王長(zhǎng)江,沈志強(qiáng)1,,金婷婷,武曰星,劉 慧,曲光剛
( 1.山東省濱州畜牧獸醫(yī)研究院,山東濱州 256600;2.山東綠都生物科技有限公司,山東濱州 256600;3.華中農(nóng)業(yè)大學(xué),湖北武漢 43000)
10.11751/ISSN.1002-1280.2017.10.03
犬新孢子蟲巨噬細(xì)胞轉(zhuǎn)移抑制因子(NcMIF)克隆表達(dá) 與其免疫調(diào)節(jié)作用鑒定
王長(zhǎng)江2,沈志強(qiáng)1,2,金婷婷2,武曰星2,劉 慧3,曲光剛1*
( 1.山東省濱州畜牧獸醫(yī)研究院,山東濱州 256600;2.山東綠都生物科技有限公司,山東濱州 256600;3.華中農(nóng)業(yè)大學(xué),湖北武漢 43000)
通過(guò)原核系統(tǒng)表達(dá)犬新孢子蟲巨噬細(xì)胞轉(zhuǎn)移抑制因子(NcMIF),并對(duì)該蛋白的免疫調(diào)節(jié)作用進(jìn)行分析。根據(jù)GenBank發(fā)表的序列,利用分子生物學(xué)軟件設(shè)計(jì)了一對(duì)特異性引物,通過(guò)RT-PCR方法擴(kuò)增出NcMIF全基因,經(jīng)測(cè)序分析后,將NcMIF亞克隆到原核表達(dá)載體pET28a(+),然后將鑒定為陽(yáng)性的重組質(zhì)粒轉(zhuǎn)化到E. coli BL21(DE3)中用IPTG進(jìn)行誘導(dǎo)表達(dá)。利用HPLC對(duì)可溶性表達(dá)的重組NcMIF蛋白進(jìn)行純化,去除內(nèi)毒素,最后對(duì)NcMIF的免疫調(diào)節(jié)作用進(jìn)行鑒定。結(jié)果顯示,NcMIF沒(méi)有明顯的抗糖皮質(zhì)激素的免疫抑制作用,也沒(méi)有上調(diào)巨噬細(xì)胞TNF-α和NO表達(dá)量的作用,為進(jìn)一步探究NcMIF的生物學(xué)功能及MIF在宿主免疫調(diào)節(jié)中的作用奠定了基礎(chǔ)。
犬新孢子蟲;巨噬細(xì)胞轉(zhuǎn)移抑制因子;原核表達(dá); 免疫調(diào)節(jié)
新孢子蟲病(Neosporiasis)是一種原生動(dòng)物性疾病,并在世界范圍內(nèi)廣泛流行[1]。犬新孢子蟲(Neosporacanium)不僅能夠?qū)е掳ㄅ?、羊、犬等孕畜流產(chǎn)、死胎和弱胎等嚴(yán)重繁殖障礙性疾病,還導(dǎo)致幼畜出現(xiàn)運(yùn)動(dòng)神經(jīng)系統(tǒng)紊亂等癥狀,對(duì)養(yǎng)殖業(yè)造成極大的經(jīng)濟(jì)損失[2-3]。同時(shí),在病人血清中也有檢測(cè)出新孢子蟲陽(yáng)性的報(bào)道[4],因此犬新孢子蟲也可能是人獸共患寄生蟲病。但是迄今為止,尚未有根除犬新孢子蟲的藥物和有效預(yù)防犬新孢子蟲的疫苗。
犬新孢子蟲感染過(guò)程中,天然免疫系統(tǒng)中的很多成分被激活,如巨噬細(xì)胞、樹突狀細(xì)胞等,這些被激活的細(xì)胞會(huì)釋放如IL-2、TNF-α等一系列細(xì)胞因子。哺乳動(dòng)物的巨噬細(xì)胞轉(zhuǎn)移抑制因子(macrophage migration inhibitory factor,MIF)作為細(xì)胞因子的一種,與機(jī)體的敗血性休克有關(guān)[5],具有調(diào)節(jié)巨噬細(xì)胞和淋巴細(xì)胞應(yīng)答[6]及內(nèi)分泌的功能[7]。它不僅能夠抑制單核巨噬細(xì)胞的隨機(jī)遷移和糖皮質(zhì)激素的抗炎癥效果,而且能夠抑制活化誘導(dǎo)的p53依賴性凋亡[8]。MIF還能夠上調(diào)免疫細(xì)胞TLR4受體和促炎癥細(xì)胞因子的表達(dá),如TNF-α、IL-β、IL-6、IL-8和IL-12等,以及促進(jìn)NO的分泌[9]。MIF能夠在多種類型的細(xì)胞中表達(dá),包括單核細(xì)胞、淋巴巨噬細(xì)胞、內(nèi)皮細(xì)胞和成纖維細(xì)胞[10]。MIF被證實(shí)能夠與細(xì)胞表面的CD74受體結(jié)合[11],同時(shí)也是趨化因子CXC受體非同源的配體[12]。
研究發(fā)現(xiàn),MIF保守的CXXC序列是氧化還原酶作用的功能域;而N端第二位的脯氨酸則是互變異構(gòu)酶活性的關(guān)鍵位點(diǎn)[13]。盡管MIF蛋白的催化活性與生物學(xué)功能之間的關(guān)系還不是很清楚,但是抑制MIF的互變異構(gòu)酶作用能夠降低LPS刺激巨噬細(xì)胞產(chǎn)生的TNF的量,提高敗血癥模型動(dòng)物的成活率[14-15]。
近年來(lái),許多的寄生蟲MIF同系物被分離到,這其中包括Strongyloides MIF、Trichinella spiralis MIF及Brugia malayi MIF等,并且這些MIF同系物具有和它們哺乳動(dòng)物宿主相似的生物學(xué)功能和免疫調(diào)節(jié)作用[16-18]。本研究克隆了NcMIF基因,利用大腸埃希菌進(jìn)行表達(dá)并對(duì)該蛋白的免疫調(diào)節(jié)作用進(jìn)行分析,為研究NcMIF在宿主免疫調(diào)節(jié)中的作用奠定基礎(chǔ)。
1.1 材料
1.1.1 質(zhì)粒、菌種E.coliDH5α、BL21(DE3)及pMD-18T載體購(gòu)自大連寶生物工程有限公司;pET28a(+)載體購(gòu)自默克公司。
1.1.2 主要試劑 限制性內(nèi)切酶NdeI和HindIII、T4 DNA連接酶及PCR反應(yīng)試劑、膠回收試劑盒、質(zhì)粒提取試劑盒及DNA Marker DL2000、卡那霉素(Kanamycin)均購(gòu)自寶生物工程(大連)有限公司產(chǎn)品;M-MuLV反轉(zhuǎn)錄試劑盒購(gòu)自NEB公司;BATEKE RNA提取試劑盒購(gòu)自百泰克生物公司;Western blot發(fā)光底物購(gòu)自Thermo公司;實(shí)驗(yàn)用綿羊由山東省濱州畜牧獸醫(yī)研究院繁育飼養(yǎng);其他常用試劑均為分析純。
1.2 NcMIF基因的克隆與測(cè)序 基于NC1 NcMIF mRNA序列(NCLIV_042400,previously known as NC_LIV_113040,www.genedb.org),利用分子生物學(xué)軟件PRIMER 5.0設(shè)計(jì)出一對(duì)特異性引物,通過(guò)PCR方法擴(kuò)增出NcMIF基因。上游引物序列為5'-ATACATATGCCAAAGTGCATGATCTAC-3',其中在5'端加入NdeI酶切位點(diǎn),下游引物序列為5'-TAAAGCTTTTAGCCAAAGGTGCGGTC-3',其中在5'端加入HindIII酶切位點(diǎn)。采用BATEKE RNA提取試劑盒從犬新孢子蟲中提取總RNA,取2 μg總RNA利用M-MuLV反轉(zhuǎn)錄試劑盒合成cDNA。以該cDNA為模板通過(guò)PCR方法擴(kuò)增出NcMIF完整的閱讀框,PCR反應(yīng)條件如下:95 ℃預(yù)變性5 min;95 ℃變性30 s,55 ℃退火30 s,72 ℃延伸40 s,共30個(gè)循環(huán),最后72 ℃延伸10 min。PCR產(chǎn)物經(jīng)PCR純化試劑盒純化后,連接到pMD-18T載體,鑒定陽(yáng)性克隆,送上海生工測(cè)序并對(duì)測(cè)序結(jié)果進(jìn)行分析比較。
1.3 NcMIF蛋白的表達(dá)、內(nèi)毒素的去除及純化 將測(cè)序結(jié)果正確的基因片段亞克隆到pET28a(+),構(gòu)建重組質(zhì)粒pET28a+NcMIF,并轉(zhuǎn)入大腸埃希菌BL21(DE3),以終濃度1 mmol/L IPTG 37 ℃進(jìn)行誘導(dǎo)表達(dá)8 h。將誘導(dǎo)產(chǎn)物4 ℃條件下4000 ×g離心20 min,取上清,用裂解緩沖液(50 mmol/L NaH2PO4,300 mmol/L NaCl,PMSF 1 mmol/L,pH8.0)重懸細(xì)菌,-80 ℃/37 ℃反復(fù)凍融三次,然后用終濃度10 μg/mL的DNase/RNase 37 ℃消化30 min,然后4 ℃條件下20000 ×g離心20 min取上清,用于可溶性蛋白的純化。
將蛋白用Triton X-114處理,以去除內(nèi)毒素。首先將Triton X-114加到上一步分離到的上清液中至1%的終濃度,渦旋10 s,然后放置冰上5 min,渦旋之后37 ℃孵育10 min,最后20000 ×g,38 ℃離心2 min,收集上清,重復(fù)上述步驟7次,直到內(nèi)毒素濃度低于50 EU/mg。重組蛋白rNcMIF經(jīng)分子篩高壓液相色譜法(SECHPLC)分離純化,通過(guò)BCA方法測(cè)定純化后的蛋白濃度,并通過(guò)SDS-PAGE對(duì)蛋白純度和分子量進(jìn)行分析。
1.4 免疫印跡分析 NcMIF樣品經(jīng)處理后在還原條件下用12% SDS-PAGE膠進(jìn)行分離,然后將蛋白轉(zhuǎn)到PVDV膜上,用3%的脫脂奶粉封閉PVDV膜后,在羊抗全新孢子蟲抗體(1∶200)中室溫下孵育1 h,然后用含有0.05% Tween20的PBS洗膜5次,每次5 min。將PVDV膜在1∶5000的羊抗兔IgG-HRP二抗中室溫條件下孵育1 h,然后用含有0.05% Tween20的PBS洗膜5次,每次5 min。用化學(xué)發(fā)光底物(SuperSignal?West Dura Exrended Duration Substrate)進(jìn)行顯色并用成像儀拍照。
1.5 rNcMIF抗地塞米松免疫抑制功能鑒定 將500 μL大約含有1.5×105個(gè)RAW264.7巨噬細(xì)胞的DMEM細(xì)胞培養(yǎng)液平鋪于48孔細(xì)胞培養(yǎng)板中,在37 ℃,5% CO2培養(yǎng)箱中培養(yǎng)20~24 h。為判定NcMIFs對(duì)糖皮質(zhì)激素的反向調(diào)節(jié)作用,向含有或不含地塞米松(DEX)的培養(yǎng)基中分別加入遞增濃度的rNcMIF,將細(xì)胞預(yù)培養(yǎng)1 h,然后加入脂多糖至終濃度10 ng/mL,繼續(xù)培養(yǎng)16 h,收集細(xì)胞培養(yǎng)液上清,4 ℃ 20000 ×g離心20 min。上清中的TNF-α和NO分別利用ELISA試劑盒(eBioscience Inc,San Diego,CA)和格里斯反應(yīng)方法進(jìn)行檢測(cè)與分析。格里斯反應(yīng)分析如Straccia等[19]所述,即將150 μL樣品加入96孔板中,另外加入20 μL 1 μmol/L的磷酸酰胺腺嘌呤二核苷酸(Sigma,St.Louis,MO)和30 μL含葡萄糖(Sigma,St.Louis,MO)、葡萄糖磷酸脫氫酶(Sigma,St.Louis,MO)和硝酸還原酶的混合液。在孔中混合半小時(shí),然后分別加入20 μL 1%的磺胺和20 μL 0.1%N-(1-萘基)二鹽酸乙二胺(Sigma,St.Louis,MO)。孵育5 min后,使用酶標(biāo)儀(Molecular Devices SpectraMaxPlus384,Ramsey,MN)分別在550 nm和650 nm處讀數(shù)。亞硝酸鹽的濃度由亞硝酸鈉標(biāo)準(zhǔn)曲線確定。
1.6 NcMIF對(duì)TNF-α和NO的免疫調(diào)節(jié)作用鑒定
為了評(píng)估NcMIF對(duì)TNF-α和NO的免疫調(diào)節(jié)作用,RAW264.7巨噬細(xì)胞用不同濃度的rNcMIF處理,然后加入脂多糖繼續(xù)培養(yǎng)16 h,收集細(xì)胞上清,凍存于-80 ℃?zhèn)溆?,同時(shí)設(shè)脂多糖空白組陰性對(duì)照,上清中的TNF-α和NO含量檢測(cè)方法同1.5。
2.1 NcMIF基因克隆 利用一對(duì)特異性的引物,通過(guò)PCR的方法擴(kuò)增到一條約348 bp的產(chǎn)物,核酸電泳結(jié)果如圖1所示。將測(cè)序正確的NcMIF亞克隆到pET28a表達(dá)載體,構(gòu)建重組表達(dá)質(zhì)粒pET28a-NcMIF,并用Nde I和Hind III雙酶切鑒定,電泳結(jié)果如圖1第2、3和4泳道所示。
2.2 重組NcMIF的表達(dá)與純化 NcMIF重組大腸埃希菌BL21(DE3)在37 ℃用1 mmol/L IPTG進(jìn)行誘導(dǎo)表達(dá),經(jīng)過(guò)SDS-PAGE電泳驗(yàn)證,蛋白在分子量11 kDa處均有條帶,且部分為可溶性表達(dá)。重組蛋白用SECHPLC進(jìn)行純化,純化后的蛋白經(jīng)SDS-PAGE電泳鑒定(圖2)。去除內(nèi)毒素后rNcMIF內(nèi)毒素含量低于0.5 EU/mL。
2.3 NcMIF的免疫印跡分析 NcMIF免疫印跡結(jié)果表明在11 kDa左右出現(xiàn)明顯的條帶(圖3),說(shuō)明rNcMIF能夠與犬新孢子蟲的陽(yáng)性血清反應(yīng)。
2.4 rNcMIF抗地塞米松免疫抑制功能鑒定 在RAW264.7巨噬細(xì)胞中驗(yàn)證rNcMIF抗地塞米松免疫抑制實(shí)驗(yàn)結(jié)果表明,不同濃度的rNcMIF均沒(méi)有表現(xiàn)出抗糖皮質(zhì)激素免疫抑制作用來(lái)上調(diào)巨噬細(xì)胞TNF-α和NO產(chǎn)量的功能(圖4A、B)。
2.5 NcMIF對(duì)TNF-α和NO的免疫調(diào)節(jié)作用 RAW264.7巨噬細(xì)胞用不同濃度的rNcMIF處理,然后加入LPS繼續(xù)培養(yǎng)16 h,收集細(xì)胞上清,檢測(cè)TNF-α和NO的含量變化,結(jié)果證明rNcMIF同樣也沒(méi)有上調(diào)巨噬細(xì)胞TNF-α和NO的表達(dá)(數(shù)據(jù)未提供)。
研究表明,馬拉布魯線蟲來(lái)源的MIF(BmMIF-1)與人類MIF一樣,具有控制巨噬細(xì)胞趨化運(yùn)動(dòng)的功能;在體外,BmMIF-1能夠增加巨噬細(xì)胞TNF-α與IL-8的表達(dá);在體內(nèi),BmMIF-1又通過(guò)誘導(dǎo)激活的巨噬細(xì)胞產(chǎn)生,促使T細(xì)胞向Th2細(xì)胞分化,使宿主產(chǎn)生Th2免疫應(yīng)答,最終抑制促炎癥細(xì)胞因子的產(chǎn)生,這成為拉布魯線蟲逃避宿主免疫應(yīng)答的重要手段[20-21]。這種免疫調(diào)節(jié)作用很可能與MIF的氧化還原酶和互變異構(gòu)酶兩種酶的功能有關(guān)。
本研究首次報(bào)道了犬新孢子蟲MIF同系物。通過(guò)分析發(fā)現(xiàn),NcMIF具有哺乳動(dòng)物MIF高度保守的氨基酸殘基,說(shuō)明NcMIF可能具有與哺乳動(dòng)物MIF相似的生物學(xué)活性。為進(jìn)一步研究NcMIF的相關(guān)功能和生物學(xué)特性,本研究利用原核表達(dá)系統(tǒng)成功表達(dá)了可溶性的rNcMIF,并通過(guò)HPLC系統(tǒng)對(duì)蛋白進(jìn)行了純化。通過(guò)對(duì)rNcMIF抗地塞米松免疫抑制功能和對(duì)TNF-α和NO的免疫調(diào)節(jié)作用進(jìn)行研究,發(fā)現(xiàn)rNcMIF沒(méi)有上述的免疫調(diào)節(jié)功能,由于NcMIF的免疫調(diào)節(jié)功能與MIF的氧化還原酶和互變異構(gòu)酶活性密切相關(guān),所以下一步將對(duì)NcMIF的酶進(jìn)行鑒定。
關(guān)于NcMIF的其他生物學(xué)活性,以及NcMIF對(duì)在寄生蟲感染宿主、逃避宿主免疫系統(tǒng)中發(fā)揮的作用有待進(jìn)一步研究,為有效防控犬新孢子蟲提供更多參考。
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Cloning,ExpressionandImmunoregulatoryActivitiesIdentificationofMacrophageMigrationInhibitoryFactorDerivedfromNeosporacanium
WANG Chang-jiang2,SHEN Zhi-qiang1,2, JIN Ting-ting2,WU Yue-xing2,LIU Hui3,QU Guang-gang1*
(1.ShandongBinzhouAnimalScience&VeterinaryMedicineAcademy,Binzhou,Shandong256600,China; 2.ShandongLvDuBio-ScienceandTechnologyCo.,LTD,Binzhou,Shandong256600,China; 3.HuazhongAgricultureUniversity,Wuhan430000,China)
WANGChang-jiang,E-mail:guanggangqu@163.com
To express macrophage migration inhibitory factor (NcMIF) ofNeosporacaniumby prokaryotic expression system and identify the enzyme activities of NcMIF, the NcMIF complete open reading frame (ORF) sequence was obtained based on sequences reported in the GenBank. And one pair of specific primers was designed to amplify the NcMIF gene from cDNA by using polymerase chain reaction (PCR) and the NcMIF ORF was sequenced and subcloned into pET28a(+). The recombinant plasmid was transformed intoE.coliBL21 and induced by IPTG. The recombinant protein was expressed in soluble way and purified by HPLC and endotoxin was removed from the recombinant protein and native proteins. Results showed that NcMIF had no effect to counter-regulate glucocorticoid activity in macrophages or to regulate TNF-α and nitric oxide production by macrophages. It will establish the foundation to explore the biological function of NcMIF and its role in host immune regulation of MIF.
Neosporacanium; macrophage migration inhibitory factor; prokaryotic expression; immunoregulatory
2017-06-26
A
1002-1280 (2017) 10-0018-06
S852.7
山東省自然科學(xué)基金資助項(xiàng)目(ZR2013CQ004)
王長(zhǎng)江,碩士,從事病原源性免疫調(diào)節(jié)因子免疫調(diào)節(jié)功能的研究。
曲光剛。E-mail:guanggangqu@163.com
(編輯:陳希)