朱樂園，劉建濤, 2，王燕玲，袁美妗，吳文碧*，楊 凱

桿狀病毒Ac110蛋白的保守氨基酸位點(diǎn)N27對(duì)病毒在中腸建立有效感染起重要作用

朱樂園1，劉建濤1, 2，王燕玲1，袁美妗1，吳文碧1*，楊 凱1

( 1．中山大學(xué)生命科學(xué)學(xué)院，有害生物控制與資源利用國(guó)家重點(diǎn)實(shí)驗(yàn)室，廣州 510275；2．江西科技師范大學(xué)生命科學(xué)學(xué)院，南昌 330013)

桿狀病毒是節(jié)肢動(dòng)物，特別是鱗翅目昆蟲的重要病原微生物，在生物防治中有著廣泛的應(yīng)用前景。其對(duì)昆蟲蟲體口服感染的過程中，需要一類稱為桿狀病毒口服感染因子的幫助才能在蟲體中腸成功建立系統(tǒng)感染。Ac110是本實(shí)驗(yàn)室最新發(fā)現(xiàn)的口服感染因子之一，但其作用機(jī)制仍是未知。在本研究中，通過定點(diǎn)突變技術(shù)成功構(gòu)建了兩株Ac110保守氨基酸位點(diǎn)突變型重組桿狀病毒，將病毒DNA轉(zhuǎn)染昆蟲細(xì)胞Sf9后，通過蛋白免疫印跡實(shí)驗(yàn)驗(yàn)證了Ac110的突變體蛋白均能夠正常表達(dá)；接著通過病毒滴度測(cè)定實(shí)驗(yàn)證明了重組病毒均能在Sf9細(xì)胞中產(chǎn)生與補(bǔ)回型病毒相當(dāng)?shù)母腥拘圆《玖Ｗ樱f明Ac110的這兩個(gè)保守氨基酸位點(diǎn)的突變并不影響病毒在細(xì)胞中的復(fù)制和在細(xì)胞之間的傳播；最后將純化的病毒多角體口服感染甜菜夜蛾幼蟲進(jìn)行生物測(cè)定實(shí)驗(yàn)，發(fā)現(xiàn)Ac110的N27氨基酸位點(diǎn)的突變對(duì)昆蟲幼蟲的口服感染能力顯著性降低，而L35氨基酸位點(diǎn)的突變則沒有影響。

桿狀病毒；口服感染因子；Ac110；生物測(cè)定

桿狀病毒 (Baculovirus)是一類寄生于節(jié)肢動(dòng)物的專一性病原微生物，其宿主主要為昆蟲綱中的鱗翅目、雙翅目、膜翅目和鞘翅目昆蟲。桿狀病毒的病毒粒子呈桿狀，基因組為雙鏈閉合環(huán)狀 DNA，病毒基因組大小介于80-180 kb 之間。桿狀病毒是目前對(duì)人類最為有益的病毒，不僅對(duì)人的健康無害，在控制農(nóng)林害蟲的種群密度方面更是起到重要作用。桿狀病毒在其生活周期中產(chǎn)生兩種類型的病毒粒子：芽生型病毒粒子BV (Budded virus)和包埋型病毒粒子ODV (Occlusion derived virus)。BV出芽時(shí)在核膜或細(xì)胞膜處獲得囊膜，而囊膜可以介導(dǎo)病毒與宿主細(xì)胞表面特定的受體相結(jié)合，對(duì)病毒粒子在蟲體內(nèi)的傳播及病毒在離體培養(yǎng)細(xì)胞間的傳播起到重要作用；ODV包埋于由多角體蛋白質(zhì)組成的包涵體蛋白晶體中，形成多角體(Occlusion body, OB)，而多角體還被多角體囊膜(polyhedron envelope, PE)包被。多角體在自然環(huán)境中非常穩(wěn)定，對(duì)包埋在其中的病毒粒子起到保護(hù)作用，利于病毒在自然界中的傳播(Braunageletal., 2003)。

桿狀病毒感染昆蟲是一個(gè)較為復(fù)雜的過程，昆蟲攝食了含有桿狀病毒多角體的食物后，多角體入宿主中腸腔體，在中腸的堿性環(huán)境下被裂解，釋放其包裹的ODV。隨后，ODV要穿越多重障礙進(jìn)入昆蟲宿主中腸上皮細(xì)胞建立初始感染，其中第一個(gè)障礙就是宿主腸道的保護(hù)屏障圍食膜(peritrophic matrix, PM)。PM是介于宿主中腸上皮細(xì)胞和腸腔間的網(wǎng)狀結(jié)構(gòu)，由幾丁質(zhì)、糖蛋白和幾丁質(zhì)結(jié)合蛋白組成(Hegedusetal., 2009)。ODV的大小遠(yuǎn)大于PM的孔徑，因此ODV除了可能借助由于宿主吞食食物過程中造成PM機(jī)械損傷導(dǎo)致其孔徑變大使得ODV順利穿越之外(Wang and Granados, 2000)，還可能借助多角體自身包裹的桿狀病毒編碼的Enhancin和金屬蛋白酶等因子對(duì)PM起到降解的作用(Leporeetal., 1996)，從而使ODV順利穿越PM到達(dá)中腸上皮細(xì)胞(Ishimweetal., 2015)。ODV到達(dá)中腸上皮細(xì)胞之后，需要穿過第二個(gè)障礙即中腸上皮細(xì)胞微絨毛而進(jìn)入中腸上皮細(xì)胞，已有研究表明ODV通過膜融合，即ODV囊膜與中腸柱狀上皮細(xì)胞的微絨毛發(fā)生結(jié)合和融合，釋放出ODV中的核衣殼，使其進(jìn)入中腸上皮細(xì)胞，從而成功建立初始感染(Faulkneretal., 1997)。而這個(gè)過程需要桿狀病毒編碼的一類稱為口服感染因子(perosinfectivity factor, PIF)的蛋白發(fā)揮至關(guān)重要的作用。

目前已發(fā)現(xiàn)的桿狀病毒PIF包括了P74 (PIF-0或Ac138) (Faulkneretal., 1997)、PIF-1 (Ac119) (Kikhnoetal., 2002)、PIF-2 (Ac22) (Fangetal., 2006; Pijlmanetal., 2003)、PIF-3 (Ac115) (Ohkawaetal., 2005)、PIF-4 (Ac96) (Fangetal., 2009)、PIF-5 (Ac148) (Harrisonetal., 2010; Xiangetal., 2011)、PIF-6 (Ac68) (Nieetal., 2012)、PIF-8 (Ac83) (Javedetal., 2017; Zhuetal., 2013)、以及本實(shí)驗(yàn)室發(fā)現(xiàn)的PIF-7 (Ac110) (Javedetal., 2017; Liuetal., 2016)。已有的研究表明，在這些PIFs中，缺失其中的任何一個(gè)都會(huì)極大降低桿狀病毒對(duì)宿主的口服感染能力。同時(shí)發(fā)現(xiàn)很多PIF之間存在相互作用，可能共同行使著促進(jìn)桿狀病毒在宿主中腸細(xì)胞建立初始感染的功能。

本實(shí)驗(yàn)室前期的研究中，通過構(gòu)建Ac110缺失型和補(bǔ)回型重組病毒，發(fā)現(xiàn)Ac110是桿狀病毒一個(gè)新的口服感染因子，而且PM并不是其作用位點(diǎn)(Liuetal., 2016)。為了對(duì)Ac110的功能進(jìn)行深入的探究，我們對(duì)Ac110的兩個(gè)保守氨基酸位點(diǎn)分別進(jìn)行了定點(diǎn)突變，構(gòu)建出兩株Ac110突變型重組病毒，研究其對(duì)Ac110功能的影響。

1 材料和方法

1.1 實(shí)驗(yàn)昆蟲，細(xì)胞和病毒

Sf9細(xì)胞：草地貪夜蛾Spodopterafrugiperda細(xì)胞Sf9購(gòu)自英濰捷基(上海)公司。甜菜夜蛾幼蟲：由中山大學(xué)昆蟲學(xué)研究所昆蟲飼養(yǎng)室飼養(yǎng)提供。甜菜夜蛾幼蟲飼料：由中山大學(xué)昆蟲學(xué)研究所昆蟲飼養(yǎng)室提供。

Ac110缺失型重組病毒vAc110KO和補(bǔ)回型重組病毒vAc110HA為本實(shí)驗(yàn)室保存(Liuetal., 2016)。

1.2 Ac110氨基酸序列分析

利用 NCBI 網(wǎng)站上的核苷酸序列 BLAST 搜索軟件(protein-protein blast, blastp)搜索Ac110同源蛋白的氨基酸序列并將序列下載。運(yùn)用Clustal W 1.83軟件對(duì) AcMNPV Ac110蛋白同源物的氨基酸序列進(jìn)行多序列比對(duì)分析，通過Genedoc軟件進(jìn)行編輯。

1.3 Ac110點(diǎn)突變重組病毒的構(gòu)建

通過定點(diǎn)突變技術(shù)(Chiuetal., 2004)和Bac-to-Bac Expression System(Invitrogen)提供特異性轉(zhuǎn)座，構(gòu)建Ac110突變型重組病毒。首先在每個(gè)需要突變的位點(diǎn)設(shè)計(jì)四條引物(表1所示)，以pUC110HA-SV40質(zhì)粒DNA (Liuetal., 2016)(此質(zhì)粒以pUC18質(zhì)粒為骨架，插入包含ac110自身啟動(dòng)子、開放閱讀框和多聚腺苷酸的片段，是構(gòu)建補(bǔ)回型重組病毒轉(zhuǎn)座載體的中間載體)為模板進(jìn)行PCR反應(yīng)，以獲得pUC18-Ac110N27A和pUC18-Ac110L35A質(zhì)粒；通過酶切將Ac110N27A和Ac110L35A片段從pUC18-Ac110N27A和pUC18-Ac110L35A中切下來并連接至pFB1-PH-GFP (Wuetal., 2006)載體上，構(gòu)建出轉(zhuǎn)座載體pFB1-Ac110N27A-PH-GFP和pFB1-Ac110L35A-PH-GFP。將轉(zhuǎn)座載體分別轉(zhuǎn)化至含有Ac110缺失型重組病毒bAc110KO (Liuetal., 2016)的大腸桿菌細(xì)胞DH10B中，通過特異性位點(diǎn)轉(zhuǎn)座將ac110點(diǎn)突變型基因轉(zhuǎn)座至缺失了ac110基因的桿狀病毒bAc110KO中，再通過藍(lán)白斑篩選出帶有綠色熒光基因蛋白gfp和多角體蛋白基因polyhedrin的Ac110點(diǎn)突變重組病毒vAc110N27A和vAc110L35A。最后通過PCR和測(cè)序?qū)χ亟M病毒進(jìn)行鑒定。

表1 Ac110點(diǎn)突變引物序列Table 1 Sequences of primers for Ac110 site mutation

1.4 病毒對(duì)細(xì)胞的轉(zhuǎn)染和感染

病毒的轉(zhuǎn)染和感染根據(jù)文獻(xiàn)(Wuetal., 2006)操作進(jìn)行：首先把1×106個(gè)處于對(duì)數(shù)生長(zhǎng)期的Sf9細(xì)胞接種于直徑為35 mm的細(xì)胞培養(yǎng)皿中，然后將重組病毒DNA與脂質(zhì)體混勻，室溫溫育45 min，再加入到細(xì)胞上孵育5 h，最后吸除孵育細(xì)胞的病毒和脂質(zhì)體混合液，給細(xì)胞換上新鮮的培養(yǎng)基，并以此時(shí)間點(diǎn)作為病毒轉(zhuǎn)染后0 h來計(jì)算病毒轉(zhuǎn)染的時(shí)間。

病毒感染實(shí)驗(yàn)的操作如下：將病毒液以一定的感染復(fù)數(shù)(multiplicity of infection, MOI)接種于細(xì)胞并孵育1 h，然后吸除孵育細(xì)胞的病毒液，給細(xì)胞換上新鮮的培養(yǎng)基，并以此時(shí)間點(diǎn)作為病毒感染后0 h來計(jì)算病毒感染的時(shí)間。

1.5 病毒滴度測(cè)定及生長(zhǎng)曲線的繪制

在病毒感染后的不同時(shí)間點(diǎn)取細(xì)胞上清液進(jìn)行病毒滴度的檢測(cè)，采用TCID50(50% tissue culture infective dose) (Wuetal., 2006)終點(diǎn)稀釋法對(duì)樣品中可感染性病毒粒子BV進(jìn)行定量。運(yùn)用GraphPad Prism 6.0軟件，根據(jù)每個(gè)時(shí)間點(diǎn)的病毒滴度繪制病毒生長(zhǎng)曲線。

1.6 蛋白免疫印跡實(shí)驗(yàn)(Western blotting)

蛋白免疫印跡實(shí)驗(yàn)的方法參考(Wuetal., 2008)文獻(xiàn)：將病毒感染后的細(xì)胞離心收集后，用PBS清洗兩次，加入蛋白裂解液制備蛋白樣品。蛋白樣品進(jìn)行SDS-PAGE膠分離后，通過半干轉(zhuǎn)膜系統(tǒng)將蛋白轉(zhuǎn)印到PVDF膜，以HA兔單抗(Cell Signal Technology)或者β-actin鼠單抗(Proteintech)為一抗和辣根過氧化物酶HRP標(biāo)記的二抗進(jìn)行蛋白雜交，最后通過ECL (Pierce ECL Western Blotting Substrate)進(jìn)行膠片顯影。

1.7 病毒的蟲體擴(kuò)增及純化

多角體的擴(kuò)增和純化參考(O’Reilly, 1992)文獻(xiàn)中的操作：將病毒多角體涂布于人工飼料表面飼喂感染3齡的昆蟲幼蟲，并于幼蟲發(fā)病死亡后收集蟲尸。首先加入PBS用勻漿器充分碾磨均勻蟲尸，勻漿液先用 4 層紗布過濾，再用 6 層紗布過濾，除去組織碎片，收集濾液。室溫下4500 rpm離心30 min，棄上清；以PBS重懸沉淀，室溫下500 rpm離心10 min，取上清，8000 rpm離心10 min，取沉淀(重復(fù)此步驟5-10 次)；以0.05% SDS重懸沉淀，室溫下 500 rpm離心10 min，取上清，8000 rpm離心10 min，取沉淀(重復(fù)此步驟5-10 次)；以ddH2O 重懸沉淀，室溫下 500 rpm離心 10 min，取上清，8000 rpm離心 10 min，取沉淀(重復(fù)此步驟5-10次)；最后沉淀重懸于ddH2O，即為較純的多角體，應(yīng)為均質(zhì)乳白狀；置于-20℃保存?zhèn)溆谩?/p>

1.8 生物測(cè)定實(shí)驗(yàn)

將收集純化的病毒多角體進(jìn)行計(jì)數(shù)并稀釋至相應(yīng)的濃度，將飼料切成1 mm見方的大小，涂抹1 μL多角體懸液于飼料表面喂食已饑餓處理的甜菜夜蛾3齡幼蟲，待幼蟲吃完涂有病毒多角體的飼料后再喂食新鮮的飼料。每天喂食足夠的飼料并觀察幼蟲的感染情況。

2 結(jié)果與分析

2.1 Ac110同源蛋白氨基酸序列同源性及保守氨基酸位點(diǎn)分析

Ac110是桿狀病毒中高度保守的基因(Garavagliaetal., 2012)，目前認(rèn)為它是桿狀病毒中第38個(gè)核心基因(Javedetal., 2017)。通過NCBI網(wǎng)站上的blastp進(jìn)行搜索，將Ac110的部分同源蛋白的氨基酸序列下載并進(jìn)行分析。表2是所下載的Ac110同源蛋白的信息。通過多序列比對(duì)軟件對(duì)Ac110同源蛋白的氨基酸序列進(jìn)行同源性分析，結(jié)果顯示Ac110序列與PlxyMNPV、BmMNPV、OpNPV和AngeNPV 對(duì)應(yīng)ORF的一致性可達(dá)到69%-98%，與其他鱗翅目桿狀病毒同源蛋白一致性在26%-48%。通過多序列比對(duì)軟件對(duì)Ac110同源蛋白的氨基酸序列進(jìn)行多序列比對(duì)，結(jié)果如圖1所示，所有進(jìn)行比對(duì)的氨基酸序列當(dāng)中，有11個(gè)氨基酸殘基是高度保守的(圖1)。

表2 桿狀病毒Ac110及其同源蛋白 Table 2 Baculovirus Ac110 and its homologs

續(xù)上表

病毒名稱Virusname縮寫Abbreviation全稱Fullname登錄號(hào)Accessionnumber對(duì)應(yīng)的蛋白CorrespondingproteinDasychirapudibundaNPVDapuNPV茸毒蛾核多角體病毒AKR14124.1hypotheticalpro-teinDendrolimuskikuchiiNPVDekiNPV思茅松毛蟲核多角體病毒AFS51927.1DekiORF49EctropisobliquaNPVEcobNPV茶尺蠖核多角體病毒YP_874277.1hypotheticalpro-teinEONV_gp084EpiphyaspostvittanaNPVEpNPV萍淺褐卷蛾核多角體病毒NP_203265.1hypotheticalpro-teinHelicoverpaarmigeraNPVHearNPV棉鈴蟲核多角體病毒AJP07380.1hypotheticalpro-teinHelicoverpazeasingleNPVHzSNPV美洲棉鈴蟲單粒包埋核多角體病毒AAL56102.1ORF96Hemileucasp.NPVHespNPV雄鹿天蠶蛾核多角體病毒YP_008378309.1ac110LonomiaobliquaMNPVLbMNPV羅奴霉素多粒包埋核多角體病毒AKN81034.1hypotheticalpro-teinLymantriadisparMNPVLdMNPV舞毒蛾多粒包埋核多角體病毒AMO27775.1hypotheticalpro-teinMamestraconfigurataNPVBMacoNPV-B蓓帶夜蛾B多角體病毒NP_689255.1hypotheticalpro-teinMarucavitrataNPVMvNPV豆莢野螟蛾核多角體病毒YP_950816.1Mv-ORF86peptideOrgyialeucostigmaNPVOrleNPV白斑天幕質(zhì)型多角體病毒YP_001651002.1hypotheticalpro-teinOrgyiapseudotsugataMN-PVOpMNPV黃杉毒蛾核多角體病毒NP_046267.1hypotheticalpro-teinOpmnVgp111PlutellaxylostellaGVPlxyGV小菜蛾顆粒體病毒NP_068260.1PxORF41peptidePlutellaxylostellaMNPVPlxyMNPV小菜蛾多粒包埋核多角體病毒ABE68494.1unknownRachiplusiaouMNPVRoMNPV薄荷灰夜蛾核型多角體病毒AAN28145.1unknownSpodopteraexiguaMNPVSeMNPV甜菜夜蛾核多角體病毒NP_037820.1ORF60SpodopterafrugiperdaMNPVSfMNPV草地貪夜蛾核多角體病毒YP_001036352.1hypotheticalpro-teinSFMNPV_gp060SpodopteralittoralisNPVSpliNPV?；页嵋苟旰硕嘟求w病毒AGE89945.1hypotheticalpro-teinSlsnVgp090ThysanoplusiaorichalceaNPVThorNPV大豆夜蛾核多角體病毒YP_007250516.1hypotheticalpro-teinUrbanusproteusNPVUrprNPV豆卷葉螟核多角體病毒YP_009250071.1hypotheticalpro-tein

圖1 Ac110同源蛋白氨基酸序列比對(duì)Fig.1 Alignment of Ac110 homologs

2.2 Ac110點(diǎn)突變重組病毒的構(gòu)建和鑒定

通過比較Ac110同源蛋白的氨基酸序列的同源性，選取2個(gè)高度保守的氨基酸位點(diǎn)(圖1中黑色箭頭所標(biāo)識(shí)的N27和L35位點(diǎn))進(jìn)行點(diǎn)突變，分別構(gòu)建重組病毒vAc110N27A和vAc110L35A。病毒構(gòu)建示意圖如圖3所示，其中bMON14272為Bac-to-Bac Expression System中缺失了多角體蛋白基因并能在該基因原位點(diǎn)提供特異性轉(zhuǎn)座的桿狀病毒基因組。

2.3 Ac110突變型蛋白的檢測(cè)

由于重組病毒構(gòu)建過程中，Ac110蛋白的C末端加上了一個(gè)HA標(biāo)簽，因此可用抗HA的標(biāo)簽抗體來檢測(cè)重組病毒中Ac110HA點(diǎn)突變蛋白是否表達(dá)。結(jié)果如圖3所示，Ac110點(diǎn)突變重組病毒感染的細(xì)胞中，能檢測(cè)到與補(bǔ)回型病毒vAc110HA感染的細(xì)胞中大小一樣的特異蛋白帶，而缺失型重組病毒vAc110KO感染的細(xì)胞中則沒有這樣的蛋白產(chǎn)生，說明Ac110突變體蛋白均能正常表達(dá)。同時(shí)以β-actin的多抗檢測(cè)了細(xì)胞宿主蛋白β-actin以作為內(nèi)參。

圖2 Ac110重組病毒構(gòu)建示意圖Fig.2 The diagram of the construction of Ac110 recombinant viruses AcMNPV polyhedrin (polh) locus was disrupted by lacZ and mini-attTn7 as showed in bMON14272.The ac110 gene was replaced by chloramphenicol resistance gene (Cm) in bAc110KO.Green fluorescent protein gene (gfp) and polh gene were transposed to the polyhedrin locus of bAc110KO to obtain the recombinant virus vAc110KO.The Ac110 with an HA tag at the C terminus was repaired in the vAc110HA.The vAc110N27A and vAc110L35A contained the mutated Ac110 N27A and L35A, respectively.All constructs were confirmed by PCR and sequencing analyses.

圖3 Ac110點(diǎn)突變蛋白表達(dá)檢測(cè)Fig.3 The detection of the Ac110 variants expression by Western blot analysis

2.4 Ac110保守氨基酸的突變不影響病毒在細(xì)胞中的正常增殖和擴(kuò)散

將vAc110N27A和vAc110L35A的bacmid DNA轉(zhuǎn)染至Sf9細(xì)胞中，在轉(zhuǎn)染后的不同時(shí)間點(diǎn)分別進(jìn)行熒光顯微鏡觀察和光學(xué)顯微鏡觀察，并拍照記錄結(jié)果。如圖4所示，在病毒轉(zhuǎn)染后24 h，不同病毒轉(zhuǎn)染的細(xì)胞中，能產(chǎn)生熒光的細(xì)胞數(shù)量相當(dāng)，說明病毒DNA的轉(zhuǎn)染效率差不多。而到了病毒轉(zhuǎn)染后72 h，所有細(xì)胞都產(chǎn)生了熒光，說明重組病毒均能在細(xì)胞中正常復(fù)制并擴(kuò)散。通過光學(xué)顯微鏡可以在白光下看到不同病毒轉(zhuǎn)染的細(xì)胞中均有部分細(xì)胞已產(chǎn)生了多角體(圖4)。收集病毒轉(zhuǎn)染后120 h上清液檢測(cè)病毒滴度，結(jié)果顯示Ac110突變型重組病毒的病毒產(chǎn)量和對(duì)照組病毒的病毒產(chǎn)量相當(dāng)(圖5A)，同時(shí)收集該時(shí)間點(diǎn)的細(xì)胞純化多角體，通過血球計(jì)數(shù)板對(duì)多角體進(jìn)行計(jì)數(shù)，發(fā)現(xiàn)突變體與補(bǔ)回型重組病毒相比，多角體產(chǎn)量差別不大(結(jié)果未展示)。將重組病毒以低MOI感染Sf9細(xì)胞，于不同時(shí)間點(diǎn)收取細(xì)胞上清液進(jìn)行病毒滴度測(cè)定，繪制病毒生長(zhǎng)曲線，發(fā)現(xiàn)Ac110突變型重組病毒與補(bǔ)回型重組病毒生長(zhǎng)趨勢(shì)一致。以上結(jié)果說明Ac110點(diǎn)突變后不影響病毒在細(xì)胞中的增殖和擴(kuò)散。

圖4 重組病毒轉(zhuǎn)染Sf9細(xì)胞Fig.4 Transfection of recombinant viruses in Sf9 cellsNote:The bacmid DNA of recombinant viruses were transfected in Sf9 cells and the cells were observed by fluorescent microscopy and light microscopy at indicated time points.

圖5 重組病毒在細(xì)胞中的復(fù)制Fig.5 Replication of recombinant viruses in Sf9 cells注：(A)重組病毒轉(zhuǎn)染Sf9細(xì)胞的滴度測(cè)定(B)重組病毒低MOI感染Sf9細(xì)胞的生長(zhǎng)曲線測(cè)定。Note: (A) Titres of Ac110 recombinant viruses harvested at 120 h post-transfection.(B) Viral growth curves of Ac110 recombinant viruses.Sf9 cells were infected with recombinant viruses at an MOI of 0.01 TCID50/cell.The supernatants of viruses infected cells were collected at designated time points and the titres were determined by using a TCID50 assay.Each data point represents the average from two independent experiments and error bars represent the standard deviations.Statistical analyses were carried out using GraphPad Prism 6.0 software.

2.5 Ac110蛋白N27位點(diǎn)的突變顯著降低了病毒對(duì)蟲體的口服感染能力

將收集的各個(gè)重組病毒多角體(OB)分別稀釋至濃度為1×106，1×107和1×108OB/mL，每頭三齡期甜菜夜蛾幼蟲喂食1 μL OB懸液，即分3種喂食劑量，每頭分別喂食1×103，1×104和1×105個(gè)OB。每種病毒的每個(gè)喂食劑量設(shè)置兩組實(shí)驗(yàn)，每組12頭幼蟲，另外喂食H2O作為空白對(duì)照。統(tǒng)計(jì)病毒感染幼蟲的死亡率(表2)，與補(bǔ)回型重組病毒vAc110HA相比，vAc110L35A用不同劑量感染時(shí)，死亡率與vAc110HA基本一致，不存在顯著性差異；而vAc110N27A感染的幼蟲死亡率會(huì)下降約20%，感染劑量為1×105和1×104OB時(shí)，二者之間存在顯著性差異，但劑量為1×103OB時(shí)不存在顯著性差異(圖6)。

表2 重組病毒生物測(cè)定Table 2 Bioassays of recombinant viruses

圖6 重組病毒生物測(cè)定分析Fig.6 Mortality analyses of bioassays with recombinant viruses

3 結(jié)論與討論

本實(shí)驗(yàn)室前期對(duì)桿狀病毒Ac110的功能研究中，發(fā)現(xiàn)Ac110是一個(gè)新的口服感染因子PIF，其缺失對(duì)病毒在細(xì)胞中的增殖和擴(kuò)散不受影響，但是病毒卻不能通過口服感染昆蟲幼蟲，并且昆蟲的圍食膜PM不是Ac110的作用靶標(biāo)(Liuetal., 2016)。為了進(jìn)一步揭示Ac110在口服感染過程中的作用機(jī)制，本文選取了Ac110蛋白兩個(gè)高度保守的氨基酸位點(diǎn)N27和L35，構(gòu)建了兩株Ac110點(diǎn)突變重組病毒，展開了相關(guān)的實(shí)驗(yàn)研究。結(jié)果顯示這兩個(gè)氨基酸位點(diǎn)的突變均不影響病毒在細(xì)胞的增殖和擴(kuò)散，并且在不同的病毒多角體的感染劑量下，L35位點(diǎn)的突變不影響病毒對(duì)昆蟲幼蟲的口服感染，但是在每頭蟲子感染1×105和1×104OB時(shí)，N27位點(diǎn)的突變卻顯著降低了病毒對(duì)昆蟲幼蟲的口服感染能力，說明N27位點(diǎn)對(duì)Ac110的功能起著重要的作用。

近年來對(duì)PIF的功能研究以及ODV如何進(jìn)入中腸上皮細(xì)胞一直是研究的熱點(diǎn)。通過電鏡觀察，有研究表明ODV在感染昆蟲中腸的過程中，會(huì)與中腸上皮細(xì)胞微絨毛結(jié)合和融合(Kawanishietal., 1972)，但對(duì)這過程中的分子機(jī)制了解不多。在目前已經(jīng)研究發(fā)現(xiàn)的眾多的PIF中，P74、PIF-1和PIF-2介導(dǎo)了病毒與細(xì)胞的結(jié)合，缺失三者中的任何一個(gè)基因都會(huì)造成ODV與微絨毛的結(jié)合能力下降(Haas-Stapletonetal., 2004; Ohkawaetal., 2005)。而PIF-3和PIF-5則不參與ODV與中腸上皮細(xì)胞的結(jié)合和融合，可能參與了ODV與細(xì)胞結(jié)合后的一些關(guān)鍵過程，比如衣殼的轉(zhuǎn)運(yùn)，細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)等(Ohkawaetal., 2005; Sparksetal., 2011)。PIF-1、PIF-2和PIF-3能在ODV的表面形成一個(gè)高度穩(wěn)定的復(fù)合體，PIF-4與該復(fù)合體有強(qiáng)的相互作用，P74和PIF-8則與該復(fù)合體有較弱的相互作用，而PIF-5則與該復(fù)合體完全沒有相互作用(Pengetal., 2012)。另外，P74與PIF-5能分別與宿主中腸蛋白中大小為35 kDa和97 kDa的兩個(gè)蛋白產(chǎn)生相互作用，但是這兩個(gè)宿主蛋白到底是什么卻仍舊未知(Sparksetal., 2011; Zhouetal., 2005)。上述的8個(gè)PIF以及本實(shí)驗(yàn)室所發(fā)現(xiàn)的Ac110都是由桿狀病毒核心基因所編碼的，表明病毒對(duì)昆蟲的口服感染機(jī)制是古老而又高度保守的。然而也有研究表明，非桿狀病毒核心基因所編碼的SF58，也參與到病毒對(duì)蟲體的口服感染過程中(Simonetal., 2012)，暗示了口服感染機(jī)制比預(yù)想中更要復(fù)雜，可能由于病毒與宿主的協(xié)同進(jìn)化，導(dǎo)致這一過程與病毒的種屬特異性有關(guān)。

目前Ac110的作用機(jī)制未明，Ac110是否通過與PIF復(fù)合體結(jié)合，或者通過與宿主蛋白相互作用而行使功能，需要進(jìn)一步的實(shí)驗(yàn)驗(yàn)證。而Ac110保守氨基酸位點(diǎn)N27的突變影響了病毒的口服感染能力，說明這個(gè)位點(diǎn)對(duì)于Ac110蛋白有效發(fā)揮功能是至關(guān)重要的。該位點(diǎn)的突變可能影響了Ac110蛋白的結(jié)構(gòu)從而影響了Ac110與其他蛋白的相互作用網(wǎng)絡(luò)，也可能是Ac110直接的功能位點(diǎn)，其中的分子機(jī)理有待深入的研究。

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The conserved amino acid N27 of baculovirus Ac110 is important for efficient establishment of midgut infection during oral infection

ZHU Le-yuan1, LIU Jian-tao1,2, WANG Yan-ling1, YUAN Mei-jin1, WU Wen-bi1, YANG Kai1

(1.State Key Laboratory of Biocontrol, Sun Yat-sen University, Guangzhou 510275, China;2.Jiangxi Key Laboratory of Bioprocess, Jiangxi Science & Technology Normal University, Nanchang, Jiangxi 330013, China)

The family Baculoviridae is a highly selective pathogen in arthropods, mainly in insects of the order Lepidoptera.Ac110 is one of theperosinfectivityfactorsthat are essential for the initial infection of lavae in midgut cells, but the mechanism by which Ac110 employs that affect the midgut infection is still unknown.In the present study, two recombinant viruses, vAc110N27A and vAc110L35A were generated in which the conserved amino acids N27 and L35 of Ac110 were mutated to alanine, respectively.The expression of the mutant protein were confirmed by Western blot analysis.Transfection and infection assays and viral growth curves analysis were done by using Sf9 cells, the results showed that both the recombinant viruses were able to produce compatible infectious budded viruses and the spread of the viruses among Sf9 cells was not affected compared with the control virus.Bioassays were performed by feedingSpodopteraexigualarvae with purified polyhedra to detect the ability to initiate midgut infection for both recombinant viruses.The mortality rate of vAc110N27A was significantly reduced compared to the control virus, while no difference was observed for vAc110L35A.Thus we conclude that N27 of Ac110 is important for the function of Ac110 duringperosinfection.

Baculovirus;perosinfectivityfactor; Ac110; Bioassay

朱樂園，劉建濤，王燕玲,等.桿狀病毒Ac110蛋白的保守氨基酸位點(diǎn)N27對(duì)病毒在中腸建立有效感染起重要作用[J].環(huán)境昆蟲學(xué)報(bào)，2017,39(3):505-514.

中央高校基本科研業(yè)務(wù)費(fèi)專項(xiàng)資金(15lgjc20)

朱樂園，女，1994年生，碩士，研究方向?yàn)闂U狀病毒功能基因，E-mail：zhuly23@mail2.sysu.edu.cn

*通訊作者 Author for correspondence, E-mail: wuwenbi3@mail.sysu.edu.cn

Received:2017-05-06; 接受日期Accepted: 2017-05-28

Q968.1;S433.4

A

1674-0858(2017)03-0505-10