中國(guó)對(duì)蝦蛋白磷酸酶1催化亞基β基因的克隆表達(dá)及特性分析*

何亮銀, 李 微, 唐小千, 邢 婧, 繩秀珍, 戰(zhàn)文斌

(中國(guó)海洋大學(xué)水產(chǎn)動(dòng)物病害與免疫實(shí)驗(yàn)室, 山東 青島 266003)

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中國(guó)對(duì)蝦蛋白磷酸酶1催化亞基β基因的克隆表達(dá)及特性分析*

何亮銀, 李 微, 唐小千, 邢 婧, 繩秀珍, 戰(zhàn)文斌**

(中國(guó)海洋大學(xué)水產(chǎn)動(dòng)物病害與免疫實(shí)驗(yàn)室, 山東 青島 266003)

本論文利用RACE(Rapid-amplification of cDNA ends)技術(shù)克隆獲得中國(guó)對(duì)蝦(Fenneropenaeuschinensis)蛋白磷酸酶1催化亞基β(Protein phosphatase 1 catalytic subunit beta isoform,PP1β)基因cDNA序列全長(zhǎng)。該基因全長(zhǎng)1 214 bp, 包含一個(gè)987 bp的開(kāi)放閱讀框，編碼328個(gè)氨基酸。同源性分析顯示，中國(guó)對(duì)蝦PP1β氨基酸序列與不同物種PP1β的相似性高達(dá)90%～91%，表現(xiàn)出高度保守性。多序列比對(duì)結(jié)果顯示，不同物種PP1β均含有絲氨酸/蘇氨酸特異性蛋白磷酸酶家族的特征基序GDxHG、GDxVDRG和GNHE。系統(tǒng)進(jìn)化樹(shù)分析顯示，甲殼動(dòng)物PP1β聚為一大支，中國(guó)對(duì)蝦PP1β和凡納濱對(duì)蝦(Litopenaeusvannamei)聚為一小支。實(shí)時(shí)熒光定量PCR分析顯示，PP1β在健康的中國(guó)對(duì)蝦各組織中均有不同程度的表達(dá)，其中在性腺中表達(dá)最高，血細(xì)胞次之。白斑癥病毒(White spot syndrome virus, WSSV)注射感染健康中國(guó)對(duì)蝦后，血細(xì)胞和性腺中PP1β基因均呈上調(diào)表達(dá)，并在12 h達(dá)到峰值，且在血細(xì)胞中上調(diào)表達(dá)更顯著。構(gòu)建了中國(guó)對(duì)蝦PP1β基因原核重組表達(dá)載體pET28a-PP1β，轉(zhuǎn)化大腸桿菌后成功誘導(dǎo)表達(dá)重組PP1β蛋白(rPP1β)，分子量為41 kDa。將親和層析純化的rPP1β免疫BALB/c小鼠制備抗血清，通過(guò)制備中國(guó)對(duì)蝦血細(xì)胞滴片，應(yīng)用間接免疫熒光法檢測(cè)PP1β在血細(xì)胞中的分布情況，結(jié)果顯示，中國(guó)對(duì)蝦PP1β在血細(xì)胞的核區(qū)及細(xì)胞質(zhì)內(nèi)均有分布。本研究結(jié)果為進(jìn)一步解析中國(guó)對(duì)蝦PP1β與WSSV感染的相互關(guān)系提供了數(shù)據(jù)。

中國(guó)對(duì)蝦；蛋白磷酸酶1；基因克隆；原核表達(dá)；免疫熒光

蛋白磷酸酶(Protein phosphatase)是控制蛋白質(zhì)去磷酸化的關(guān)鍵酶，參與細(xì)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)途徑的調(diào)控，從而在生物體的生長(zhǎng)發(fā)育、新陳代謝、細(xì)胞的分裂分化、細(xì)胞間通訊、基因表達(dá)、離子通道活性和免疫反應(yīng)等多方面發(fā)揮重要作用[1-3]。根據(jù)蛋白磷酸酶作用底物的特異性可將其分為兩大類：絲氨酸/蘇氨酸蛋白磷酸酶和酪氨酸蛋白磷酸酶，其中，根據(jù)酶對(duì)底物的特異性、對(duì)抑制物的敏感程度差異和對(duì)不溶離子的需要，真核生物的絲氨酸/蘇氨酸蛋白磷酸酶可分為蛋白磷酸酶1(Protein phosphatase 1，PP1)，2A和2B三個(gè)主要亞型[4]。

在高等哺乳動(dòng)物中已有較多報(bào)道顯示，宿主細(xì)胞的蛋白磷酸酶可以和病毒之間存在密切聯(lián)系，參與病毒的轉(zhuǎn)錄、復(fù)制和生命周期調(diào)控[5-8]。然而，目前在甲殼動(dòng)物中蛋白磷酸酶與病毒間的互作研究十分有限，近年來(lái)有研究發(fā)現(xiàn)凡納濱對(duì)蝦(Litopenaeusvannamei)的一種蛋白磷酸酶被證實(shí)可以和白斑癥病毒(White spot syndrome virus，WSSV)開(kāi)放閱讀框(ORF)427[9]和ORF403[10]編碼蛋白相結(jié)合，該酶與已報(bào)道物種的PP1催化亞基β(PP1β)具有較高的同源性。中國(guó)對(duì)蝦(Fenneropenaeuschinensis)廣泛分布于中國(guó)黃、渤海，東海和南海也有少量分布，是中國(guó)北方沿海地區(qū)重要的海水養(yǎng)殖品種。對(duì)蝦白斑病(White spot disease, WSD)自1990年代暴發(fā)以來(lái)，已成為全球甲殼動(dòng)物養(yǎng)殖產(chǎn)業(yè)最主要的威脅，給對(duì)蝦養(yǎng)殖產(chǎn)業(yè)造成了重大的經(jīng)濟(jì)損失[11]。本實(shí)驗(yàn)室的前期研究發(fā)現(xiàn)，中國(guó)對(duì)蝦在感染W(wǎng)SSV后，其血細(xì)胞中的蛋白磷酸酶發(fā)生了顯著上調(diào)表達(dá)[12]，推測(cè)其可能與WSSV感染之間存在密切關(guān)系。

為進(jìn)一步解析中國(guó)對(duì)蝦蛋白磷酸酶在WSSV感染之間的關(guān)系，本研究克隆并分析了中國(guó)對(duì)蝦PP1β基因，利用實(shí)時(shí)熒光定量PCR檢測(cè)其在各組織中的表達(dá)情況以及對(duì)蝦在感染W(wǎng)SSV后該基因的應(yīng)答表達(dá)情況。同時(shí)，對(duì)中國(guó)對(duì)蝦PP1β進(jìn)行了原核重組表達(dá)，并制備了重組PP1β蛋白(rPP1β)的多克隆抗體，應(yīng)用間接免疫熒光技術(shù)檢測(cè)了PP1β在血細(xì)胞中的分布情況。

1 材料與方法

1.1 總RNA提取及cDNA第一鏈的合成

性成熟的健康海捕中國(guó)對(duì)蝦成蝦購(gòu)自青島水產(chǎn)品市場(chǎng)，均為雄蝦，體長(zhǎng)15～17 cm，經(jīng)PCR檢測(cè)為WSSV陰性[12]；按照Z(yǔ)hong等所述方法[13]，以血淋巴∶抗凝劑(27 mmol/L sodium citrate, 336 mmol/L NaCl, 115 mmol/L glucose, 9 mmol/L EDTA, pH=4.2)為1∶1的體積比從中國(guó)對(duì)蝦圍心腔抽取血淋巴，分離血細(xì)胞，使用Trizol法提取總RNA，1%瓊脂糖凝膠電泳檢測(cè)RNA的完整性，nano-drop測(cè)定其濃度和純度，取2 μg總RNA經(jīng)RT-PCR合成cDNA第一鏈。

1.2PP1β基因的全長(zhǎng)克隆

根據(jù)凡納濱對(duì)蝦、印度跳蟻(Harpegnathossaltator)和佛羅里達(dá)弓背蟻(Camponotusfloridanus)的PP1β序列，設(shè)計(jì)簡(jiǎn)并引物PP1β-F1和PP1β-R1(見(jiàn)表1)擴(kuò)增中國(guó)對(duì)蝦PP1β保守序列，根據(jù)獲得的PP1β保守基因片段設(shè)計(jì)3’和5’RACE特異性引物PP1β-3’1、PP1β-3’2、PP1β-5’1、PP1β-5’2(見(jiàn)表1)，分別與SMART cDNA合成試劑盒(Takara，Japan)內(nèi)的引物UPM和NUP聯(lián)合使用擴(kuò)增中國(guó)對(duì)蝦PP1βcDNA的3'和5'端序列。將所得到的3個(gè)片段拼接，獲得PP1βcDNA全長(zhǎng)，設(shè)計(jì)引物PP1β-F2和PP1β-R2(見(jiàn)表1)進(jìn)行基因全長(zhǎng)的克隆驗(yàn)證。PCR擴(kuò)增條件為：94 ℃ 5 min；94 ℃ 30 s，58 ℃ 40 s，72 ℃ 1 min 30 s，35個(gè)循環(huán)；72 ℃ 10 min。PCR產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳檢測(cè)，回收純化，連接到pMD-19T載體，轉(zhuǎn)化感受態(tài)細(xì)胞DH5α，利用含氨芐青霉素的LB培養(yǎng)平板進(jìn)行篩選，PCR檢測(cè)陽(yáng)性菌落后，送上海桑尼生物技術(shù)有限公司測(cè)序。

1.3PP1β基因的生物信息學(xué)分析

利用ORF Finder分析中國(guó)對(duì)蝦PP1β的開(kāi)放閱讀框(Open reading frame，ORF)并推導(dǎo)其氨基酸序列，Smart在線軟件(http://smart.embl-heidelberg.de/)預(yù)測(cè)蛋白功能域，ProtParam tool (http://web.expasy.org/protparam/)分析蛋白等電點(diǎn)與分子量，NetOGlyc 3.1 Server (http:// www.cbs.dtu.dk/services/NetOGlyc-3.1/)和NetPhos 2.0 Server(http://www.cbs.dtu.dk/ services/NetPhos/)分析O-糖基化位點(diǎn)和磷酸化位點(diǎn)。使用BLAST程序進(jìn)行PP1β的同源性比對(duì)，將不同物種的PP1β氨基酸序列通過(guò)Clustal X 2.0軟件進(jìn)行多序列比對(duì)分析，用MEGA 4.0軟件進(jìn)行系統(tǒng)發(fā)生和進(jìn)化分析，采用鄰位相連法構(gòu)建系統(tǒng)進(jìn)化樹(shù)。

1.4 實(shí)時(shí)熒光定量PCR檢測(cè)PP1βmRNA的組織分布情況

為檢測(cè)PP1βmRNA在中國(guó)對(duì)蝦各組織中的表達(dá)差異，隨機(jī)選取3尾健康中國(guó)對(duì)蝦，取其血細(xì)胞、心臟、肝胰腺、腸、性腺、淋巴器官、鰓和肌肉，分別提取各組織總RNA，反轉(zhuǎn)錄合成cDNA鏈，測(cè)定并調(diào)整cDNA濃度到一致，以此為模板，以PP1β-F3和PP1β-R3(見(jiàn)表1)為引物擴(kuò)增目的片段，每個(gè)樣品做3個(gè)平行，18S rRNA作為內(nèi)參。PCR擴(kuò)增條件為：95℃ 2min；95℃ 10s，58℃ 10s，72℃ 20s，45個(gè)循環(huán)。根據(jù)測(cè)得的Ct值，利用2-Ct法計(jì)算不同組織中PP1β基因的相對(duì)表達(dá)量。

1.5 實(shí)時(shí)熒光定量PCR分析PP1β基因?qū)SSV感染的應(yīng)答表達(dá)

取凍存于-80℃患白斑綜合征病毒病的中國(guó)對(duì)蝦的鰓，按照Li等所述方法[12]制備WSSV粗提液并進(jìn)行病毒濃度的測(cè)定，0.01mol/L無(wú)菌磷酸鹽緩沖液(PBS, 137mmol/L NaCl, 2.7mmol/L KCl, 8.09 mmol/L Na2HPO4, 1.47mmol/L KH2PO4, pH=7.4)調(diào)整WSSV濃度至108拷貝/mL，實(shí)驗(yàn)對(duì)蝦分為感染組和對(duì)照組，分別注射100μL WSSV 粗提液 (107拷貝)或PBS。各組于感染后0、6、12、24、36、48和72h隨機(jī)抽取中國(guó)對(duì)蝦6尾，收集血細(xì)胞和性腺，進(jìn)行RNA提取和實(shí)時(shí)熒光定量PCR實(shí)驗(yàn)，具體方法同1.4。利用2-ΔΔCt法計(jì)算WSSV感染后對(duì)蝦血細(xì)胞和性腺組織中PP1β基因的相對(duì)表達(dá)量的變化情況。

1.6PP1β的原核表達(dá)與純化

設(shè)計(jì)分別帶有酶切位點(diǎn)KpnI和Hind Ⅲ的原核表達(dá)用引物PP1β-F4和PP1β-R4(見(jiàn)表1)，擴(kuò)增PP1β編碼基因全長(zhǎng)。對(duì)擴(kuò)增片段和pET-28a(+)載體分別進(jìn)行雙酶切，切膠純化，以T4 DNA連接酶連接目的片段和載體，構(gòu)建重組表達(dá)質(zhì)粒pET-28a-PP1β，轉(zhuǎn)化至大腸桿菌BL21(DE3)感受態(tài)細(xì)胞，隨后將陽(yáng)性菌株進(jìn)行測(cè)序確認(rèn)。

挑取陽(yáng)性重組菌株的單菌落接種至LB液體培養(yǎng)基中(含50μg/mL卡那霉素)，37℃，220r/min振蕩培養(yǎng)，培養(yǎng)菌液的OD600為0.6時(shí)，向菌液中加入1.0mmol/L的異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)大腸桿菌表達(dá)目的蛋白，繼續(xù)振蕩培養(yǎng)4h后，離心收集菌體，加PBS超聲破碎30min，離心取沉淀，利用SDS-PAGE檢測(cè)目的蛋白表達(dá)情況，以未誘導(dǎo)的菌體總蛋白為對(duì)照。將重組菌大量培養(yǎng)，離心破碎收集菌體，參照Qiagen鎳瓊脂糖親和層析柱蛋白純化操作步驟，在變性條件下純化以包涵體形式表達(dá)的融合蛋白，尿素梯度透析復(fù)性，SDS-PAGE 電泳檢測(cè)重組PP1β蛋白(r PP1β)純化結(jié)果。

表1 中國(guó)對(duì)蝦 PP1β cDNA全長(zhǎng)擴(kuò)增、實(shí)時(shí)熒光定量PCR和原核表達(dá)所用引物

1.7 鼠抗rPP1β血清的制備

取純化復(fù)性后的rPP1β，按照Tang等所述方法[14]分4次免疫購(gòu)自山東大學(xué)實(shí)驗(yàn)動(dòng)物中心的SPF級(jí)BLAB/c小鼠。第3次加強(qiáng)免疫后第7天心臟一次性采血，室溫傾斜放置2h，置于4℃過(guò)夜；次日5 000g離心20min得抗血清。間接ELISA方法測(cè)得鼠抗rPP1β血清效價(jià)。同時(shí)，參考Tang等報(bào)道的方法[15]利用Western blotting檢測(cè)制備鼠抗血清的特異性。

1.8 間接免疫熒光實(shí)驗(yàn)(IIFA)檢測(cè)PP1β在血細(xì)胞上的定位

用吸有4 ℃預(yù)冷抗凝劑的注射器從WSSV感染12 h后的中國(guó)對(duì)蝦圍心腔抽取血淋巴液，按抗凝劑與血淋巴3∶1 比例混勻，800g離心20min，棄上清；沉淀用抗凝劑重懸，800g離心20min，棄上清；沉淀用PBS重懸，滴于干凈載玻片上，室溫沉降1h，丙酮固定。以制備的鼠抗rPP1β血清為第一抗體，滴加在上述制備的血細(xì)胞滴片上，37℃濕盒中孵育45min，以正常鼠血清為陰性對(duì)照；PBST(PBS containing 0.05% Tween 20)洗3次，每次5min；以FITC標(biāo)記的羊抗鼠IgG(Sigma,1∶256)為第二抗體，滴加在血細(xì)胞滴片上，37℃濕盒中孵育45min；PBST洗3次，每次5min；甘油封片，熒光顯微鏡下觀察。

1.9 數(shù)據(jù)處理

所得數(shù)據(jù)使用SPSS 19.0進(jìn)行統(tǒng)計(jì)分析，采用單因子方差分析及Duncan多重比較處理，以P<0.05作為差異顯著水平。

2 結(jié)果

2.1 中國(guó)對(duì)蝦PP1β基因序列分析

中國(guó)對(duì)蝦PP1βcDNA全長(zhǎng)及相應(yīng)的氨基酸序列如圖1所示。中國(guó)對(duì)蝦PP1β完整的cDNA全長(zhǎng)1 214 bp(GenBank登錄號(hào)：KF773851)，包含一個(gè)987 bp的開(kāi)放閱讀框，編碼328個(gè)氨基酸。PP1β編碼蛋白由PP2Ac結(jié)構(gòu)域和低復(fù)雜度區(qū)域組成，其理論等電點(diǎn)為5.68，理論分子量為37.6 kDa，氨基酸序列中包含14個(gè)磷酸化位點(diǎn)和1個(gè)O-糖基化位點(diǎn)。成熟肽中Leu所占比例最高(11.9%)，其次是Asp(7.3%)和Gly(7.3%)。5’非編碼區(qū)13 bp，3’非編碼區(qū)214 bp，其中包含1個(gè)終止密碼子，1個(gè)多聚腺苷酸加尾信號(hào)(AATAAA)。

(下劃線：PP2Ac結(jié)構(gòu)域；陰影：低復(fù)雜度區(qū)域；方框：多聚腺苷酸加尾信號(hào)(AATAAA)。PP2Ac domain was shown with underlines, and low complexity region was shaded in grey. The polyadenylation signals(AATAAA)in the 3’-UTR were also boxed.)

圖1 中國(guó)對(duì)蝦PP1βcDNA及推導(dǎo)的氨基酸序列

Fig.1 Full length cDNA sequence and deduced amino acid sequences ofF.chinensisPP1β

2.2 中國(guó)對(duì)蝦PP1β與其他物種的同源性分析

將中國(guó)對(duì)蝦PP1β的氨基酸序列通過(guò)BLAST在線分析顯示，該序列與不同物種的PP1β序列的相似度高達(dá)90%～91%，表現(xiàn)出高度保守性。需要指出的是，中國(guó)對(duì)蝦PP1β氨基酸序列與GenBank中一條C端缺失的凡納濱對(duì)蝦PP1β序列完全一致，相似性為100%。多序列比對(duì)結(jié)果顯示，不同物種的PP1β均含有絲氨酸/蘇氨酸特異性蛋白磷酸酶家族的特征基序GDxHG、GDxVDRG和GNHE(見(jiàn)圖2)。構(gòu)建的系統(tǒng)進(jìn)化樹(shù)顯示，中國(guó)對(duì)蝦PP1β與凡納濱對(duì)蝦聚為一小支，二者與其他甲殼動(dòng)物PP1β聚為一大支(見(jiàn)圖3)。

2.3PP1β基因在各組織中的表達(dá)

熒光定量PCR檢測(cè)結(jié)果顯示，PP1βmRNA在健康中國(guó)對(duì)蝦8種組織中均有不同程度的表達(dá)，其中在性腺中的表達(dá)量最高，其次是血細(xì)胞，然后依次為淋巴器官、腸、鰓、心臟和肝胰腺，在肌肉中的表達(dá)量最低(見(jiàn)圖4)。

2.4 WSSV感染后中國(guó)對(duì)蝦血細(xì)胞和性腺中PP1β基因的表達(dá)變化

實(shí)時(shí)熒光定量PCR檢測(cè)結(jié)果顯示，在WSSV感染后中國(guó)對(duì)蝦后血細(xì)胞和性腺中的PP1β基因均呈顯著性上調(diào)表達(dá)，在感染后12 h，2個(gè)組織中PP1β表達(dá)量均達(dá)到峰值，其中血細(xì)胞中該基因的上調(diào)倍數(shù)顯著大于性腺中，為(2.71±0.16)倍。峰值過(guò)后，PP1β基因的相對(duì)表達(dá)量在2個(gè)組織中均呈現(xiàn)逐漸降低的趨勢(shì)但仍高于對(duì)照組水平，而與血細(xì)胞相比，性腺中的下調(diào)較為平緩，2種組織中該基因的相對(duì)表達(dá)量均于感染后72 h恢復(fù)至接近對(duì)照組水平(見(jiàn)圖5)。

2.5 中國(guó)對(duì)蝦PP1β基因的原核表達(dá)與抗體特異性分析

SDS-PAGE圖譜顯示，經(jīng)誘導(dǎo)的含pET28a-PP1β表達(dá)質(zhì)粒的大腸桿菌全蛋白中出現(xiàn)了一條分子量為41 kDa的特異性條帶(見(jiàn)圖6，泳道2)，與理論預(yù)測(cè)值相符，而未經(jīng)誘導(dǎo)的大腸桿菌沒(méi)有出現(xiàn)相應(yīng)的條帶(見(jiàn)圖6，泳道1)。重組蛋白經(jīng)鎳瓊脂糖親和層析純化后獲得了條帶單一的高純度rPP1β(見(jiàn)圖6，泳道3)。應(yīng)用制備的鼠抗rPP1β多克隆抗體結(jié)合Western blotting技術(shù)分析顯示，多抗能特異性識(shí)別分子量為41 kDa的條帶(見(jiàn)圖6，泳道4)，而陰性對(duì)照組未見(jiàn)條帶(見(jiàn)圖6，泳道5)，表明制備的鼠抗rPP1β多克隆抗體特異性良好。

(序列比對(duì)所用PP1β物種來(lái)源及相應(yīng)GenBank登錄號(hào)如下：太平洋牡蠣(C.gigas), EKC31188；凡納濱對(duì)蝦(L.vannamei), AAT37505；印度跳蟻(H.saltator)，EFN76901；佛羅里達(dá)弓背蟻(C.floridanus)，EFN68092；斑馬魚(yú)(D.rerio)，NP_001004527；熱帶爪蟾(X.tropicalis)，CAJ81891；原雞(G.gallus)，NP_990453；褐家鼠(R.norvegicus)，NP_037197；牛(B.taurus)，NP_001029825；人(H.sapiens)，NP_996759； “”：相同的氨基酸；“：”和“.”：相似的氨基酸；藍(lán)色字體：PP2Ac結(jié)構(gòu)域；陰影：絲氨酸/蘇氨酸特異性蛋白磷酸酶家族的特征基序GDxHG，GDxVDRG和GNHE。GenBank accession number:C.gigas, EKC31188;L.vannamei, AAT37505;H.saltator，EFN76901;C.floridanus，EFN68092;D.rerio，NP_001004527;X.tropicali，CAJ81891;G.gallus，NP_990453;R.norvegicus，NP_037197;B.taurus，NP_001029825;H.sapiens，NP_996759. “” represents the same amino acid, “:” and “.” represent the similar amino acids. The PP2Ac domain was shown in blue font. The conserved catalytic domains which were specific for Ser/Thr phosphatases (GDxHG，GDxVDRG and GNHE) were shaded in gray.)

圖2 中國(guó)對(duì)蝦PP1β氨基酸序列與其他物種的多序列比對(duì)

Fig.2 Multiple alignment of the deduced amino acid sequences of PP1β fromF.chinensiswith those from other species

(系統(tǒng)進(jìn)化樹(shù)所用序列除圖2中的11種之外，其他6種包括：獼猴(M.mulatta)，NP_001247581；小家鼠(M.musculus)，NP_766295；綠海龜(C.mydas)，EMP24516；大西洋鮭(S.salar)，ACN58678；埃及伊蚊(A.aegypti)，XP_001663366；致倦庫(kù)蚊(C.quinquefasciatus)，XP_001843526。GenBank accession number (not including the ones in Figure 2):M.mulatta, NP_001247581;M.musculus, NP_766295;C.mydas, EMP24516;S.salar, ACN58678;A.aegypti, XP_001663366;C.quinquefasciatus, XP_001843526.)

圖3 不同物種PP1β氨基酸序列系統(tǒng)進(jìn)化樹(shù)

Fig.3 Phylogenetic analysis of the deduced amino acid sequences ofF.chinensisPP1β

(小寫(xiě)字母表示各組織中PP1β mRNA相對(duì)表達(dá)量顯著性差異水平(P<0.05)。Different letters indicates significant difference between different tissues (P<0.05).)

圖4PP1βmRNA在中國(guó)對(duì)蝦不同組織中的分布

Fig.4 Quantitative real-time RT-PCR analysis of tissues distribution ofPP1βtranscripts in healthyF.chinensis

(小寫(xiě)字母表示感染組對(duì)蝦PP1β基因表達(dá)量與對(duì)照組相比差異顯著(P<0.05)。Different letters indicates significant difference between different tissues (P<0.05).)

圖5 WSSV感染后中國(guó)對(duì)蝦PP1β基因在血細(xì)胞和性腺中的時(shí)空表達(dá)分析

Fig.5 Expression profiles ofPP1βin hemocytes and gonad ofF.chinensispost WSSV infection

(M：標(biāo)準(zhǔn)分子量蛋白；1：未誘導(dǎo)的重組菌總蛋白；2：誘導(dǎo)后的重組菌總蛋白；3：純化后的重組蛋白，4：鼠抗rPP1β血清與誘導(dǎo)后重組菌總蛋白反應(yīng)；5：正常鼠血清與誘導(dǎo)后重組菌總蛋白反應(yīng)。 M: Marker; 1: Total proteins from uninduced bacterial cell lysate; 2: Total proteins from induced bacterial cell lysate; 3: Purified recombinant protein; 4: Antisera against rPP1β reacted with induced bacterial cell proteins; 5: Negative control.)

圖6 PP1β重組蛋白的SDS-PAGE及Western blotting分析

Fig.6 SDS-PAGE and Western blotting analysis of PP1β recombinant protein

2.6 中國(guó)對(duì)蝦PP1β在血細(xì)胞上的定位

IIFA檢測(cè)結(jié)果顯示，鼠抗rPP1β血清能與中國(guó)對(duì)蝦血細(xì)胞發(fā)生陽(yáng)性反應(yīng)，熒光顯微鏡下可見(jiàn)黃綠色熒光信號(hào)在血細(xì)胞的核區(qū)及細(xì)胞質(zhì)內(nèi)均有分布(見(jiàn)圖 7B)，而以正常小鼠血清作為對(duì)照時(shí)，未顯示其與血細(xì)胞發(fā)生結(jié)合，血細(xì)胞中無(wú)黃綠色熒光信號(hào)，血細(xì)胞僅被伊文斯蘭襯染為紅色(見(jiàn)圖 7A)。

3 討論

PP1作為絲氨酸/蘇氨酸蛋白磷酸酶家族中的一員，是一種高度保守的蛋白質(zhì)，在真菌與哺乳動(dòng)物間的同源性高達(dá)72%[16]，本文克隆得到的中國(guó)對(duì)蝦PP1β基因編碼328個(gè)氨基酸，以其氨基酸序列進(jìn)行BLAST比對(duì)，發(fā)現(xiàn)中國(guó)對(duì)蝦PP1β蛋白與已報(bào)道的不同物種的PP1β蛋白序列相似度極高，為90%以上，這一結(jié)果證明了PP1β在不同物種間高度保守，與上述報(bào)道相一致。不同物種的PP1不僅在序列上極保守，而且在功能方面也具有一定的保守性，有研究指出，在真菌中由PP1突變而引起的表型變化在一定程度上能通過(guò)誘導(dǎo)外源哺乳動(dòng)物的PP1基因從而得到緩解[17-18]，由此本文推測(cè)，相較真菌而言與哺乳動(dòng)物在進(jìn)化上更為接近的對(duì)蝦，其PP1分子也具有與高等動(dòng)物相似的功能，參與到對(duì)蝦生命活動(dòng)調(diào)控的各個(gè)方面。

在哺乳動(dòng)物中，蛋白磷酸酶參與生殖細(xì)胞的分化、精子移動(dòng)和減數(shù)分裂等過(guò)程的調(diào)控，其在性腺組織中呈高豐度表達(dá)[19-20]。目前，無(wú)脊椎動(dòng)物的蛋白磷酸酶是否具有上述功能還不清楚，但是有報(bào)道指出果蠅(Drosophilamelanogaster)體內(nèi)存在多種特異性的蛋白磷酸酶(PpY-55A，PpN58A，PpD5，PpD6)，這些酶在果蠅精巢中表達(dá)量極高[21-25]。本文以實(shí)時(shí)熒光定量PCR法檢測(cè)到PP1βmRNA在對(duì)蝦性腺中表達(dá)量最高，也與上述結(jié)論相符合，推測(cè)對(duì)蝦性腺中的PP1β承擔(dān)著與高等動(dòng)物PP1β相似的功能。PP1β在中國(guó)對(duì)蝦血細(xì)胞中表達(dá)量也比較高，并且在WSSV感染后，血細(xì)胞中PP1β基因的上調(diào)表達(dá)程度較性腺中更顯著，這可能是由于血細(xì)胞是對(duì)蝦免疫功能的主要承擔(dān)者[26]，PP1β參與了血細(xì)胞應(yīng)答WSSV的反應(yīng)所致。

(A：血細(xì)胞與正常鼠血清反應(yīng)；B：血細(xì)胞與鼠抗PP1β重組蛋白血清反應(yīng)。A：Normal sera reacted with the haemocytes; B: Antisera against rPP1β reacted with the haemocytes.)

圖7 PP1β在血細(xì)胞上的定位

Fig.7 Localization of PP1β in Chinese shrimp haemocytes by IIFA

在真核細(xì)胞內(nèi)，幾乎所有的信號(hào)轉(zhuǎn)導(dǎo)過(guò)程都是通過(guò)由蛋白激酶和蛋白磷酸酶催化的磷酸化和去磷酸化作用來(lái)調(diào)節(jié)的。因此，蛋白磷酸酶被認(rèn)為廣泛分布于細(xì)胞的核區(qū)及細(xì)胞質(zhì)內(nèi)[27]。本文對(duì)PP1β重組蛋白進(jìn)行了純化，獲得了純度較高的目的蛋白，此純化產(chǎn)物經(jīng)簡(jiǎn)單的透析和凍干后，直接作為抗原免疫小鼠獲得抗血清，免疫印跡實(shí)驗(yàn)證實(shí)制備的抗血清可與rPP1β發(fā)生特異性結(jié)合反應(yīng)，顯示其具有較好的特異性，應(yīng)用間接免疫熒光實(shí)驗(yàn)檢測(cè)發(fā)現(xiàn)PP1β存在于中國(guó)對(duì)蝦血細(xì)胞的核區(qū)及細(xì)胞質(zhì)內(nèi)，該結(jié)果與上述觀點(diǎn)相吻合，推測(cè)對(duì)蝦PP1β與其他高等真核生物類似，在介導(dǎo)胞內(nèi)信號(hào)轉(zhuǎn)導(dǎo)等重要生物學(xué)過(guò)程中發(fā)揮著重要作用。

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責(zé)任編輯 朱寶象

Cloning, Expression and Characterization of Protein Phosphatase 1 Catalytic Subunit Beta Isoform in ShrimpFenneropenaeuschinensis

HE Liang-Yin, LI Wei, TANG Xiao-Qian, XING Jing, SHENG Xiu-Zhen, ZHAN Wen-Bin

(Laboratory of Pathology and Immunology of Aquatic Animals，Ocean University of China，Qingdao 266003，China)

Protein phosphatase 1 (PP1) of host cells was documented to play a crucial role in virus infection in mammals, participating in the transcription, replication and life cycle regulation of virus. PP1 in hemocytes ofFenneropenaeuschinensiswas up-regulated significantly after WSSV infection as was demonstrated in our previous work, which indicated that PP1 ofF.chinensiswas involved in WSSV infection. To further illustrate the role of PP1 in WSSV infection, in present work, protein phosphatase 1 catalytic subunit beta isoform (PP1β) gene ofF.chinensiswas cloned and sequenced by rapid amplification of cDNA ends approaches (RACE). The full-length cDNA sequence ofPP1βgene was 1,214 bp, and contained an open reading frame (ORF) of 987 bp that encoded for a polypeptide of 328 amino acids. Homology comparison showed that PP1β ofF.chinensisshared 90%～91% amino acids with thst of other species, indicating the high conservation ofPP1βgene. Multiple sequence alignment was performed using the ClustalW Multiple Alignment program. It was demonstrated that all amino acid sequences of PP1β from various species contained three conserved catalytic domains which were specific for Ser/Thr phosphatases, GDxHG，GDxVDRG and GNHE. A neighbor-joining (NJ) tree was constructed based on the protein sequences of PP1β from 17 species by the NJ algorithm using MEGA 4.0 software package and Clustal X using α-lactalbumin as outgroup. The result showed thatF.chinensiswas clustered withL.vannamei, and PP1β of crustaceans gathered in one branch. By quantitative real-time RT-PCR,PP1βgene mRNA was observed in all the eight tissues of healthyF.chinensis, with the high transcription level in gonad and hemocytes. The high transcription level ofPP1βgene in gonad suggested that PP1β possibly involved in germ cell differentiation and spermatogenesis as was reported in mammals. Moreover, the gene in the above two tissues was up-regulated after WSSV infection, with the peak value found at 12 h, while the fold change ofPP1βgene transcript abundance in hemocytes was higher than that in gonad. The higher transcription level ofPP1βgene observed in hemocytes suggested that PP1β might play an important role in resistance to WSSV infection. ThePP1βgene ORF was cloned into pET-28a expression plasmid and the recombinant plasmid was transformed intoE.coliBL21 (DE3). SDS-PAGE analysis showed that the molecular weight of recombinant protein was 41 kDa. Subsequently, rPP1β was purified by using affinity chromatography, and the polyclonal antibody against rPP1β was produced by immunizing mouse. The localization of PP1β inF.chinensishaemocytes was determined by indirect immunofluorescence assay (IIFA). The results showed that PP1β was synthesized in cell nucleus and cytoplasm, which implied that PP1β might mediate kinds of biological processes such as the signal transduction inF.chinensis. Overall, this study provided important data for illustrating the relationship betweenF.chinensisPP1β and WSSV infection.

Fenneropenaeuschinensis; protein phosphatase 1 catalytic subunit beta isoform; gene cloning; prokaryotic expression; immunofluorescence

國(guó)家重點(diǎn)基礎(chǔ)研究發(fā)展規(guī)劃項(xiàng)目 (2012CB114405)；“泰山學(xué)者特聘專家”項(xiàng)目；青島海洋科學(xué)與技術(shù)國(guó)家實(shí)驗(yàn)室鰲山科技創(chuàng)新計(jì)劃項(xiàng)目(2015ASKJ01)；山東省科技發(fā)展計(jì)劃項(xiàng)目(2014GNC111015)；山東省自主創(chuàng)新及成果轉(zhuǎn)化專項(xiàng)項(xiàng)目(2014ZZCX06205)資助

2016-01-27；

2016-04-21

何亮銀(1987-)，男，博士生，主要從事水產(chǎn)動(dòng)物病害與免疫學(xué)研究。

** 通訊作者： E-mail：wbzhan@ouc.edu.cn

S91

A

1672-5174(2016)11-073-09

10.16441/j.cnki.hdxb.20160024

何亮銀， 李微， 唐小千， 等. 中國(guó)對(duì)蝦蛋白磷酸酶1催化亞基β基因的克隆表達(dá)及特性分析[J]. 中國(guó)海洋大學(xué)學(xué)報(bào)(自然科學(xué)版)， 2016, 46(11)： 73-81.

HE Liang-Yin, LI Wei, TANG Xiao-Qian， et al. Cloning, expression and characterization of protein phosphatase 1 catalytic subunit beta isoform in shrimpFenneropenaeuschinensis[J]. Periodical of Ocean University of China, 2016, 46(11)： 73-81.

Supported by National Basic Research Program of China(2012CB114405); “Taishan Scholar Program of Shangdong Province”; The Scientific and Technological Innovation Project Financially Supported by Qingdao National Laboratory for Marine Science and Technology(2015ASKJ01); Science and technology development project of Shandong Province(2014GNC111015); Independent innovation and achievement transformation project in Shandong Province(2014ZZCX06205)