼必須打破核纖層方可出核；二是核纖層在核膜上不連續(xù)分布,核衣殼直接穿過分布盲區(qū)到達核膜；三是核纖層層數(shù)較少,桿狀的核衣殼可穿過核纖層纖維之間的網(wǎng)格而到達核膜.本研究對Sf9細胞的核纖層進行了初步分析,我們正利用cDNA末端快速擴增技術(rapid-amplificatioin of cDNA ends,RACE)來克隆Dm0-likelamin基因全長,并通過電鏡深入觀察核纖層的精密結構,以探討桿狀病毒感染Sf9細胞后核纖層的分布、數(shù)量的變化,分析核纖層對子代病毒核衣殼出核的影響.此外,Sf9細胞與其他物種的Lamin在進化上的關系,將在克隆Sf9Dm0-likelamin基因全長并分析內含子結構的差異后得知.Lamin是真核細胞普遍存在的一個重要結構蛋白.Dm0-like Lamin在草地貪夜蛾生長發(fā)育中的功能及作用機制也是一個值得研究的問題.

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