劉銳銳，李聰慧，虞 游，陳建明

(杭州師范大學(xué)生命與環(huán)境科學(xué)學(xué)院，浙江 杭州 310036)

Egr-1基因反義干擾表達(dá)載體的構(gòu)建與鑒定

劉銳銳，李聰慧，虞 游，陳建明

(杭州師范大學(xué)生命與環(huán)境科學(xué)學(xué)院，浙江 杭州 310036)

為構(gòu)建可在人肝癌細(xì)胞中穩(wěn)定表達(dá)Egr-1基因短發(fā)夾RNA(shRNA)的表達(dá)載體，設(shè)計(jì)合成Egr-1基因shRNA片段，連接到經(jīng)BamHⅠ和EcoRⅠ雙酶切的pGreenPuroTMshRNA真核表達(dá)載體中，連接產(chǎn)物轉(zhuǎn)化大腸桿菌后挑取陽(yáng)性菌落擴(kuò)大培養(yǎng)，經(jīng)菌液PCR法初步鑒定為pGreenPuro-Egr-1 shRNA重組子的重組子DNA，用測(cè)序法進(jìn)一步鑒定，結(jié)果顯示成功構(gòu)建了Egr-1基因shRNA的反義干擾表達(dá)載體pGreenPuro-Egr-1 shRNA.

Egr-1基因；shRNA；反義干擾；載體構(gòu)建

0 引言

前期研究發(fā)現(xiàn)，姜黃素能通過誘導(dǎo)早期生長(zhǎng)反應(yīng)因子1(Early growth response factor 1)基因Egr-1表達(dá)而抑制肝癌細(xì)胞HepG-2增殖[1]，且Egr-1基因外來過表達(dá)也能抑制HepG-2細(xì)胞增殖[2]，提示Egr-1基因可能參與姜黃素抑制肝癌細(xì)胞HepG-2的增殖作用.姜黃素是一種天然抗氧化劑，能抑制多種腫瘤細(xì)胞增殖并能誘導(dǎo)腫瘤細(xì)胞發(fā)生凋亡[3-5].雖然有報(bào)道發(fā)現(xiàn)姜黃素抑制腫瘤細(xì)胞增殖可能與細(xì)胞周期阻滯有關(guān)[6-7]，而誘導(dǎo)腫瘤細(xì)胞凋亡可能與多信號(hào)通路的相互作用有關(guān)[8]，但具體分子機(jī)制尚未清楚.Egr-1基因編碼產(chǎn)物是一種早期生長(zhǎng)反應(yīng)因子，當(dāng)細(xì)胞受到刺激之后Egr-1基因能夠在短時(shí)間內(nèi)迅速被激活而表達(dá)，從而直接或間接地調(diào)控一系列下游基因的表達(dá)[9-10].現(xiàn)已知Egr-1基因能被多種刺激源包括抗癌藥物所激活，激活后的Egr-1基因產(chǎn)物可能參與細(xì)胞增殖及細(xì)胞凋亡等過程[11-12].本文利用RNAi Designer設(shè)計(jì)Egr-1 基因siRNA序列，并根據(jù)pGreenPuroTMshRNA表達(dá)載體的要求設(shè)計(jì)合成Egr-1基因shRNA序列，構(gòu)建Egr-1基因反義干擾表達(dá)載體，為深入研究Egr-1基因參與姜黃素抑制HepG-2細(xì)胞增殖作用的機(jī)制奠定基礎(chǔ).

1 材料和方法

1.1 主要儀器

My Gene PCR儀(杭州朗基科學(xué)儀器有限公司)，Tanon Gis System凝膠成像系統(tǒng)(上海天能科技有限公司)，電熱恒溫水槽、電熱恒溫培養(yǎng)箱(上海精宏實(shí)驗(yàn)設(shè)備有限公司)，小型臺(tái)式冷凍式離心機(jī)、分光光度計(jì)(Eppendorf)，電泳儀(Amersham Biosciences).

1.2 主要試劑

PCR擴(kuò)增試劑盒(上海生工生物工程股份有限公司)，AxyPrep DNA凝膠回收試劑盒(Axygen)，質(zhì)粒提取試劑盒(Biomiga)，瓊脂糖(Biowest)，DNA marker(Takara)，BamHⅠ、EcoRⅠ(Thermo Scientific)，T4連接酶、X-gal及IPTG(碧云天生物技術(shù)研究所)，氨芐青霉素(北京全式金生物技術(shù)有限公司).

1.3 質(zhì)粒、菌種

大腸桿菌DH 5α 感受態(tài)細(xì)胞(北京全式金生物技術(shù)有限公司)，pGreenPuroTM克隆表達(dá)載體由浙江大學(xué)醫(yī)學(xué)院提供.

1.4 Egr-1基因shRNA的設(shè)計(jì)合成

利用RNAi Designer(http://bioinfo.clontech.com/rnaidesigner/frontpage.jsp)設(shè)計(jì)Egr-1 siRNA序列(5’-GATGAACGCAAGAGGCATA-3’).根據(jù)pGreenPuroTMshRNA表達(dá)載體的要求，首先在Egr-1基因siRNA序列的5’-端加上BamHⅠ酶切位點(diǎn)，隨后在其3’-端加上Loop環(huán)序列TTCAAGAGA，接下來再加上其反向互補(bǔ)序列TATGCCTCTTGCGTTCATC，最后加上轉(zhuǎn)錄終止子序列TTTTT及EcoRⅠ酶切位點(diǎn)，此為Egr-1基因shRNA的正義鏈序列(5’-GATCCGATGAACGCAAGAGGCATATTCAAGAGATATGCCTCTTGCGTTCATCTTTTTG).再按同樣方法設(shè)計(jì)Egr-1基因shRNA的反義鏈序列(5’-GCTACTTGCGTTCTCCGTATAAGTTCTCTATACGGAGAACGCAAGTAGAAA AACTTAA).將設(shè)計(jì)好的Egr-1基因shRNA的正義鏈及反義鏈交由上海生工合成.

1.5 Egr-1基因shRNA反義干擾表達(dá)載體pGreenPuro-Egr-1 shRNA的構(gòu)建

將合成好的Egr-1基因shRNA的正義鏈和反義鏈退火成雙鏈DNA.pGreenPuroTMshRNA載體經(jīng)BamHⅠ和EcoRⅠ雙酶切后，凝膠電泳純化回收目的酶切產(chǎn)物.按照pGreenPuroTMshRNA表達(dá)載體構(gòu)建要求，將雙鏈Egr-1基因shRNA和pGreenPuroTMshRNA載體酶切產(chǎn)物在T4連接酶的作用下，于16 ℃連接過夜，將連接產(chǎn)物轉(zhuǎn)化到DH 5α 感受態(tài)大腸桿菌中并涂到含有氨芐的篩選培養(yǎng)板上.挑取若干陽(yáng)性菌落，搖菌擴(kuò)大培養(yǎng)并用菌液PCR進(jìn)行初步篩選.

1.6 pGreenPuro-Egr-1 shRNA表達(dá)載體的鑒定

以P-F和P-R為引物，用菌液PCR進(jìn)行初步篩選.PCR反應(yīng)體系(20 μL)：2×PCR Master 10 μL；P-F(10 μmol/L)1 μL；P-R(10 μmol/L)1 μL；菌液1 μL；加滅菌水至20 μL.載體鑒定引物P-F：5’-AATGTCTTTGGATTTGGGAATCTTAT-3’；P-R：5’-TGGTCTAACCAGAGAGACCCAGTA-3’.反應(yīng)條件：94 ℃ 4 min，94 ℃ 30 s，58 ℃ 30 s，72 ℃ 30 s，循環(huán)18次，72 ℃延伸10 min，4 ℃保存.以ddH2O和pGreenPuroTMshRNA空載體為對(duì)照，2%瓊脂糖凝膠電泳，凝膠成像系統(tǒng)成像拍照.連入正確目的片段陽(yáng)性菌落的PCR產(chǎn)物應(yīng)為154 bp，未連入目的片段的空載體可產(chǎn)生105 bp的PCR產(chǎn)物.對(duì)PCR鑒定正確的菌液進(jìn)行質(zhì)粒提取并交由上海生工測(cè)序.

2 結(jié)果

2.1 pGreenPuro-Egr-1 shRNA反義干擾表達(dá)載體構(gòu)建策略

設(shè)計(jì)合成好的Egr-1基因shRNA，與經(jīng)BamHⅠ和EcoRⅠ酶切后的pGreenPuroTMshRNA載體，用T4連接酶連接，16 ℃過夜，構(gòu)建pGreenPuro-Egr-1 shRNA反義干擾表達(dá)載體，圖1所示為構(gòu)建策略.

圖中上方顯示的BamH I與EcoR I酶切位點(diǎn)之間的DNA序列為Egr-1基因shRNA序列.圖1 pGreenPuro-Egr-1 shRNA反義干擾表達(dá)載體構(gòu)建策略圖Fig. 1 The schematic diagram of the construction strategy for pGreenPuro-Egr-1 shRNA antisense interfering expression vector

2.2 pGreenPuro-Egr-1 shRNA重組子PCR篩選鑒定

M：DNA 分子量標(biāo)記；泳道1：ddH2O；泳道2：pGreenPuro shRNA空載體；泳道3～8：含pGreenPuro-Egr-1 shRNA 重組子的陽(yáng)性菌落.圖 2 pGreenPuro-Egr-1 shRNA重組子PCR鑒定結(jié)果Fig. 2 The identification result of pGreenPuro-Egr-1 shRNA recombinant vector with the PCR method

按上述構(gòu)建策略將Egr-1 shRNA片段與pGreenPuroTMshRNA載體連接后的連接產(chǎn)物，轉(zhuǎn)化DH5α菌后培養(yǎng)過夜，挑取幾個(gè)抗性菌落擴(kuò)大培養(yǎng)并用菌液PCR法進(jìn)行篩選.含有Egr-1基因shRNA片段插入的pGreenPuro-Egr-1 shRNA目的重組子PCR擴(kuò)增產(chǎn)物為154 bp，而pGreenPuroTMshRNA空載體PCR擴(kuò)增產(chǎn)物為105 bp，PCR篩選鑒定結(jié)果(圖2)顯示泳道3～8的陽(yáng)性克隆中含有目的重組子DNA.

2.3 pGreenPuro-Egr-1 shRNA重組子測(cè)序鑒定

對(duì)經(jīng)PCR篩選鑒定結(jié)果含有目的重組子pGreenPuro-Egr-1 shRNA的陽(yáng)性克隆進(jìn)行質(zhì)粒提取，將提取后的質(zhì)粒DNA送予上海生工生物工程有限公司進(jìn)行DNA測(cè)序鑒定.測(cè)序結(jié)果(圖3)顯示目的重組子中插入片段的DNA序列與設(shè)計(jì)合成的Egr-1 基因shRNA序列一致，說明Egr-1基因shRNA反義干擾表達(dá)載體pGreenPuro-Egr-1 shRNA構(gòu)建成功.

第28到第79之間的堿基序列即為Egr-1基因shRNA序列(見圖1).圖 3 目的重組子pGreenPuro-Egr-1 shRNA質(zhì)粒DNA測(cè)序鑒定結(jié)果Fig. 3 The sequencing result of the desired recombinant pGreenPuro-Egr-1 shRNA plasmid DNA

3 討論

肝癌等惡性腫瘤的傳統(tǒng)治療方法主要包括手術(shù)、放療和化療3種，但療效并不理想且毒副作用大.因此，近年來興起的綜合療法倍受關(guān)注，以保健品治療腫瘤就是其中的一種方法.姜黃素是具有抗炎、抗氧化、降血脂、抗腫瘤作用的一種化合物，多項(xiàng)研究報(bào)道稱，姜黃素處理后可使一系列基因的表達(dá)發(fā)生變化，例如p53、Bcl-2、BAX[3,5]等.Egr-1是一種核轉(zhuǎn)錄因子，屬于Cys2His2型鋅指結(jié)構(gòu)EGR蛋白(Early growth response proteins)家族中的一員，其編碼的蛋白質(zhì)有3個(gè)重復(fù)的鋅指結(jié)構(gòu)域，可調(diào)控細(xì)胞增殖和細(xì)胞凋亡.長(zhǎng)期以來Egr-1就作為一種腫瘤抑制因子存在，近年來的研究報(bào)道[13-15]已經(jīng)證實(shí)，Egr-1基因可抑制乳腺癌、胃癌、肺癌細(xì)胞的生長(zhǎng).

本課題組的前期研究[1-2]發(fā)現(xiàn)，Egr-1基因在HepG-2細(xì)胞內(nèi)處于低表達(dá)狀態(tài)，姜黃素處理后能誘導(dǎo)其表達(dá)升高并能抑制HepG-2細(xì)胞增殖，而Egr-1基因的外來表達(dá)能抑制HepG-2細(xì)胞增殖，提示Egr-1基因可能在HepG-2細(xì)胞中起增殖抑制作用.本文構(gòu)建了Egr-1基因shRNA的表達(dá)載體pGreenPuro-Egr-1 shRNA，該載體在細(xì)胞內(nèi)能轉(zhuǎn)錄表達(dá)出Egr-1基因的小干擾性RNA，能特異性靶向沉默細(xì)胞內(nèi)源性Egr-1基因.利用該載體，可以在后續(xù)研究中通過轉(zhuǎn)染篩選，建立穩(wěn)定的細(xì)胞內(nèi)源性Egr-1基因靶向沉默的HepG-2細(xì)胞系，在內(nèi)源性Egr-1基因靶向沉默狀態(tài)下研究姜黃素處理對(duì)HepG-2細(xì)胞增殖及凋亡等的影響，為進(jìn)一步研究Egr-1基因在姜黃素抑制HepG-2細(xì)胞增殖中的作用奠定基礎(chǔ).

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TheConstructionandIdentificationofEgr-1GeneAntisenceInterferingExpressionVector

LIU Ruirui, LI Conghui, YU You, CHEN Jianming

(College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China)

To construct the vector stably expressingEgr-1 shRNA in human hepatic carcinoma cells, the well designed and correctly synthesizedEgr-1 shRNA oligonucleotides were used to ligate into pGreenPuroTMshRNA eukaryotic expressing vector predigested withBamHⅠandEcoRⅠenzymes. The ligation product was then used to transform theE.coliand several positive colonies were picked out for further culture. And subsequently the positive colonies were checked with bacteria liquid PCR method to identify the pGreenPuro-Egr-1 shRNA DNA recombinant. The PCR preliminary identified DNA recombinant was further checked with DNA sequencing method. And finally, the sequencing results showed theEgr-1 shRNA recombinant expression vector termed pGreenPuro-Egr-1 shRNA was successfully and correctly constructed.

Egr-1 gene; shRNA; antisense interference; vector construction

2017-04-17

杭州市社會(huì)發(fā)展自主申報(bào)項(xiàng)目(20160533B01).

陳建明(1964—)，男，副教授，博士，主要從事腫瘤細(xì)胞分子生物學(xué)研究.E-mail：jianmingchen@hznu.edu.cn

10.3969/j.issn.1674-232X.2017.06.009

Q253；R363.1

A

1674-232X(2017)06-0618-05