余宏偉+廖志偉+莊雅靖+喻芳+周同沖
[摘要] 目的 構建并鑒定USP22基因ShRNA慢病毒載體,為進一步研究USP22基因在鼻咽癌中的作用機制奠定基礎。 方法 針對USP22基因的編碼序列設計并合成2條特異性干擾序列,序列兩端含有限制性內(nèi)切酶位點HpaⅠ和XhoⅠ。寡核苷酸鏈退火生成寡核苷酸雙鏈,5′端磷酸化后將含有酶切位點的寡核苷酸雙鏈克隆到pLL3.7慢病毒表達載體。連接產(chǎn)物經(jīng)轉(zhuǎn)化、培養(yǎng),提取其質(zhì)粒,提取出來的質(zhì)粒經(jīng)HpaⅠ和XhoⅠ酶切電泳鑒定,鑒定正確的質(zhì)粒進行測序。構建成功的慢病毒表達載體pLL-USP22-shRNA與包裝載體質(zhì)?;靹蚬厕D(zhuǎn)染于293T細胞。通過熒光顯微鏡下觀察綠色熒光蛋白(GFP)情況,對病毒滴度和感染效率進行檢測。 結果 成功構建慢病毒表達載體pLL-USP22-shRNA。與包裝載體質(zhì)粒共轉(zhuǎn)染293T細胞后測定慢病毒滴度為4×107 TU/ml。 結論 本實驗應用相關技術成功構建USP22 ShRNA慢病毒載體,為進一步研究USP22基因的生物學功能奠定了基礎。
[關鍵詞] USP22;慢病毒載體;構建;鑒定
[中圖分類號] R34 [文獻標識碼] A [文章編號] 1674-4721(2014)11(b)-0009-05
腫瘤細胞中基因表達具有組織特異性,USP22泛素水解酶屬去泛素化酶DUB基因家族成員,其普遍表達表明其功能的保守性,因此,USP22被歸類為腫瘤干細胞的標記基因而引起高度關注[1]。國內(nèi)外學者研究發(fā)現(xiàn),USP22基因過表達與結直腸癌[2]、肺癌[3]、胃癌[4]、食管癌[5]、乳腺癌[6]等惡性腫瘤的浸潤、轉(zhuǎn)移和預后差高度相關。沉默USP22基因表達,能顯著抑制膀胱癌[7]、結直腸癌[8]細胞增殖,由此推測USP22基因可能成為腫瘤治療的一個新靶點。本研究通過基因工程技術構建USP22 ShRNA慢病毒載體,為進一步研究USP22基因在人鼻咽癌細胞中的作用機制提供實驗基礎。
1 材料與方法
1.1 實驗材料、試劑及儀器
pLL3.7慢病毒表達載體及包裝載體質(zhì)粒購自廣州永諾生物科技有限公司。T4磷酸化酶、T4連接酶、HpaⅠ酶及XhoⅠ酶均購自NEB(New England BioLabs)公司。瓊脂糖購自Biowest公司。高純質(zhì)粒小量提取試劑盒和Tran5α感受態(tài)細胞購自北京全式金生物公司。質(zhì)粒大提試劑盒(QIAGEN試劑盒)購自QIAGEN公司。溴化乙錠(EB)及LB培養(yǎng)基購自上海生工生物工程有限公司。293T細胞購自中國科學院上海細胞庫。Lipofectamine(脂質(zhì)體)2000購自Life Technologies公司。主要儀器包括水浴箱、恒溫振蕩器、臺式離心機、Eppendorf加樣槍和臺式冷凍離心機、恒溫搖床、電熱恒溫CO2培養(yǎng)箱、Millipore超純水系統(tǒng)、Bio-Rad瓊脂糖凝膠電泳儀、SynGene凝膠成像系統(tǒng)、Olympus倒置熒光顯微鏡、-4℃冰箱、-20℃冰箱等。
1.2 方法
1.2.1 SiRNA的設計與合成
運用Promega、Invitrogen和Dharmaco公司提供的RNA干擾設計工具,并結合RNA干擾設計的原則,尋找19個堿基的靶序列。將編碼SiRNA的DNA片段設計成發(fā)夾結構,發(fā)夾結構的兩端含限制性內(nèi)切酶位點HpaⅠ及XhoⅠ。SiUSP22-1:CACGGACAGTCTCAACAAT,5′-AACTCACGGACAGTCTCAACAATTT-CAAGAGAATTGTTGAGACTGTCCGTGTTTTTTC-3′,3′-TTGAGTGCCTGTCAGAGTTGTTAAAGTTCTCTTA-ACAACTCTGACAGGCACAAAAAAGAGCT-5′;SiUS-P22-2:GAAGCATATTCACGAGCAT,5′-AACTGAAGCATATTCACGAGCATTTCAAGAGAATGCTCGTGAA-TATGCTTCTTTTTTC-3′,3′-TTGACTTCGTATAAGTG-CTCGTAAAGTTCTCTTACGAGCACTTATACGAAGA-AAAAAGAGCT-5′。
1.2.2 慢病毒載體的構建
1.2.2.1 寡核苷酸鏈的退火 反應體系:1 μg/μl正義寡核苷酸鏈,1 μg/μl反義寡核苷酸,1 μl 20×SSC Buffer,加ddH2O至20 μl。反應條件:95℃,10 min后自然冷卻至室溫。
1.2.2.2 寡核苷酸雙鏈5′端的磷酸化 因合成的寡核苷酸鏈的5′端沒有被磷酸化,不利于載體的連接,故需要磷酸化。反應體系:7 μl寡核苷酸雙鏈,2.5 μl 10×T4 PNK Buffer,2.5 μl ATP,0.5 μl T4磷酸化酶,加ddH2O至25 μl。反應條件:37℃,30 min。
1.2.2.3 將含有酶切位點的寡核苷酸雙鏈克隆到pLL3.7載體 連接體系:1 μl pLL3.7載體,1 μl寡核苷酸雙鏈,1 μl 10×連接Buffer,1 μl T4連接酶,加ddH2O至10 μl。反應條件:室溫,30 min。
1.2.2.4 連接產(chǎn)物的轉(zhuǎn)化 ①冰浴中將50~100 ng連接產(chǎn)物分別加入至50 μl Tran5α感受態(tài)細胞中,然后輕輕地旋轉(zhuǎn)使其混合均勻,冰浴時間30 min;②放置42℃的水浴箱中熱休克90 s;③快速地將管轉(zhuǎn)移至冰浴中,冰浴時間2 min;④分別加入500 μl LB培養(yǎng)基,混勻,37℃、150 r/min振蕩培養(yǎng)40 min;⑤將150 μl菌液涂布于含氨芐青霉素(Amp)(100 μg/ml)的LB平板表面,室溫下放置,至液體吸收。倒置平板,轉(zhuǎn)移入37℃生化培養(yǎng)箱過夜培養(yǎng)。
1.2.2.5 質(zhì)粒提取及鑒定 從平板上面各接挑取6個菌落進行擴增培養(yǎng),加入含有相應抗生素的3 ml LB培養(yǎng)液中37℃過夜培養(yǎng),提取其質(zhì)粒(高純質(zhì)粒小量提取試劑盒)。提取出來的質(zhì)粒經(jīng)HpaⅠ和XhoⅠ酶切鑒定,鑒定正確的質(zhì)粒送中美泰和公司測序。測序正確的質(zhì)粒經(jīng)大提(QIAGEN試劑盒)后保存于-20℃冰箱。
1.2.3 慢病毒載體的包裝、濃縮及滴度測定
1.2.3.1 慢病毒的包裝 ①轉(zhuǎn)染前24 h,用胰酶消化對數(shù)生長期的293T細胞,傳代到10 cm細胞培養(yǎng)皿中,在37℃、5%CO2的培養(yǎng)箱內(nèi)進行培養(yǎng)。培養(yǎng)24 h后待細胞密度達70%~80%時即可用于轉(zhuǎn)染。細胞狀態(tài)對于病毒包裝很重要,需保證良好的細胞狀態(tài)及較少的傳代次數(shù)。②轉(zhuǎn)染前將細胞培養(yǎng)基更換成無血清培養(yǎng)基,再將稀釋后的DNA(pLV-gene載體10 μg,pGag/Pol載體5 μg、pRev載體5 μg、pVSV-G載體5 μg)與稀釋后的Lipofectamine 2000混合,慢慢地顛倒以使相互混勻,但不要震蕩,然后在室溫下培育20 min,以形成DNA和Lipofectamine 2000稀釋液的轉(zhuǎn)染復合物。③將DNA與Lipofectamine 2000混合液轉(zhuǎn)移至培養(yǎng)293T細胞的培養(yǎng)液中,充分混勻,放置于37℃,5%CO2的細胞培養(yǎng)箱中培養(yǎng)。培養(yǎng)6 h后吸去含有轉(zhuǎn)染混合物的培養(yǎng)基,在每瓶細胞培養(yǎng)液中加入含10%血清的培養(yǎng)基10 ml,再放置于37℃、5%CO2的細胞培養(yǎng)箱內(nèi)繼續(xù)培養(yǎng)48 h。
1.2.3.2 病毒的收獲和濃縮 ①收集轉(zhuǎn)染后48 h以及72 h的293T細胞上清液。在4℃,4000×g下離心10 min,以除去細胞碎片。在0.45 μm過濾器上過濾上清液于50 ml的離心管中。②將病毒粗提液樣品加入過濾杯中(最多19 ml),蓋上蓋子,在5000×g離心,達到需要的病毒濃縮體積(通常需要10~15 min)。離心結束后過濾杯中即為病毒濃縮液。將病毒濃縮液移出,分裝后保存在病毒管中,可在4℃下保存1周,或在-80℃冰箱中長期保存。
1.2.3.3 慢病毒滴度測定 ①在測定滴度的前一天,將293T細胞鋪于96孔板中,每個孔加入約4×104個細胞,體積為100 μl。然后根據(jù)病毒的預期滴度,準備6~10個無菌的Ep管,在每個Ep管中加入90 μl的無血清培養(yǎng)基。②吸取待測定的病毒原液10 μl加入第一個管中,記為1E+1 μl;充分混勻后,取10 μl加入第二個管中,記為1E+0 μl。以此類推,重復相同的操作直至最后一個管。③選取所需的細胞孔,吸去90 μl的培養(yǎng)基,丟棄,加入90 μl稀釋好的病毒溶液,放于培養(yǎng)箱中培養(yǎng)。④培養(yǎng)24 h后,加入完全培養(yǎng)基100 μl,4 d后,通過熒光顯微鏡拍照觀察其熒光表達情況。⑤根據(jù)熒光圖片中GFP表達情況,如在觀察到熒光表達的最后一個濃度1E-4 μl組中看到有5個帶熒光的細胞,則說明該孔中至少有5個病毒顆粒感染了細胞,則該病毒的滴度等于帶有熒光的細胞數(shù)除以病毒原液量,就是5/(1E-4)=5×104,單位為TU/μl,也就等于5×107 TU/ml。
2 結果
2.1 慢病毒表達載體質(zhì)粒的酶切鑒定及測序
提取出來的質(zhì)粒經(jīng)HpaⅠ和XhoⅠ酶切鑒定,進行凝膠電泳形成一條約7.6 kb的DNA條帶(圖1),與預期結果相符。鑒定正確的質(zhì)粒被送至北京中美泰和公司測序,測序結果顯示含有與先前設計相同的干擾序列,表明USP22慢病毒表達載體質(zhì)粒構建成功。測序結果如下。
2.2 慢病毒載體的包裝以及滴度的測定
慢病毒轉(zhuǎn)染293T細胞后,通過孔稀釋法來測定其滴度。在熒光顯微鏡下觀察綠色熒光蛋白(GFP)的表達情況,觀察到的熒光細胞數(shù)隨稀釋倍數(shù)的增加而減少(圖2)。
通過圖2中GFP的表達情況,可以觀察到最后一個濃度為1E-4 μl組的孔中有4個帶有熒光的細胞,說明該孔中至少有4個病毒顆粒感染了細胞,那么該病毒的滴度就觀察到的熒光細胞數(shù)除以病毒原液量,測定滴度為4×104 TU/μl(4×107 TU/ml),適合感染目的細胞。
3 討論
RNAi作為一種簡單有效的替代基因敲除的方法,其特異性及很好的表達抑制效果成為基因功能研究的有力手段[9-10]。慢病毒載體是在人類免疫缺陷病毒1型(HIV-1)基礎上改造而成的病毒載體系統(tǒng),它能高效將目的基因(或RNAi)導入動物和人的原代細胞或細胞系[11]。慢病毒載體基因組是正鏈RNA,其基因組進入細胞后,在細胞質(zhì)中被其自身攜帶的反轉(zhuǎn)錄酶反轉(zhuǎn)為DNA,形成DNA整合前復合體,進入細胞核后,DNA整合到細胞基因組中。整合后的DNA轉(zhuǎn)錄mRNA,回到細胞質(zhì)中,表達目的蛋白;或產(chǎn)生RNAi干擾。慢病毒載體介導的基因表達或RNAi干擾作用持續(xù)且穩(wěn)定,原因是目的基因整合到宿主細胞基因組中,并隨細胞基因組的分裂而分裂[12]。
USP22屬于泛素水解酶家族定位于人17號染色體,由14個外顯子組成,編碼525個氨基酸,USP22在腫瘤組織如肝癌和宮頸癌中異常地高表達,而在正常組織中呈現(xiàn)弱表達[13]。Zhang等[14-15]的研究發(fā)現(xiàn),RNA干擾技術特異性沉默USP22基因可阻止Myc及其下游基因的表達及作用抑制癌細胞的異常增殖。有研究發(fā)現(xiàn)USP22在多種惡性腫瘤中呈現(xiàn)高表達,且表達水平與患者預后、化療耐藥等密切相關。因此USP22也被認為可能是腫瘤早期診斷及基因治療的新靶點,是腫瘤干細胞的標志基因之一[16]。但至目前,仍未有相關文獻報道USP22基因在鼻咽癌的發(fā)生發(fā)展中是否起著關鍵作用。
本實驗通過RNA干擾技術成功設計了USP22基因的SiRNA,并成功構建了USP22 ShRNA慢病毒載體。通過熒光顯微鏡下觀察GFP的表達情況測定慢病毒滴度為4×107 TU/ml,表明慢病毒載體質(zhì)粒成功轉(zhuǎn)染293T細胞,適合感染目的細胞。本研究利用相關技術成功構建了USP22 ShRNA慢病毒載體,為進一步研究USP22基因在鼻咽癌的發(fā)生、發(fā)展中的生物學作用奠定了實驗基礎。
[參考文獻]
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(收稿日期:2014-07-09 本文編輯:郭靜娟)
[2] Liu YL,Yang YM,Xu H,et al.Increased expression of ubiquitin-specific protease 22 can promote cancer progression and predict therapy failure in human colorectal cancer[J].J Gastroenterol Hepatol,2010,25(11):1800-1805.
[3] Ning J,Zhang J,Liu W,et al.Overexpression of ubiquitin-specific protease 22 predicts poor survival in patients with early-stage non-small cell lung cancer[J].Eur J Histochem,2012,56(4):e46.
[4] Yang DD,Cui BB,Sun LY,et al.The co-expression of USP22 and BMI-1 may promote cancer progression and predict therapy failure in gastric carcinoma[J].Cell Biochem Biophys,2011,61(3):703-710.
[5] Li J,Wang Z,Li Y.USP22 nuclear expression is significantly associated with progression and unfavorable clinical outcome in human esophageal squamous cell carcinoma[J].J Cancer Res Clin Oncol,2012,138(8):1291-1297.
[6] Zhang Y,Yao L,Zhang X,et al.Elevated expression of USP22 in correlation with poor prognosis in patients with invasive breast cancer[J].J Cancer Res Clin Oncol,2011,137(8):1245-1253.
[7] Lv L,Xiao XY,Gu ZH,et al.Silencing USP22 by asymmetric structure of interfering RNA inhibits proliferation and induces cell cycle arrest in bladder cancer cells[J].Mol Cell Biochem,2011,346(1-2):11-21.
[8] Xu H,Liu YL,Yang YM,et al.Knock-down of ubiquitin-specific protease 22 by micro-RNA interference inhibits colorectal cancer growth[J].Int J Colorectal Dis,2012,27(1):21-30.
[9] Tusch IT,Borkhardt A.Small interfering RNAs:arevolutionary tool for the analysis of gene function and gene therapy[J].Mol Intervent,2002,2(3):158-167.
[10] Verma NK,Dey CS.RNA-mediated gene silencing:mechanisms and its therapeutic applications[J].J Clin Pharm Ther,2004,29(5):395-404.
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(收稿日期:2014-07-09 本文編輯:郭靜娟)
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(收稿日期:2014-07-09 本文編輯:郭靜娟)