曲 璇,李 軍,馬曉軍,韓洛川,史海龍

(陜西中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院生物學(xué)教研室,咸陽 712046;*通訊作者,E-mail:quxuan519@163.com)

SKP2突變載體S72A和S72D的構(gòu)建以及生物學(xué)功能研究

曲 璇*,李 軍,馬曉軍,韓洛川,史海龍

(陜西中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)院生物學(xué)教研室,咸陽 712046;*通訊作者,E-mail:quxuan519@163.com)

目的 構(gòu)建SKP2突變載體SKP2S72A和SKP2S72D并驗(yàn)證其生物學(xué)功能。 方法 利用一步法PCR合成含有SKP2S72A和SKP2S72D突變序列,通過酶切連接將序列插入pBabe載體中,將克隆載體轉(zhuǎn)入大腸桿菌后,篩選陽性克隆進(jìn)行序列測定,將鑒定正確的克隆轉(zhuǎn)染HEK293細(xì)胞,并利用Western blot實(shí)驗(yàn)鑒定基因表達(dá)及其對靶蛋白P27的影響。 結(jié)果 PCR擴(kuò)增得到序列測定結(jié)果與預(yù)期序列完全一致;構(gòu)建的SKP2S72A和SKP2S72D突變質(zhì)粒,用于基因轉(zhuǎn)染純度較好,在真核細(xì)胞中成功表達(dá),并且影響了P27的蛋白水平。 結(jié)論 構(gòu)建獲得SKP2突變載體,為今后SKP2泛素連接酶活性的功能研究提供良好的基礎(chǔ)。

S期激酶相關(guān)蛋白; 突變載體; 過表達(dá)載體

SKP2(S-phase kinase-associated protein 2)稱為S期激酶相關(guān)蛋白2,是F-box家族成員之一。研究表明,在蛋白泛素化降解過程中,SKP2發(fā)揮E3泛素連接酶的作用,通過募集泛素分子來標(biāo)記靶蛋白,并實(shí)現(xiàn)對蛋白的降解過程,如細(xì)胞周期調(diào)控蛋白P27、P21等[1,2]。SKP2在多種腫瘤細(xì)胞中呈高表達(dá),通過調(diào)控細(xì)胞周期、細(xì)胞增殖和遷移等過程,促進(jìn)腫瘤的發(fā)生發(fā)展,如結(jié)腸癌[3]、乳腺癌[4]、黑素瘤[5]等。在細(xì)胞內(nèi),SKP2能夠被PI3K/AKT信號通路的激活,其一級結(jié)構(gòu)中的絲氨酸72位點(diǎn)能夠被AKT識別并磷酸化,因此,PI3K/AKT信號通路在SKP2的活化中扮演重要角色[6,7]。為了深入研究SKP2活化對細(xì)胞生物學(xué)行為的影響,我們構(gòu)建了SKP2點(diǎn)突變體:失活型突變體SKP2S72A和組成性活化突變體SKP2S72D,并對其表達(dá)和功能進(jìn)行了驗(yàn)證。

1 材料與方法

1.1 細(xì)胞株及主要試劑

HEK293細(xì)胞源自美國模式培養(yǎng)物收集中心(American Type Culture Collection,ATCC),并由陜西中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)實(shí)驗(yàn)室凍存;大腸桿菌XL10由陜西中醫(yī)藥大學(xué)基礎(chǔ)醫(yī)學(xué)實(shí)驗(yàn)室保存;一步法快速定點(diǎn)突變試劑盒購自南京諾唯贊生物科技有限公司;質(zhì)粒提取試劑盒及凝膠回收純化試劑盒為天根生化科技(北京)有限公司公司產(chǎn)品;Lipofectamine2000及TRIzol試劑均為美國Invitrogen公司產(chǎn)品;反轉(zhuǎn)錄試劑盒為美國Promega公司產(chǎn)品;兔抗人SKP2:sc-7164為美國SANTA CRUZ公司產(chǎn)品;兔抗人p27(C-19):sc-528為美國SANTA CRUZ公司產(chǎn)品;小鼠抗人β-肌動蛋白(actin)單抗及Western blot試劑盒為德國Sigma公司產(chǎn)品。

1.2 pBabe-SKP2突變載體的構(gòu)建與測序

根據(jù)已構(gòu)建成功的pBabe-SKP2的過表達(dá)載體為模板,分別設(shè)計(jì)S72A和S72D的突變載體引物。S72A:上游引物:5′-ACGGAAACGGCTGAAGGCCAAAGGGA-3′;下游引物:5′-TTGTCACTCCCTTTGGCCTTCA-3′(下劃線為突變堿基);S72D:上游引物:5′-ACGGAAACGGCTGAAGGACAAAGGGA-3′;下游引物:5′-TTGTCACTCCCTTTGTCCTTCA-3′(下劃線為突變堿基)。PCR擴(kuò)增條件為:95 ℃預(yù)變性5 min、95 ℃變性30 s、57 ℃退火30 s、72 ℃延伸90 s,共30個(gè)循環(huán),72 ℃再延伸7 min;得到的PCR產(chǎn)物經(jīng)DpnⅠ消化、ClonExpress TM重組環(huán)化后直接進(jìn)行轉(zhuǎn)化。待單克隆形成肉眼可見的菌落時(shí),挑取10個(gè)單克隆,將單克隆接種于液體LB培養(yǎng)基中,37 ℃培養(yǎng)12 h,所得的菌液進(jìn)行測序。

1.3 細(xì)胞轉(zhuǎn)染

細(xì)胞轉(zhuǎn)染操作前進(jìn)行實(shí)驗(yàn)分4組:陰性對照組(轉(zhuǎn)染pBabe陰性對照質(zhì)粒)，野生型SKP2過表達(dá)組(轉(zhuǎn)染pBabe-SKP2過表達(dá)質(zhì)粒)，突變型SKP2S72A過表達(dá)組(轉(zhuǎn)染SKP2S72A)，突變型SKP2S72D過表達(dá)組(轉(zhuǎn)染SKP2S72D)。細(xì)胞轉(zhuǎn)染過程如下:按Lipofectamine2000脂質(zhì)體轉(zhuǎn)染試劑盒說明書進(jìn)行操作。將5×105HEK293細(xì)胞/孔接種于6孔板,接種隔夜后取4 μg pBabe陰性對照載體、pBabe-SKP2過表達(dá)載體和pBabe-SKP2S72A和pBabe-SKP2S72D兩個(gè)突變載體分別與10 μl Lipofectamine2000混勻,室溫靜置20 min后進(jìn)行瞬時(shí)轉(zhuǎn)染。

1.4 Western blot法檢測蛋白表達(dá)

分別收集25 cm2培養(yǎng)瓶中的親本HEK293細(xì)胞、pBabe陰性對照載體、pBabe-SKP2過表達(dá)以及pBabe-SKP2S72A和pBabe-SKP2S72D兩個(gè)突變載體。首先使用冰浴的PBS清洗細(xì)胞兩次,之后加入遇冷的RIPA裂解液。反復(fù)吹打細(xì)胞,直至細(xì)胞完全溶解,最后將RIPA裂解液轉(zhuǎn)移至1.5 ml離心管中,放置于冰盒中,期間不時(shí)渦旋震蕩,裂解細(xì)胞30 min。低溫高速離心15 min,離心后將上層液體轉(zhuǎn)移至新的1.5 ml離心管中,吸取一定量的樣品進(jìn)行蛋白定量,測出樣品的蛋白濃度,余下的樣品按比例加入5×loading buffer,煮沸法處理樣品。取30 μg蛋白樣品進(jìn)行蛋白電泳(SDS-PAGE),根據(jù)目的蛋白不同的分子量,選取不同濃度的分離膠。在蛋白電泳過程中濃縮膠電壓為100 V,分離膠電壓為120 V。蛋白電泳完成之后將分離膠上的蛋白轉(zhuǎn)移至硝酸纖維素膜上,轉(zhuǎn)膜過程中電壓為100 V。將NC膜用5%脫脂奶封閉2 h,然后分別加入SKP2多抗(1 ∶1 000)、p27多抗(1 ∶1 000)和β-actin單抗(1 ∶1 000),室溫孵育1 h,4 ℃過夜。次日用含0.5% Tween-20的TBST洗3次,5 min/次;再分別加入1 ∶5 000)HRP標(biāo)記的二抗室溫?fù)u床孵育1 h。TBST洗3次,5 min/次,ECL發(fā)光檢測,膠片顯色,分析結(jié)果。以β-actin為內(nèi)參照。

1.5 統(tǒng)計(jì)學(xué)分析

利用灰度掃面軟件ImageJ對Western blot結(jié)果進(jìn)行蛋白定量分析,通過Student’sT檢驗(yàn)進(jìn)行比較,P<0.05為差異具有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 pBabe-SKP2突變載體的構(gòu)建與鑒定

利用重組DNA技術(shù)以野生型pBabe-SKP2為模板,分別構(gòu)建了SKP2失活型突變體pBabe-SKP2S72A和組成性活化突變體pBabe-SKP2S72D,并對三者進(jìn)行了測序比對。野生型載體測序結(jié)果見圖1,pBabe-SKP2S72A突變型載體測序結(jié)果見圖2,pBabe-SKP2S72D突變型載體測序結(jié)果見圖3。

2.2 pBabe-SKP2野生型和突變質(zhì)粒在HEK293細(xì)胞中的表達(dá)鑒定

為了進(jìn)一步驗(yàn)證pBabe-SKP2能否在細(xì)胞中正確表達(dá),本研究進(jìn)行了細(xì)胞轉(zhuǎn)染和Western blot實(shí)驗(yàn)。結(jié)果顯示,相對于pBabe陰性對照組,轉(zhuǎn)染過表達(dá)載體pBabe-SKP2組、突變載體p-BabeSKP2S72A組和pBabeSKP2S72D組的HEK293細(xì)胞中SKP2的蛋白表達(dá)水平明顯升高(P<0.05,見圖4)。

圖1 重組質(zhì)粒pBabe-SKP2測序結(jié)果Figure 1 Sequencing result of recombinant plasmidpBabe-SKP2

圖2 重組質(zhì)粒pBabe-SKP2S72A測序結(jié)果(下劃線為突變堿基)Figure 2 Sequencing result of pBabe-SKP2S72A(mutations were marked with the underscore)

圖3 重組質(zhì)粒pBabe-SKP2S72D測序結(jié)果(下劃線為突變堿基)Figure 3 Sequencing result of pBabe-SKP2S72D(mutations were marked with the underscore)

1.pBabe;2.pBabe-Skp2;3.pBabe-Skp2S72A;4.pBabe-Skp2S72D與pBabe組比較，*P<0.05圖4 Western blot法檢測SKP2的表達(dá)Figure 4 Detection of SKP2 expression by Western blot

2.3 SKP2野生型和突變型載體在HEK293細(xì)胞對P27蛋白水平的影響

為了進(jìn)一步驗(yàn)證pBabe-SKP2真核表達(dá)載體的功能,通過Western blot實(shí)驗(yàn)對SKP2下游底物蛋白P27表達(dá)進(jìn)行了檢測。結(jié)果顯示,相對于pBabe陰性對照組,轉(zhuǎn)染野生型SKP2組的P27表達(dá)水平顯著降低(P<0.05),而轉(zhuǎn)染失活突變載體pBabe-SKP2S72A組的P27水平?jīng)]有受到抑制(P=0.736 8),轉(zhuǎn)染組成性活化pBabe-SKP2S72D組的P27的蛋白表達(dá)水平明顯降低(P<0.05,見圖5)。以上結(jié)果證明pBabe-SKP2S72A和pBabe-SKP2S72D突變表達(dá)載體構(gòu)建成功并可以正確表達(dá)并發(fā)揮功能,可用于后期實(shí)驗(yàn)研究。

1.pBabe;2.pBabe-Skp2;3.pBabe-Skp2S72A;4.pBabe-Skp2S72D與pBabe組比較，*P<0.05圖5 Western blot法檢測SKP2對P27蛋白水平表達(dá)的影響Figure 5 Effect of SKP2 on P27 protein expression levels by Western blot

3 討論

SKP2是泛素依賴性蛋白水解途徑中(ubiquitin-proteasome system,UPS)泛素連接酶E3的組成部分,SKP2定位于5p13,編碼的蛋白質(zhì)由436個(gè)氨基酸組成,分子量約47 kDa。SKP2主要通過泛素化(ubiquitin)降解細(xì)胞周期相關(guān)蛋白如P27等,使其通過泛素-蛋白酶體途徑降解,促進(jìn)細(xì)胞周期的轉(zhuǎn)換,已成為癌變過程中不可忽視的重要因子[8-11]。在乳腺癌、結(jié)腸癌、肺癌等多種腫瘤中,SKP2的表達(dá)和活性都增強(qiáng)[12-18]。

SKP2的兩個(gè)重要的磷酸化位點(diǎn),分別位于64位和72位的絲氨酸。絲氨酸72位點(diǎn)在M期的磷酸化程度最高。不僅絲氨酸72位的磷酸化誘導(dǎo)P27蛋白的磷酸化降解,此外,SKP2蛋白在絲氨酸72位點(diǎn)的磷酸化在促進(jìn)細(xì)胞的增殖和腫瘤的發(fā)生過程中至關(guān)重要[19,20]。因此,構(gòu)建SKP2的72位點(diǎn)絲氨酸的突變載體,對于研究SKP2的功能具有十分重要的意義。

SKP2蛋白有可能成為未來腫瘤治療的新靶點(diǎn),它的檢測對腫瘤的預(yù)防、診斷和預(yù)后評估具有重要的參考價(jià)值,可作為一種探索癌癥及癌前病變發(fā)生機(jī)制的重要生物學(xué)指標(biāo)。本實(shí)驗(yàn)成功構(gòu)建了SKP2的兩個(gè)真核突變載體pBabe-SKP2S72A和SKP2S72D,首先通過測序證明載體序列正確,并在蛋白質(zhì)水平證明SKP2突變載體組的SKP2蛋白表達(dá)明顯升高。為了進(jìn)一步研究SKP2的功能,在過表達(dá)野生型或突變型SKP2的實(shí)驗(yàn)組中,其下游靶基因P27蛋白水平均發(fā)生顯著變化。以上結(jié)果顯示,我們成功構(gòu)建了SKP2的突變載體,為進(jìn)一步研究SKP2在腫瘤的發(fā)生和發(fā)展中的作用及其分子機(jī)制奠定了一定的實(shí)驗(yàn)基礎(chǔ)。

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Construction and biological functional study of SKP2 mutant vector SKP2S72A and SKP2S72D

QU Xuan*,LI Jun,MA Xiaojun,HAN Luochuan,SHI Hailong

(DepartmentofBiology,ShaanxiUniversityofChineseMedicine,Xianyang712046,China;*Correspondingauthor,E-mail:quxuan519@163.com)

ObjectiveTo construct the SKP2 mutant vectors SKP2S72A and SKP2S72D, and verify their biological function.MethodsSKP2 mutant sequence was obtained with one-step PCR assay, and then the fragments were inserted into pBabe vector by digestion and DNA ligation. The constructed plasmid was transformed into DH5α.The positive clones were selected and sequenced, and then transfected into HEK293 cells to determine the expression and effects on P27 expression.ResultsThe sequences of the SKP2S72A and SKP2S72D were consistent with that of the SKP2 mutant sequence. The SKP2S72A and SKP2S72D vectors were purified for gene transfection. The constructs successfully expressed in mammal cells and affected the P27 expression.ConclusionSKP2 mutant constructs are successfully obtained.The results provide the basis for the further studies of the biological function of SKP2.

SKP2; mutant vector; overexpression plasmid

國家自然科學(xué)基金青年基金資助項(xiàng)目(81502370)；陜西省高?？茀f(xié)青年人才托舉計(jì)劃項(xiàng)目(20170407)

曲璇,女,1983-05生,博士,講師

2017-05-16

Q782

A

1007-6611(2017)08-0813-04

10.13753/j.issn.1007-6611.2017.08.012