繭蜂病毒調(diào)控寄主細(xì)胞的NF-κB信號(hào)通路

徐 沙，劉樾彤，羅開珺

(云南大學(xué)生命科學(xué)學(xué)院，云南省高校動(dòng)物遺傳多樣性和進(jìn)化重點(diǎn)實(shí)驗(yàn)室，昆明 650091)

繭蜂病毒調(diào)控寄主細(xì)胞的NF-κB信號(hào)通路

徐 沙，劉樾彤，羅開珺*

(云南大學(xué)生命科學(xué)學(xué)院，云南省高校動(dòng)物遺傳多樣性和進(jìn)化重點(diǎn)實(shí)驗(yàn)室，昆明 650091)

多分DNA病毒(Polydnaviruses，PDVs)是寄生蜂的共生病毒。已有研究報(bào)道PDVs能感染寄主血細(xì)胞并有特異性表達(dá)，但是，哪些病毒的DNA片段能進(jìn)入寄主細(xì)胞，并參與調(diào)控NF-κB信號(hào)通路仍然還不清楚。本研究結(jié)果初步表明，在雙斑側(cè)溝繭蜂病毒(Microplitisbicoloratusbracovirus，MbBV)感染細(xì)胞后24 h能導(dǎo)致細(xì)胞凋亡，vank86、vank92、ptp109病毒基因片段均能在被感染后的Spli221(斜紋夜蛾)、Sf9(草地貪夜蛾)、High Five(粉紋夜蛾)細(xì)胞的DNA中檢測到，但轉(zhuǎn)錄水平有差異。進(jìn)一步對(duì)這3個(gè)基因與NF-κB調(diào)控的下游抗菌肽基因(attacin、defensin)和免疫基因酚氧化酶原激活酶(ppA3)的相互關(guān)系研究發(fā)現(xiàn)：在病毒感染Spli221、Sf9、High Five細(xì)胞24 h后，檢測到Spli221細(xì)胞中attacin、defensin、ppA3的轉(zhuǎn)錄本，attacin基因顯著下調(diào)；過表達(dá)Vank86、Vank92、PTP109的Spli221細(xì)胞，由LPS刺激后檢測到只表達(dá)單個(gè)病毒蛋白的Spli221細(xì)胞中的attacin、defensin、ppA3的 mRNA表達(dá)水平無顯著變化。研究初步揭示了MbBV病毒感染Spli221細(xì)胞后抑制NF-κB信號(hào)通路，但單個(gè)病毒蛋白不能抑制NF-κB信號(hào)通路，實(shí)驗(yàn)結(jié)果為進(jìn)一步深入研究多個(gè)病毒基因的共同作用及其在害蟲生物防治中的應(yīng)用打下了基礎(chǔ)。

多分DNA病毒；脂多糖；NF-κB；抗菌肽；酚氧化酶原激活酶

多分DNA病毒(Polydnaviruses，PDVs)是與寄生鱗翅目的膜翅目姬蜂科Iehneumoniae和繭蜂科Bracoindae寄生蜂共生的一類昆蟲病毒(Dupuyetal., 2006; Amico and Slavicek, 2012)。這兩類病毒的DNA以原病毒的形式整合到寄生蜂的基因組中并以垂直傳播的方式傳給子代寄生蜂(Desjardinsetal., 2008)。病毒在雌蜂卵巢中的萼細(xì)胞的細(xì)胞核中進(jìn)行復(fù)制和組裝，成熟的病毒粒子儲(chǔ)存在卵巢萼液中，當(dāng)雌蜂產(chǎn)卵時(shí)，病毒粒子隨同寄生蜂的卵一起被注射到寄主血腔中，已有報(bào)道表明，進(jìn)入寄主血腔的病毒DNA能整合到寄主細(xì)胞中開始轉(zhuǎn)錄(Becketal., 2011)。實(shí)驗(yàn)室前期的研究結(jié)果發(fā)現(xiàn)13個(gè)基因的mRNA可以在寄生后的寄主血淋巴中檢測到，其中包括有：編碼蛋白絡(luò)氨酸磷酸酯酶(Protein tyrosine phosphatase，PTP)的基因和編碼病毒錨蛋白重復(fù)序列結(jié)構(gòu)(Viral Ankyrin repeat，vank)的基因(Lietal., 2014)。繭蜂病毒中的PTPs蛋白通過抑制免疫細(xì)胞的細(xì)胞骨架來阻止寄主卵細(xì)胞被包囊(Whitfield, 1990; Espagneetal., 2004)。Vanks蛋白屬于IκB-like家族，這類蛋白的錨蛋白重復(fù)結(jié)構(gòu)序列和果蠅NF-κB信號(hào)通路中IκB家族成員的cactus蛋白具有相似的結(jié)構(gòu)域(Belvin and Anderson, 1996; Sankaretal., 1998; Bae and Kim, 2009)。這兩種蛋白是否具有抑制寄主免疫反應(yīng)的功能，仍然需要詳細(xì)的研究去證實(shí)。

免疫刺激因子脂多糖(lipopolysaccharide，LPS)是革蘭氏陰性菌細(xì)胞壁成分，會(huì)激發(fā)昆蟲的先天免疫反應(yīng)(劉甜和羅開珺，2011)。已有研究報(bào)道LPS可以激活小鼠肺損傷中的NF-κB信號(hào)通路(Guha and Mackman, 2001; Tianzhu and Shumin, 2015)。NF-κB信號(hào)通路是Toll和IMD兩條信號(hào)通路下游的一個(gè)關(guān)鍵分子，果蠅中Toll信號(hào)通路被病原體激活后促使NF-κB核轉(zhuǎn)錄因子Dorsal/Dif從IκB家族成員Cactus上釋放，從而進(jìn)入細(xì)胞核(Lehmingetal., 1995; Nicolasetal., 1998)。IMD信號(hào)通路激活后會(huì)誘導(dǎo)Relish蛋白上caspase 8調(diào)控的片段降解，導(dǎo)致形成NF-κB的N端Rel-68片段進(jìn)入細(xì)胞核而C端Rel-49留在細(xì)胞質(zhì)中與ank蛋白結(jié)合，發(fā)揮抑制作用(Stovenetal., 2000; Stovenetal., 2003; 盧新民和葉恭銀，2006)。激活Toll和IMD信號(hào)通路中的NF-κB核轉(zhuǎn)錄因子，調(diào)控抗菌肽基因(AMPs)和其他基因的表達(dá)(Lemaitreetal., 1996; Chevignonetal., 2014)，保護(hù)寄主抵御微生物(病毒、細(xì)菌、真菌、原生動(dòng)物)的入侵(Rutschmannetal., 2002; Panetal., 2012)。然而繭蜂病毒中的ptps和vanks是否參與寄主細(xì)胞NF-κB信號(hào)通路的調(diào)控，目前研究尚不清楚。

本文以雙斑側(cè)溝繭蜂病毒(Microplitisbicoloratusbracovirus，MbBV)和Spli221(斜紋夜蛾Spodopteralitura)、Sf9(草地貪夜蛾Spodopterafrugiperda)、High Five(粉紋夜蛾Trichoplusiani)細(xì)胞為研究對(duì)象，對(duì)MbBV-ptp和MbBV-vank病毒片段的功能進(jìn)行研究。研究表明：MbBV-vank86，vank92和ptp109的病毒基因片段能在被感染的昆蟲細(xì)胞的DNA中被檢測到，但轉(zhuǎn)錄水平有差異。并且MbBV感染Spli221細(xì)胞后抑制了NF-κB信號(hào)通路活性，但用LPS刺激過表達(dá)Vank86、Vank92的Spli221細(xì)胞后發(fā)現(xiàn)不能抑制NF-κB下游抗菌肽attacin、防御素defensin的表達(dá)，表明MbBV病毒可以抑制NF-κB信號(hào)通路，但只表達(dá)Vanks中單個(gè)病毒蛋白不能調(diào)控NF-κB信號(hào)通路。這為實(shí)驗(yàn)室進(jìn)一步深入研究多個(gè)病毒基因共同參與作用及其在害蟲生物防治中的應(yīng)用打下了基礎(chǔ)。

1 材料和方法

1.1 昆蟲細(xì)胞培養(yǎng)

Spli221斜紋夜蛾細(xì)胞系(Yanaseetal., 1998)、High Five粉紋夜蛾細(xì)胞系(Robertetal., 1994)(由中山大學(xué)有害生物控制與資源利用國家重點(diǎn)實(shí)驗(yàn)室贈(zèng)與)；Sf9細(xì)胞系(Vaughnetal., 1977)(由云南省出入境檢驗(yàn)檢疫局動(dòng)檢處贈(zèng)與)，以上細(xì)胞系均由本實(shí)驗(yàn)室培養(yǎng)、傳代和凍存。培養(yǎng)的培養(yǎng)基為含10%胎牛血清的Hyclone TNM-FH昆蟲培養(yǎng)基，培養(yǎng)于27℃細(xì)胞培養(yǎng)箱中。

1.2 病毒粒子的純化及感染

依照實(shí)驗(yàn)室前期的純化方法將病毒粒子進(jìn)行純化(Luo and Pang, 2006)，病毒粒子純化好以后，將培養(yǎng)好的不同細(xì)胞均勻鋪在細(xì)胞瓶中，按1×105細(xì)胞用一個(gè)雌蜂病毒量進(jìn)行感染(Luo and Pang, 2006; Becketal., 2011)，分別在感染后(post infection，p.i.)觀察細(xì)胞形態(tài)。

1.3 感染寄主細(xì)胞中病毒DNA片段的檢測

提取感染后72 h、2周、1個(gè)月的細(xì)胞DNA進(jìn)行病毒DNA片段檢測，DNA提取試劑盒購買于北京天根生化科技有限公司。

1.4 感染寄主細(xì)胞中病毒mRNA的轉(zhuǎn)錄檢測

提取感染后72 h、2周、1個(gè)月的細(xì)胞mRNA反轉(zhuǎn)錄為cDNA后進(jìn)行病毒mRNA的轉(zhuǎn)錄檢測。RNA提取試劑盒(RNAisoTM Plus)；RT-PCR(Perfect Real Time)反轉(zhuǎn)錄試劑盒購于大連寶生物有限公司。

1.5 引物設(shè)計(jì)和合成

設(shè)計(jì)引物時(shí)，根據(jù)MbBV病毒轉(zhuǎn)錄組測序發(fā)現(xiàn)的雙斑側(cè)溝繭蜂vank86、vank92、ptp109基因序列和attacin、defensin和ppA3的序列(Lietal., 2014)。本論文中所用的引物，均由英濰捷基(上海)貿(mào)易有限公司合成，具體序列及說明見表1。

1.6 繭蜂病毒的vank86、vank92、ptp109的真核表達(dá)載體構(gòu)建

取本實(shí)驗(yàn)室已構(gòu)建好的E-pMD-19T菌液，加A+抗性過夜培養(yǎng)，用試劑盒提取質(zhì)粒，對(duì)vank86、vank92、ptp109進(jìn)行雙酶切，用同樣的酶切位點(diǎn)對(duì)真核pIZT/V5-His載體進(jìn)行雙酶切。隨后將T載體上的目的基因片段和真核表達(dá)載體片段進(jìn)行膠回收，連接，轉(zhuǎn)化大腸桿菌DH5α，鑒定(劉信毅和羅開珺，2015)。過夜培養(yǎng)鑒定連接正確的菌液，用無內(nèi)毒素質(zhì)粒提取試劑盒提取質(zhì)粒，用于真核細(xì)胞系的轉(zhuǎn)染和表達(dá)。質(zhì)粒提取試劑盒E.Z.N.A Plasmid Mini Kit I和Endo-free Plasmid Mini Kit II購于OMEGA生物公司，大腸桿菌DH5α購于昆明云科生物技術(shù)有限公司。

1.7 細(xì)胞轉(zhuǎn)染和LPS的免疫刺激

用實(shí)驗(yàn)室已建立的轉(zhuǎn)染方法(Liuetal., 2013)轉(zhuǎn)染寄主Spli 221細(xì)胞48 h后用LPS刺激24 h，LPS的終濃度為15 μg/mL，對(duì)照組加入等量的PBS。

1.8 Western blot檢測

真核表達(dá)中，細(xì)胞轉(zhuǎn)染72 h后，提取細(xì)胞總蛋白，用BCA法測定蛋白濃度。取50 ug蛋白上樣電泳，300 mA轉(zhuǎn)膜1.5 h，5%的脫脂牛奶室溫封閉1 h，洗膜后真核表達(dá)用V5標(biāo)簽抗體(1 ∶2000)孵育過夜和二抗(1 ∶5000)孵育1 h，洗膜后用ECL發(fā)光液曝光顯影(劉信毅和羅開珺，2015)。Tubulin抗體、蛋白Marker、HRP偶聯(lián)的羊抗鼠二抗及BeyoECL Plus 購于碧云天公司，V5抗體購于Invitrogen公司。

表1 本研究所用到的引物Table 1 Primers used in this study

1.9 實(shí)時(shí)熒光定量PCR(qRT-PCR)檢測

MbBV病毒感染Spli 221、Sf 9、High Five細(xì)胞和過表達(dá)Vank86、Vank92、PTP109蛋白的Spli221細(xì)胞，提取RNA進(jìn)行反轉(zhuǎn)錄，然后進(jìn)行實(shí)時(shí)熒光定量PCR(qRT-PCR)檢測attacin、defensin、ppA3轉(zhuǎn)錄水平的變化，最終使用非參數(shù)T檢驗(yàn)分析。

2 結(jié)果與分析

2.1 MbBV感染導(dǎo)致昆蟲細(xì)胞形成凋亡小體

實(shí)驗(yàn)室先前的研究表明，雙斑側(cè)溝繭蜂自然寄生斜紋夜蛾后能夠誘導(dǎo)血細(xì)胞發(fā)生凋亡(Luo and Pang, 2006)。為研究MbBV能否直接誘導(dǎo)其它昆蟲細(xì)胞凋亡，我們用純化后的MbBV病毒粒子感染Spli221、Sf9、High Five細(xì)胞。發(fā)現(xiàn)感染24 h后 Spli221細(xì)胞出現(xiàn)凋亡小體，并一直延續(xù)到48 h仍能觀察到凋亡小體，但是，隨著時(shí)間的延長，在感染后72 h，就不能觀察到細(xì)胞的凋亡小體(圖1A)；在Sf9細(xì)胞中，凋亡小體的出現(xiàn)從感染后24 h一直延續(xù)到72 h(圖1B)；同樣，在High Five細(xì)胞中，凋亡小體的出現(xiàn)從感染后24 h一直延續(xù)到72 h(圖1C)。結(jié)果發(fā)現(xiàn)，MbBV病毒粒子的感染能誘導(dǎo)昆蟲細(xì)胞發(fā)生凋亡，而感染后24 h在3種昆蟲細(xì)胞中都出現(xiàn)了穩(wěn)定的凋亡癥狀。

圖1 MbBV病毒感染不同昆蟲細(xì)胞后的形態(tài)Fig. 1 Morphology of different cells after MbBV infected注：A，MbBV感染Spli221細(xì)胞24 h、48 h、72 h時(shí)的細(xì)胞形態(tài)；B，MbBV感染Sf9細(xì)胞24 h、48 h、72 h時(shí)的細(xì)胞形態(tài)；C，MbBV感染High Five細(xì)胞24 h、48 h、72 h時(shí)細(xì)胞形態(tài)觀察；比例尺= 10 μm。Note: A, morphology of Spli221cell after MbBV infected 24 h, 48 h, 72 h; B, morphology of Sf9 cell after MbBV infected 24 h, 48 h, 72 h; C, morphology of High Five cell after MbBV infected 24 h, 48 h, 72 h; bar=10 μm.

2.2 MbBV感染的昆蟲細(xì)胞內(nèi)，病毒DNA片段的檢測

實(shí)驗(yàn)室的數(shù)據(jù)顯示，在感染后短時(shí)間內(nèi)(5 h)，均能在被感染的細(xì)胞中檢測到上述3個(gè)基因的DNA片段和轉(zhuǎn)錄mRNA(Yuetal., 2016)，為進(jìn)一步探明隨感染時(shí)間的變化被病毒感染的細(xì)胞中是否還存在病毒DNA片段，本研究提取了感染后72 h、2周、1個(gè)月的細(xì)胞基因組DNA，用vank86、vank92、ptp109引物(表1)進(jìn)行PCR檢測，結(jié)果在細(xì)胞的DNA中發(fā)現(xiàn)了vank86、vank92、ptp109基因片段(圖2A、B、C)，但是在感染后72 h，ptp109只在Spli221細(xì)胞的DNA中檢測到(圖2B)，結(jié)果表明被MbBV感染的細(xì)胞DNA中都存在有vank86、vank92、ptp109基因片段。

圖2 MbBV病毒感染不同昆蟲細(xì)胞后瓊脂糖凝膠電泳檢測Fig.2 Analysis of MbBV infected different cells by agarose gel electrophoresis注：圖通過Image J反轉(zhuǎn)；A，MbBV感染Spli221、Sf9、High Five細(xì)胞72 h后PCR電泳檢測；B，MbBV感染Spli221、Sf9、High Five細(xì)胞2周后PCR電泳檢測；C，MbBV感染Spli221、Sf9、High Five細(xì)胞1個(gè)月后PCR電泳檢測。Note: The picture Inverting by Image J; A, analysis of PCR product are amplified vank86, vank92, ptp109 when MbBV infected different insect cells(Spli221、Sf9、High Five) 72 h; B, analysis of PCR product are amplified vank86, vank92, ptp109 when MbBV infected different insect cells(Spli221, Sf9, High Five) 2 weeks; C, analys of PCR product is amplified vank86, vank92, ptp109 when MbBV infected different insect cells(Spli221, Sf9, High Five) 1 month.

2.3 MbBV感染后，昆蟲細(xì)胞內(nèi)病毒基因的轉(zhuǎn)錄檢測

為了進(jìn)一步探明隨感染時(shí)間的變化，在感染細(xì)胞DNA中存在的病毒基因片段vank86、vank92、ptp109是否能在細(xì)胞內(nèi)進(jìn)行轉(zhuǎn)錄，本研究提取了MbBV感染后72 h、2周、1個(gè)月的細(xì)胞總RNA，轉(zhuǎn)錄為cDNA，進(jìn)行RT-PCR檢測。結(jié)果發(fā)現(xiàn)在感染后72 h三種昆蟲細(xì)胞均不能檢測到vank86、vank92、ptp109基因片段的mRNA轉(zhuǎn)錄(圖3A)。但2周時(shí)vank86基因片段在Spli221、Sf9、Hive Five細(xì)胞中有mRNA的轉(zhuǎn)錄，vank92基因片段在Sf9、Hive Five細(xì)胞中有mRNA的轉(zhuǎn)錄(圖3B)。1個(gè)月時(shí)vank86和vank92基因片段只在Sf9細(xì)胞中有mRNA的轉(zhuǎn)錄(圖3C)；相反，ptp109基因片段在整個(gè)感染過程中都未能檢測到mRNA的轉(zhuǎn)錄。結(jié)果表明，vank86、vank92、ptp109在MbBV感染不同昆蟲細(xì)胞時(shí)隨感染時(shí)間的延長有轉(zhuǎn)錄水平的差異。

2.4 MbBV病毒調(diào)控凋亡細(xì)胞的NF-κB信號(hào)通路，抑制抗菌肽基因attacin的轉(zhuǎn)錄

實(shí)驗(yàn)室前期報(bào)道了，雙斑側(cè)溝繭蜂影響寄主血細(xì)胞凋亡的主要信號(hào)通路，包括NF-κB信號(hào)通路(Lietal., 2014)。Bitra等2012年報(bào)道了毀側(cè)溝繭蜂病毒能調(diào)控NF-κB信號(hào)通路(Bitraetal., 2012)。為檢測MbBV病毒感染Spli221、Sf9、High Five細(xì)胞后是否能調(diào)控NF-κB信號(hào)通路，于是檢測了NF-κB信號(hào)通路調(diào)控的下游抗菌肽基因attacin和defensin的轉(zhuǎn)錄(Shubha，1999)。結(jié)果發(fā)現(xiàn)病毒感染細(xì)胞Sf9、High Five細(xì)胞后NF-κB信號(hào)通路未被激活(未檢測到抗菌肽基因attacin和defensin的轉(zhuǎn)錄)。檢測到病毒感染Spli221細(xì)胞24 h后attacin顯著下調(diào)(圖4)。結(jié)果表明，在病毒感染Spli221細(xì)胞以后，MbBV病毒抑制Spli221細(xì)胞中的NF-κB信號(hào)通路。

圖3 MbBV感染不同昆蟲細(xì)胞后RT-PCR檢測Fig.3 RT-PCR detection of MbBV infected different cells注：圖通過Image J反轉(zhuǎn)；A，MbBV感染Spli221、Sf9、High Five細(xì)胞72 h后RT-PCR檢測；B，MbBV感染Spli221、Sf9、High Five細(xì)胞2周后RT-PCR檢測；C，MbBV感染Spli221、Sf9、High Five細(xì)胞1個(gè)月后RT-PCR檢測。Note: The picture Inverting by Image J; A, analysis of RT-PCR product is amplified vank86, vank92, ptp109 when MbBV infected different insect cells(Spli221, Sf9, High Five) 72 h; B, analysis of RT-PCR product is amplified vank86, vank92, ptp109 when MbBV infected different insect cells (Spli221, Sf9, High Five) 2 weeks; C, analysis of RT-PCR product is amplified vank86，vank92，ptp109 when MbBV infected different insect cells(Spli221, Sf9, High Five) 1 month.

圖4 MbBV病毒感染Spli221細(xì)胞后抗菌肽基因和免疫基因的檢測Fig.4 Amps and immune gene transcription detection after MbBV infected Spli221注：非參數(shù)T檢驗(yàn)，*表示差異顯著(P<0.05)，**表示差異顯著(P<0.01)，NS表示沒有顯著差異(P>0.05)。Note: Nonparametric tests; *, means significant difference between treatment and control; **, means extremely significant difference between treatment and control; NS, means no significant difference between treatment and control.

2.5 表達(dá)單個(gè)病毒蛋白不能抑制LPS刺激下的NF-κB信號(hào)通路

MbBV病毒感染細(xì)胞后，會(huì)表達(dá)多個(gè)病毒蛋白一起參與免疫反應(yīng)，為檢測只表達(dá)單個(gè)病毒蛋白的細(xì)胞是否也能抑制NF-κB信號(hào)通路，于是本研究構(gòu)建只表達(dá)單一病毒蛋白的Vank86、Vank92、PTP109真核表達(dá)載體(圖5A)，將驗(yàn)證正確的質(zhì)粒轉(zhuǎn)染進(jìn)Spli221細(xì)胞，72 h后提取細(xì)胞總蛋白，用能識(shí)別載體本身表達(dá)的V5序列的標(biāo)簽抗體進(jìn)行Western blot實(shí)驗(yàn)，發(fā)現(xiàn)Vank86、Vank92蛋白在大于23 kDa處有一條特異性的條帶，與預(yù)期的融合蛋白大小一致。Vank86的融合蛋白大小為24 kDa，Vank92的融合蛋白大小為23 kDa，然而PTP109在44 kDa處出現(xiàn)一條主要帶(圖5B)。

在Spli221細(xì)胞中過表達(dá)Vank86、Vank92、PTP109 48 h后，加入LPS刺激細(xì)胞24 h后，檢測attacin、defensin、ppA3的mRNA水平，發(fā)現(xiàn)表達(dá)Vank86蛋白后，attacin基因表達(dá)上調(diào)，而表達(dá)Vank92蛋白、PTP109蛋白后沒有引起attacin、defensin、ppA3在mRNA水平的變化，以上結(jié)果表明：Vank86、Vank92、PTP109不能抑制Spli221細(xì)胞中由LPS刺激引起的NF-κB信號(hào)通路的變化，調(diào)控抗菌肽基因attacin和defensin的轉(zhuǎn)錄，暗示只表達(dá)單個(gè)病毒蛋白不能抑制NF-κB信號(hào)通路。

圖5 MbBV基因片段在昆蟲細(xì)胞中的表達(dá)及對(duì)抗菌肽相關(guān)基因表達(dá)的影響(兩次獨(dú)立實(shí)驗(yàn))Fig.5 MbBV segments expression in insect cells and the influence of Amps and immune gene(two independent experiment)注：非參數(shù)T檢驗(yàn)，*表示差異顯著(P<0.05)，NS表示沒有顯著差異(P>0.05)；A，vank86、vank92、ptp109真核表達(dá)結(jié)構(gòu)；B，Western blot檢測Vank86、Vank92、PTP109在Spli221中融合蛋白的表達(dá)；C，Vank86不能抑制attacin、ppA3的表達(dá)；D，Vank92不能抑制attacin、defensin、ppA3的表達(dá)；E，PTP109不能抑制attacin、defensin、ppA3的表達(dá)。Note: Nonparametric tests; *, means significant difference between treatment and control; NS, means no significant difference between treatment and control. A, vank86, vank92, ptp109 eukaryotic expression structure; B, expression of fusion protein Vank86-V5-His, Vank92-V5-His, PTP109-V5-His determined by Western blot in Spli221 cells; C, Vank86 didn’t inhibite attacin’s, ppA3’s expression; D, Vank92 didn’t inhibite attacin’s, defensin’s, ppA3’s expression; E, PTP109 didn’t inhibite attacin’s,defensin’s,ppA3’s expression.

3 結(jié)論與討論

PDVs可以誘導(dǎo)寄主細(xì)胞凋亡，但感染除寄主細(xì)胞外的細(xì)胞是否會(huì)導(dǎo)致細(xì)胞凋亡，目前研究尚少。本文研究表明MbBV病毒感染Spli221、High Five、Sf9細(xì)胞后會(huì)誘導(dǎo)細(xì)胞發(fā)生短期的凋亡(圖1)，Sf9細(xì)胞在病毒感染后的凋亡細(xì)胞的出現(xiàn)時(shí)間長于High Five和Spli221細(xì)胞，可能與Sf9中存在低水平的激活型Caspase3凋亡蛋白有關(guān)(Liuetal., 2013)。High Five和Spli221細(xì)胞凋亡可能與細(xì)胞中的PI3K信號(hào)通路失活有關(guān)(Liuetal., 2010)。Spli221細(xì)胞感染24 h和48 h細(xì)胞表面出現(xiàn)膨泡，但感染Spli221細(xì)胞72 h未觀察到細(xì)胞凋亡。非常有趣的發(fā)現(xiàn)是，在感染72 h后，未能觀察到Spli221細(xì)胞、High Five細(xì)胞、Sf9細(xì)胞凋亡，這可能是由于病毒隨著感染時(shí)間的推移，失去轉(zhuǎn)錄能力。

病毒DNA可以整合到細(xì)胞中(Burke and Strand, 2012)。本研究結(jié)果顯示，MbBV病毒vank86、vank92、ptp109基因片段可以在被感染的Spli221細(xì)胞、High Five細(xì)胞、Sf9細(xì)胞中檢測到。本文研究發(fā)現(xiàn)vank86、vank92基因片段在Spli221、High Five、Sf9穩(wěn)定細(xì)胞系中檢測到，然而ptp109基因片段不能在穩(wěn)定細(xì)胞系中檢測到。已有研究報(bào)道Tranosemarostraleichnovirus(TrIV)病毒基因組DNA能整合到Tranosemarostralevirus萼液細(xì)胞的細(xì)胞系中(Gundersen-Rindal and Pedroni, 2006；Doucetetal., 2007)。本研究中，僅用了病毒基因進(jìn)行PCR檢測，并不能確定這些基因片段整合進(jìn)了被病毒感染的細(xì)胞中，但已經(jīng)建立了能穩(wěn)定檢測到病毒DNA的細(xì)胞系，正在進(jìn)行下一步的探索研究。

為了進(jìn)一步驗(yàn)證寄主細(xì)胞中病毒基因的轉(zhuǎn)錄表達(dá)，本研究進(jìn)行RT-PCR檢測發(fā)現(xiàn)vank86、vank92、ptp109基因片段轉(zhuǎn)錄水平有差異，vank86、vank92、在2周和1個(gè)月時(shí)在Sf9細(xì)胞中都有轉(zhuǎn)錄，但在Spli221細(xì)胞和High Five細(xì)胞中ptp109未檢測到轉(zhuǎn)錄，未轉(zhuǎn)錄的現(xiàn)象可能需要做更精確的實(shí)時(shí)熒光定量水平的檢測。以上結(jié)果表明MbBV病毒基因片段進(jìn)入寄主細(xì)胞后可能行使功能。

在Glyptapantelesindiensisbracovirus(GiBV)病毒中的ptp和vanks，ptp可能會(huì)和信號(hào)轉(zhuǎn)導(dǎo)有關(guān)，而vanks和抑制NF-κB信號(hào)途徑有關(guān)(Gundersen-Rindal and Pedroni, 2006)。于是推測ptp和vanks基因編碼的蛋白可能會(huì)參與到寄主的免疫系統(tǒng)中。病毒Vank蛋白缺少N端和C端的調(diào)控結(jié)構(gòu)域，這些蛋白可以有效的抑制寄主昆蟲的NF-κB信號(hào)通路(Espagneetal., 2004; Gundersen-Rindal and Pedroni, 2006)。PDV抑制寄主血淋巴的黑化(melanizadoll)時(shí)，寄主免疫系統(tǒng)的抗菌抗病毒能力也會(huì)受到影響(Ashida and Brey, 1995)。然而在檢測MbBV病毒感染Spli221、Sf9、High Five細(xì)胞后，未檢測到Sf9和High Five細(xì)胞中抗菌肽基因和酚氧化還原激活酶的變化，這可能與細(xì)胞的種類有關(guān)，不同的細(xì)胞對(duì)外界信號(hào)的敏感度存在差異，導(dǎo)致細(xì)胞中信號(hào)通路的激活之間存在差異。然而檢測到Spli221細(xì)胞中的NF-κB信號(hào)通路活性受到抑制，這可能是由于病毒蛋白和Spli221細(xì)胞中NF-κB蛋白發(fā)生相互作用(Hongladaetal.，2005)，具體原因還需進(jìn)一步研究。通過LPS刺激Spli221細(xì)胞后并未檢測到Vank86、Vank92、PTP109會(huì)對(duì)NF-κB信號(hào)通路產(chǎn)生影響，猜測可能是因?yàn)镸bBV病毒粒子感染細(xì)胞時(shí)，存在多個(gè)病毒蛋白一起發(fā)揮抑制作用，而用LPS刺激表達(dá)單個(gè)病毒蛋白的細(xì)胞時(shí)，病毒蛋白并不能很好的發(fā)揮作用，從而不能抵御外界刺激?？赡苄枰黄鸨磉_(dá)多個(gè)病毒蛋白才能發(fā)揮作用，具體原因還需要更進(jìn)一步的實(shí)驗(yàn)進(jìn)行探究。

本實(shí)驗(yàn)室通過病毒感染細(xì)胞后發(fā)現(xiàn)病毒會(huì)促進(jìn)昆蟲細(xì)胞產(chǎn)生凋亡小體，并在被感染的細(xì)胞中檢測到了病毒基因片段，同時(shí)MbBV病毒可以抑制Spli221細(xì)胞中的NF-κB信號(hào)通路，該研究結(jié)果為后期實(shí)驗(yàn)室開展深入的病毒基因的功能研究打下了基礎(chǔ)。

References)

Amico VD, Slavicek J. Interactions between nucleopolyhedroviruses and polydnaviruses in larval lepidoptera [J].MolecularVirology, 2012: 126-146.

Ashida M, Brey PLT. Role of the integument in insect defense: Pro-phenol oxidase cascade in the cuticular matrix [J].ProceedingsoftheNationalAcademyofSciencesoftheUSA, 1995, 92(23): 10698-10702.

Bae S, Kim Y. IκB genes encoded inCotesiaplutellaebracovirus suppress an antiviral response and enhance baculovirus pathogenicity against the diamondback moth,Plutellaxylostella[J].Invertebr.Pathol., 2009, 102(1): 79-87

Bai SF, Li X, Sun SJ. Polydnavirus genome organization and gene expression in parasitoid [J].JournalofHenanAgriculturalUniversity, 2004, 38(3): 313-318. [白素芬, 李欣, 孫淑君. 寄生蜂多分DNA病毒基因組結(jié)構(gòu)及基因表達(dá)[J]. 河南農(nóng)業(yè)大學(xué)學(xué)報(bào), 2004, 38(3): 313-318]

Beck MH, Zhang S, Bitra K,etal. The encapsidated genome ofMicroplitisdemolitorbracovirus integrates into the hostPseudoplusiaincludens[J].JournalofVirology, 2011, 85(22): 11685-11696.

Belvin MP, Anderson KV. Interactions between nucleopolyhedroviruses and polydnaviruses in larval lepidoptera [J].CellDev.Biol., 1996, 12: 393-416.

Bitra K, Suderman RJ, Strand MR. Polydnavirus ank proteins bind NF-kappaB homodimers and inhibit processing of Relish [J].PLoSPathog., 2012, 8(5): e1002722.

Burke GR, Strand MR. Polydnaviruses of parasitic wasps: Domestication of viruses to act as gene delivery vectors [J].Insects, 2012, 3(1): 91-119.

Chevignon G, Theze J, Cambier S,etal. Functional annotation ofCotesiacongregatabracovirus: Identification of viral genes expressed in parasitized host immune tissues [J].JournalofVirology, 2014, 88(16): 8795-8812.

Desjardins CA, Gundersen-Rindal DE, Hostetler JB,etal. Comparative genomics of mutualistic viruses ofGlyptapantelesparasitic wasps [J].GenomeBiol., 2009, 9(12): R183.

Doucet D, Levasseur A, Beliveau C,etal. In vitro integration of an ichnovirus genome segment into the genomic DNA of lepidopteran cells [J].JournalofGeneralVirology, 2007, 88(1): 105-113.

Dupuy C, Huguet E, Drezen JM.Unfolding the evolutionary story of polydnaviruses [J].VirusResearch, 2006, 117(1): 81-89.

Espagne E, Dupuy C, Huguet E,etal. Genome sequence of a polydnavirus: Insights into symbiotic virus evolution [J].Science, 2004, 306(5694): 286-289.

Fleming J-AGW. Polydnaviruses: Mutualists and pathogens [J].AnnualReviewofEntomology, 1992, 37(1): 401-425.

Guha M, Mackman N. LPS induction of gene expression in human monocytes [J].CellularSignalling, 2001, 13(2): 85-94.

Gundersen-Rindal D, Pedroni M.Characterization and transcriptional analysis of protein tyrosine phosphatase genes and an ankyrin repeat gene of the parasitoidGlyptapantelesindiensispolydnavirus in the parasitized host [J].JournalofGeneralVirology, 2006, 87(2) 311-322.

Honglada T, Beck MH, and Strand MR. Inhibitor κB -like proteins from a polydnavirus inhibit NF-κB activation and suppress the insect immune response [J].DepartmentofEntomology, 2005, 102(32):11426-11431.

Ibrahim A, Kim Y. Transient expression of protein tyrosine phosphatases encoded inCotesiaplutellaebracovirus inhibits insect cellular immune responses [J].Naturwissenschaften, 2008, 95(1): 25-32.

Lehming N, Mcguire S, Brickman J,etal. Interactions of a Rel protein with its inhibitor [J].Biochemistry, 1995, 92(22): 10242-10246.

Lemaitre B, Nicolas E, Michaut L,etal. The dorsoventral regulatory gene cassettespatzle/Toll/cactuscontrols the potent antifungal response inDrosophilaadults [J].Cell, 1996, 86(6): 973-983.

Li G, Chen Q, Pang Y. Studies of artificial diets for the beet armyworm,Spodopteraexigua[J].Actascientiaecircumstantiae、HuanjingKexueXuebao, 1998, 37: 1-5.

Lemaitre B, Hoffmann J. The host defense ofDrosophilamelanogaster[J].Annu.Rev.Immunol., 2007, 25(1): 697-743.

Li M, Pang Z, Xiao W,etal. A transcriptome analysis suggests apoptosis-related signaling pathways in hemocytes ofSpodopteralituraafter parasitization byMicroplitisbicoloratus[J].PLoSONE, 2014, 9(10): e110967.

Liu JJ, Liu WD, Yang HZ,etal. Inactivation of PI3k/Akt signaling pathway and activation of caspase-3 are involved in tanshinone I-induced apoptosis in myeloid leukemia cells in vitro [J].AnnalsofHematology, 2010, 89(11): 1089-1097.

Liu T, Li M, Zhang Y,etal. A role for innexin2 and innexin3 proteins fromSpodopteraliturain apoptosis [J].PLoSONE, 2013, 8(7): e70456.

Liu T, Luo KJ. Immunity-related domains of Toll and IMD signaling pathways inDrosophila[J].JournalofEnvironmentalEntomology, 33(3): 388-395. [劉甜, 羅開珺, 果蠅Toll和IMD信號(hào)通路中的功能結(jié)構(gòu)[J]. 環(huán)境昆蟲學(xué)報(bào), 2011, 33(3): 388-395]

Liu X, Luo KJ. Molecular cloning and construction of expression vector of innexin1,innexin4 fromSpodopteralitura[J].JournalofEnvironmentalEntomology, 37(5): 970-978. [劉信毅, 羅開珺. 斜紋夜蛾inx1,inx4基因的克隆及表達(dá)載體構(gòu)建[J]. 環(huán)境昆蟲學(xué)報(bào), 2015, 37(5): 970-978]

Lu XM, Ye GG. The NF-κB signal pathway of insects [J].ChineseJournalofCellBiolog, 2006, 28: 561-565. [盧新民, 葉恭銀. 昆蟲的NF-kB信號(hào)通路[J]. 細(xì)胞生物學(xué)雜志, 2006, 28:561-565 ]

Luo K, Pang Y. Disruption effect ofMicroplitisbicoloratuspolydnavirus EGF-like protein, MbCRP, on actin cytoskeleton in lepidopteran insect hemocytes [J].ActaBiochimicaetBiophysicaSinica, 2006, 38(8): 577-585.

Luo K, Pang Y.Spodopteralituramulticapsid nucleopolyhedrovirus inhibitsMicroplitisbicoloratuspolydnavirus-induced host granulocytes apoptosis [J].JournalofInsectPhysiol, 2006, 52(8): 795-806.

Luo K, Trumble J, Pang Y. Development ofMicroplitisbicoloratusonSpodopteralituraand implications for biological control [J].BioControl, 2006, 52(3): 309-321.

Nicolas E, Reichhar J, Hoffmann J,etal. In vivo regulation of the IκB homologuecactus during the immune response ofDrosophila[J].JournalofBiologicalChemistry, 1998, 273(17): 10463-10469.

Pan X, Zhou G, Wu J,etal.Wolbachiainduces reactive oxygen species(ROS) -dependent activation of the Toll pathway to control dengue virus in the mosquitoAedesaegypti[J].ProceedingsoftheNationalAcademyofSciencesoftheUSA, 2012, 109(1): E23-31.

Robert RG, Li G, Anja C,etal. A new insect cell line fromTrichoplusiani(BTI-Tn-5B1-4) susceptible toTrichoplusianisingle enveloped nuclear polyhedrosis virus [J].JournalofInvertebratePathology, 1994, 64(3): 260-266.

Rutschmann S, Kilinc A, Ferrandon D. Cutting Edge: TheTollpathway is required for resistance to gram-positive bacterial infections inDrosophila[J].TheJournalofImmunology, 2002, 168(4): 1542-1546.

Sankar Ghosh, Michael J. May, Kopp. EB. NF-κB and Rel proteins: Evolutionarily conserved mediators of immune responses [J].Annu.Rev.Immunol, 1998, 16(1): 225-260.

Shubha Govind. Control of development and immunity by Rel transcription factors inDrosophila[J].DepartmentofBiology, 1999, 18(49): 6875-6887.

Stoven S, Ando I, Kadalayil L,etal. Activation of theDrosophilaNF-kB factor Relish by rapid endoproteolytic cleavage [J].EMBOReports, 2000, 1(4): 347-352.

Stoven, Silverman N, Junell A,etal. Caspase-mediated processing of theDrosophilaNF-kappaB factor Relish [J].ProceedingsoftheNationalAcademyofSciencesoftheUSA, 2003, 100(10): 5991-5996.

Tianzhu Z, Shumin W. Esculin inhibits the inflammation of LPS-induced acute lung injury in mice via regulation of TLR/NF-kappaB pathways [J].Inflammation, 2015, 38(4): 1529-1536.

Vaughn JL, Goodwin RH, Tompkins GJ,etal. The establishment of two cell lines from the insectSpodopterafrugiperda(lepidoptera; noctuidae) [J].InVitroCellular&DevelopmentalBiology-Plant, 1977, 13(4): 213-217.

Whitfield JB. Parasitoids, polydnaviruses and endosymbiosis [J].ParasitolToday, 1990, 6(12): 381.

Yanase T, Yasunaga C, Kawarabata T. Replication ofSpodopteraexiguanucleopolyhedrovirus in permissive and non-permissive lepidopteran cell lines [J].ActaVirologica, 1998, 42(5): 293-298.

Yu DS, Chen YB, Li M,etal. A polydnaviral genome of fMicroplitisbicoloratusbracovirus and molecular interactions between host and virus with respect to NF-κB singal [J].ArchivesofVirology, 2016, 161(11): 3095-3124.

MbBVregulateshostcell’sNF-κBsignalpathway

XU Sha, LIU Yue-Tong, LUO Kai-Jun*

(Yunnan Province Key Laboratory for University Zoological Genetic Diversity and Evolution, School of Biosciences, Yunnan University, Kunming 650091, China)

Polydnaviruses(PDVs) are symbiotic virus of parasitoid wasps. It has been reported that PDVs can infected and specifically express on the host hemocytes. However, which virus DNA segments can enter the host cell and participate in the regulation of NF-κB signal pathway remain unclear. Our preliminary research results showed thatMicroplitisbicoloratusbracovirus(MbBV) can induce cells apoptosis after infected 24 h. MbBV-vank86，vank92，ptp109 segments were detected in insect cells(Spli221, Sf9, High Five) after infected with MbBV, but there were differences on transcription levels. Further more, We explore the relationship between three genes and downstream of NF-κB signal pathway antibacterial peptide(attacin，defensin) and immune gene prophenolosidase-activating enzyme(ppA3): When MbBV infected Spli221, Sf9, High Five 24 hours, we detect transcription level ofattacin，defensin,ppA3. We findattacindown-regulation extremely. Vank86, Vank92, PTP109 over-expression were detected in Spli221, attacin,defensin, ppA3 mRNA level have no significant difference after LPS stimulated. These results suggested that MbBV can regulate NF-κB signal pathway in Spli221 cell, but single virul protein cann’t inhibit NF-κB signal pathway. The results of this study could offer a reliable experiment basis for exploringmultiple genes function together and insect pests biological control in the future.

Polydnaviruses; lipopolysaccharide; NF-κB; antibacterial peptide; prophenolosidase-activating enzyme

徐沙，劉樾彤，羅開珺.繭蜂病毒調(diào)控寄主細(xì)胞的NF-κB信號(hào)通路[J].環(huán)境昆蟲學(xué)報(bào)，2017,39(5):1090-1099.

Q963;S476

A

1674-0858(2017)05-1090-10

國家自然科學(xué)基金項(xiàng)目(31060251，31260448，31471823)；云南省應(yīng)用基礎(chǔ)研究計(jì)劃重點(diǎn)項(xiàng)目(2013FA003)

徐沙，女，1991年生，云南宣威人，碩士，主要從事昆蟲的先天免疫研究

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

Received: 2016-08-02；接受日期Accepted: 2016-12-08