Sf9細(xì)胞存在Dm0-like核纖層蛋白的證據(jù)

衛(wèi)文強(qiáng)?, 季少平?*, 張銀燕

(河南大學(xué)醫(yī)學(xué)院,河南 開(kāi)封475004)

摘要為了確定Sf9細(xì)胞是否存在核纖層(lamina)及其性狀,該文首先用已知的昆蟲(chóng)的核纖層蛋白(Lamin)的基因序列在Spodobase數(shù)據(jù)庫(kù)搜索Sf9細(xì)胞的同源序列,并將推導(dǎo)的氨基酸序列與其他物種的同源蛋白進(jìn)行比對(duì)。再利用抗果蠅Lamin Dm0抗體ADL67通過(guò)免疫印跡法(Western blotting)對(duì)Sf9細(xì)胞的蛋白裂解物進(jìn)行檢測(cè),并通過(guò)免疫熒光技術(shù)(immunofluore-scence)對(duì)Sf9細(xì)胞進(jìn)行染色。在Spodobase數(shù)據(jù)庫(kù)搜索到1條Sf9細(xì)胞的Dm0-like lamin EST序列,同源比對(duì)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(duì)較高。免疫印跡結(jié)果表明Sf9細(xì)胞裂解物中存在大小約為70 ku的蛋白,免疫熒光檢測(cè)表明Sf9細(xì)胞核周呈現(xiàn)陽(yáng)性反應(yīng),這些特征與已知的其他物種的核纖層的性狀相似。結(jié)果表明,Sf9細(xì)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(guò)核纖層的機(jī)制之依據(jù).

關(guān)鍵詞Sf9細(xì)胞； 核纖層； 免疫熒光； 免疫印跡

中圖分類號(hào)Q 96文獻(xiàn)標(biāo)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(xì)胞核膜的組成部分,位于內(nèi)核膜的核質(zhì)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(zhì)的有序性、基因表達(dá)、細(xì)胞分裂、核膜的解體和重建等過(guò)程中發(fā)揮重要作用[1-4]。人類lamin的突變與多種疾病有關(guān),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(kuò)張性心肌病等[5-10].lamin突變?cè)谌橄侔?、結(jié)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(diào)控。細(xì)胞或病毒編碼的蛋白激酶可磷酸化Lamin導(dǎo)致其溶解[14-15]；JIL-1激酶與果蠅Dm0蛋白相互作用,影響細(xì)胞核的形態(tài)與完整性[16]。對(duì)各種生物L(fēng)amin結(jié)構(gòu)與功能的研究,豐富了人們對(duì)核纖層的認(rèn)識(shí),為人類lamin突變所致疾病的治療奠定了理論基礎(chǔ)[17-18]。目前已知Lamin在人、小鼠、果蠅等真核生物的細(xì)胞內(nèi)均存在。

核纖層附著在內(nèi)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(guò)它。而病毒則通過(guò)某種機(jī)制打破核纖層,以促進(jìn)子代病毒的出核。對(duì)單純皰疹病毒(herpes simplex virus,HSV)、巨細(xì)胞病毒(human cytomegalovirus,HCMV)、鼻咽癌病毒(epstein-barr virus,EBV)等動(dòng)物細(xì)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(chóng)細(xì)胞Sf9是來(lái)自草地貪夜蛾(Spodopterafrugiperda)蛹期卵巢組織的商業(yè)細(xì)胞系,是研究桿狀病毒模式株AcMNPV感染機(jī)制的常用細(xì)胞。研究表明,桿狀病毒感染后在細(xì)胞核內(nèi)裝配后的核衣殼沿著內(nèi)核膜排列,通過(guò)出芽的方式進(jìn)入核周間隙[24-25]。通過(guò)麥胚凝集素-膠體金(WGA-gold)標(biāo)記核膜技術(shù)發(fā)現(xiàn),桿狀病毒感染致核膜向核質(zhì)內(nèi)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(xì)胞質(zhì)[26]。這使我們想到Sf9細(xì)胞是否存在核纖層,以及桿狀病毒如何穿越核纖層這個(gè)有趣的問(wèn)題。但對(duì)Sf9細(xì)胞核纖層的研究目前還比較少,限制了對(duì)該問(wèn)題的理解。

本研究利用抗果蠅Lamin Dm0單抗ADL67通過(guò)免疫印跡法對(duì)Sf9細(xì)胞裂解物進(jìn)行檢測(cè);再利用抗體ADL67通過(guò)免疫熒光技術(shù)染色Sf9細(xì)胞并在熒光共聚焦顯微鏡下觀察。同時(shí),用其他昆蟲(chóng)的已知lamin序列在Spodobase[27]數(shù)據(jù)庫(kù)(專門存放來(lái)自草地貪夜蛾的EST序列數(shù)據(jù)庫(kù))搜索Sf9細(xì)胞的lamin同源序列,并進(jìn)行核苷酸和氨基酸序列比對(duì)。結(jié)果初步表明,Sf9細(xì)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(chóng)細(xì)胞Sf9細(xì)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(gòu)自Gibco公司.Grace培養(yǎng)基購(gòu)自Invitrogen公司.

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

1.2方法

1.2.1序列比對(duì)用已知的lamin基因搜索Spodobase數(shù)據(jù)庫(kù),通過(guò)Multalian在線軟件(http://multalin.toulouse.inra.fr/multalin/)將搜到的Sf9細(xì)胞Dm0-likelaminEST序列及推導(dǎo)出的氨基酸序列與其他物種的lamin進(jìn)行比對(duì),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(xì)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(jìn)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(jīng)濕轉(zhuǎn)法轉(zhuǎn)到PVDF膜上。5%脫脂奶粉室溫孵育1 h。以單抗ADL67(1∶100)4 ℃過(guò)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(guò)氧化物酶標(biāo)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(shí),取表達(dá)Dm0的細(xì)菌裂解物(由Nico Stuurman教授饋贈(zèng)的質(zhì)粒pETDmLFL在BL21細(xì)胞中誘導(dǎo)表達(dá)果蠅Dm0蛋白)作為陽(yáng)性對(duì)照。最后以化學(xué)發(fā)光法檢測(cè)Lamin.

1.2.3免疫熒光檢測(cè)Sf9細(xì)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(guò)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(biāo)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(xì)胞置于熒光共聚焦顯微鏡下,觀察細(xì)胞核內(nèi)核纖層的分布、形態(tài)特征.

2結(jié)果

2.1Sf9細(xì)胞Dm0-like Lamin的大小

為了確定Dm0-like Lamin的分子質(zhì)量,取Sf9細(xì)胞總蛋白,經(jīng)SDS-PAGE后轉(zhuǎn)移到PVDF膜上,然后用抗體ADL67通過(guò)免疫印跡進(jìn)行檢測(cè).結(jié)果顯示,在大約70 ku處有1條明顯的陽(yáng)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(xì)胞的Dm0-like Lamin;M:蛋白標(biāo)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(cè)Sf9細(xì)胞Dm0-like Lamin Fig.1　Detection of Dm0-like Lamin of Sf9 cells by Western blotting

2.2Sf9細(xì)胞Dm0-like Lamin與其他物種同源蛋白序列比對(duì)

為了確定Sf9細(xì)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(kù).結(jié)果搜到1條來(lái)自Sf9細(xì)胞的Dm0-likelamin同源序列(Sf9LR450003-5-1-C1112),再由其核苷酸序列推導(dǎo)出氨基酸序列.核苷酸序列比對(duì)結(jié)果顯示,Sf9Dm0-likelamin與其他物種的lamin核苷酸序列同源性達(dá)56%～80%,其中,與家蠶的同源性達(dá)80%,與果蠅的同源性達(dá)64%(圖2A),但與其他物種lamin同源性相對(duì)較低.氨基酸序列比對(duì)結(jié)果顯示,Sf9 Dm0-like Lamin與其他物種的同源蛋白同源性達(dá)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(dǎo)的氨基酸(B)序列比對(duì) Fig.2　Comparison of nucleotide (A) and deduced amino acid (B) sequences of Lamin among Sf9 cells and other species

2.3Sf9細(xì)胞Dm0-like Lamin的定位

為了觀察Sf9細(xì)胞是否存在核纖層及核纖層蛋白的細(xì)胞定位,使用抗體ADL67對(duì)Sf9細(xì)胞進(jìn)行免疫熒光染色.同時(shí),用Hoechst 33258對(duì)細(xì)胞核進(jìn)行染色.熒光共聚焦顯微鏡觀察顯示在Sf9細(xì)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(cè)Dm0-like Lamin在Sf9細(xì)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(guò)生物信息學(xué)分析、免疫印跡、免疫熒光等方法初步證明Sf9細(xì)胞存在Dm0-likelamin.這為下一步研究桿狀病毒核衣殼穿過(guò)核纖層的機(jī)制提供了依據(jù).

利用已知的昆蟲(chóng)lamin序列,在Spodobase[27]數(shù)據(jù)庫(kù)進(jìn)行序列同源性搜索,結(jié)果找到1條Sf9細(xì)胞的Dm0-likelaminEST序列.生物信息學(xué)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(guò)對(duì)Lamin內(nèi)含子的進(jìn)化分析認(rèn)為,目前在昆蟲(chóng)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(dú)有.而家蠶僅有1種Lamin蛋白.Sf9細(xì)胞也很可能只存在1種Lamin蛋白[28-29].所以,抗體ADL67檢測(cè)到的細(xì)胞蛋白可能是由搜到的這個(gè)Dm0-likelamin基因表達(dá)。隨后,根據(jù)這段EST序列設(shè)計(jì)特異性引物,利用從Sf9細(xì)胞提取的RNA進(jìn)行反轉(zhuǎn)錄PCR(reverse transcription PCR,RT-PCR),擴(kuò)增到預(yù)期大小的條帶(未展示),測(cè)序結(jié)果也符合預(yù)期.該段序列的發(fā)現(xiàn)為下一步克隆Sf9Dm0-likelamin基因全長(zhǎng)并深入研究其蛋白結(jié)構(gòu)與功能奠定了基礎(chǔ).

雖然在Spodobase數(shù)據(jù)庫(kù)搜到1個(gè)Sf9Dm0-likelaminEST序列,但其完整的開(kāi)放閱讀框(open reading frame,ORF)尚未獲得,故其蛋白大小也無(wú)法預(yù)測(cè).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(méi)有.而針對(duì)果蠅Dm0的單抗ADL67目前已經(jīng)存在,而且Sf9 Dm0-like Lamin與果蠅的Dm0 Lamin的同源性也達(dá)到59%.所以,我們嘗試用該抗體對(duì)Sf9細(xì)胞的Dm0-like Lamin的性狀進(jìn)行分析.利用ADL67對(duì)Sf9全細(xì)胞裂解物進(jìn)行免疫印跡檢測(cè),結(jié)果檢測(cè)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(shuō)明Lamin蛋白大小在不同昆蟲(chóng)間可能是相對(duì)保守的.根據(jù)氨基酸序列比對(duì)的結(jié)果可知,獲得的Sf9細(xì)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(shí)別表位對(duì)應(yīng)果蠅的Dm0的C端的548～620位氨基酸,故目前無(wú)法對(duì)果蠅ADL67抗原表位與Sf9細(xì)胞Dm0-like Lamin的序列同源性進(jìn)行比對(duì).但ADL67能識(shí)別Sf9細(xì)胞的Dm0-like Lamin,說(shuō)明ADL67識(shí)別表位在不同昆蟲(chóng)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(xì)胞核膜的核質(zhì)面,熒光顯微鏡下觀察呈環(huán)形.為了確定抗體ADL67檢測(cè)到的70 ku左右的蛋白在Sf9細(xì)胞內(nèi)是否也定位于核膜,再次利用該抗體進(jìn)行了免疫熒光實(shí)驗(yàn).結(jié)果顯示,在細(xì)胞核的周圍觀察到環(huán)狀的陽(yáng)性熒光信號(hào)，與其他物種的細(xì)胞核纖層形態(tài)、分布相似.免疫熒光實(shí)驗(yàn)直觀地說(shuō)明Sf9細(xì)胞存在核纖層蛋白.

基于上述實(shí)驗(yàn)結(jié)果,初步認(rèn)為在Sf9細(xì)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(yīng)先通過(guò)核纖層再靠近內(nèi)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(jié)構(gòu),推測(cè)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(guò)分布盲區(qū)到達(dá)核膜；三是核纖層層數(shù)較少,桿狀的核衣殼可穿過(guò)核纖層纖維之間的網(wǎng)格而到達(dá)核膜.本研究對(duì)Sf9細(xì)胞的核纖層進(jìn)行了初步分析,我們正利用cDNA末端快速擴(kuò)增技術(shù)(rapid-amplificatioin of cDNA ends,RACE)來(lái)克隆Dm0-likelamin基因全長(zhǎng),并通過(guò)電鏡深入觀察核纖層的精密結(jié)構(gòu),以探討桿狀病毒感染Sf9細(xì)胞后核纖層的分布、數(shù)量的變化,分析核纖層對(duì)子代病毒核衣殼出核的影響.此外,Sf9細(xì)胞與其他物種的Lamin在進(jìn)化上的關(guān)系,將在克隆Sf9Dm0-likelamin基因全長(zhǎng)并分析內(nèi)含子結(jié)構(gòu)的差異后得知.Lamin是真核細(xì)胞普遍存在的一個(gè)重要結(jié)構(gòu)蛋白.Dm0-like Lamin在草地貪夜蛾生長(zhǎng)發(fā)育中的功能及作用機(jī)制也是一個(gè)值得研究的問(wèn)題.

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