結(jié)核分枝桿菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表達(dá)載體的構(gòu)建與表達(dá)

李武，鄧光存，劉曉明，王玉炯寧夏大學(xué) 西部特色生物資源保護(hù)與利用教育部重點(diǎn)實(shí)驗(yàn)室，寧夏 銀川 750021

醫(yī)學(xué)與免疫生物技術(shù)

結(jié)核分枝桿菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表達(dá)載體的構(gòu)建與表達(dá)

李武，鄧光存，劉曉明，王玉炯
寧夏大學(xué) 西部特色生物資源保護(hù)與利用教育部重點(diǎn)實(shí)驗(yàn)室，寧夏 銀川 750021

CFP10、ESAT6、Ag85A和Ag85B是結(jié)核分枝桿菌的主要免疫優(yōu)勢(shì)抗原。為了構(gòu)建一種可同時(shí)表達(dá)這4種抗原的真核多基因共表達(dá)載體pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB)，并利用HEK 293T細(xì)胞對(duì)其體外表達(dá)進(jìn)行檢測(cè)。采用酶切、連接的方法，將CFP10和ESAT6編碼基因以 (Gly4Ser) 3蛋白Linker連接，插入至質(zhì)粒pcDNA3.1(+)多克隆位點(diǎn)CMV啟動(dòng)子與加尾信號(hào)BGH pA之間，使兩者融合表達(dá)，將Ag85A和Ag85B編碼基因以內(nèi)部核糖體進(jìn)入位點(diǎn) (Internal ribosome entry site, IRES) 序列連接，并賦予RSV啟動(dòng)子和加尾信號(hào)BGH pA，使兩者在RSV啟動(dòng)子作用下獨(dú)立表達(dá)。重組質(zhì)粒經(jīng)酶切及測(cè)序驗(yàn)證后轉(zhuǎn)染至HEK 293T細(xì)胞中進(jìn)行體外表達(dá)實(shí)驗(yàn)，48 h后提取總蛋白，利用CFP10、ESAT6、Ag85A和Ag85B特異性抗體進(jìn)行Western blotting檢測(cè)。結(jié)果顯示多基因共表達(dá)載體pcDNA-CEAB在真核細(xì)胞HEK 293T中得到表達(dá)，且CFP10、ESAT6、Ag85A和Ag85B抗原能被相應(yīng)的特異性抗體所識(shí)別，表明質(zhì)粒pcDNA-CEAB構(gòu)建正確，這為進(jìn)一步研究其免疫原性和免疫保護(hù)效果奠定了基礎(chǔ)。

結(jié)核分枝桿菌，優(yōu)勢(shì)抗原，共表達(dá)，蛋白免疫印跡

結(jié)核病 (Tuberculosis, TB) 是由結(jié)核分枝桿菌 (Mycobacterium tuberculosis, MTB) 引起的慢性致死性人獸共患性傳染病。據(jù)世界衛(wèi)生組織估計(jì)，全世界大約有1/3的人群感染MTB。近年來，隨著多重耐藥性菌株的不斷出現(xiàn)，MTB與艾滋病共感染等情況更加劇了全球結(jié)核病負(fù)擔(dān)[1-2]。我國是世界上22個(gè)結(jié)核病高負(fù)擔(dān)國家之一，結(jié)核病人數(shù)僅次于印度。對(duì)TB的防治目前仍然沒有有效的手段，卡介苗 (Bacillus Calmette-Guerin, BCG) 雖然在全世界范圍內(nèi)廣泛使用，但其免疫保護(hù)率在不同地區(qū)或人群中差異極大，從0–80%不等[3-4]，而且研究證實(shí)其保護(hù)時(shí)間不超過15年，加強(qiáng)免疫的效果也不太明顯[5]。因此，關(guān)于TB預(yù)防疫苗及其免疫機(jī)理的研究一直是個(gè)熱點(diǎn)研究領(lǐng)域。但是，到目前為止，仍然沒有取得理論及實(shí)踐的突破。

近年來，隨著CFP10、ESAT6、Ag85A、Ag85B等一些結(jié)核分枝桿菌優(yōu)勢(shì)抗原基因被克隆，這些基因隨之被用于DNA疫苗、重組BCG疫苗、納米疫苗等多種形式的結(jié)核病預(yù)防疫苗的研究中，且被證實(shí)具有很好的免疫保護(hù)效果[6-9]。但是，現(xiàn)有的研究中，多數(shù)研究者將這些基因串聯(lián)起來進(jìn)行融合表達(dá)?？紤]到多基因融合表達(dá)可能對(duì)抗原的免疫原性產(chǎn)生影響。因此，本研究以質(zhì)粒pcDNA3.1(+)為基礎(chǔ)，引入雙啟動(dòng)子和IRES序列，構(gòu)建共表達(dá)MTB免疫保護(hù)性抗原CFP10、ESAT6、Ag85A和Ag85B的重組質(zhì)粒pcDNA-CEAB，最終實(shí)現(xiàn)在一個(gè)表達(dá)載體中共表達(dá)4種MTB優(yōu)勢(shì)抗原的目的。其中，Ag85A和Ag85B獨(dú)立表達(dá)，CFP10和ESAT6以融合蛋白的形式進(jìn)行表達(dá)，以期為進(jìn)一步研究它們的免疫原性和免疫保護(hù)效果奠定基礎(chǔ)。

1 材料與方法

1.1 菌株、質(zhì)粒及細(xì)胞

MTB毒株H37Rv 基因組DNA，HEK 293T細(xì)胞，質(zhì)粒 pcDNA3.1(+)、pREP10、pIRES2-EGFP等均由寧夏大學(xué)西部特色生物資源保護(hù)與利用教育部重點(diǎn)實(shí)驗(yàn)室保存；Trans5α感受態(tài)細(xì)胞購自北京全式金生物技術(shù)有限公司。

1.2 試劑

EasyPfu DNA聚合酶、DNA分子量marker、蛋白marker等購自北京全式金生物技術(shù)有限公司；限制性內(nèi)切酶 AflⅡ、BamHⅠ、EcoRⅠ、NotⅠ、AscⅠ、XhoⅠ、NheⅠ、XbaⅠ、BstXⅠ、PmeⅠ等購自Fermentas公司；質(zhì)粒小量提取試劑盒購自深圳市艾思進(jìn)生物科技有限公司；無內(nèi)毒素質(zhì)粒大提試劑盒購自天根生化科技有限公司；PCR產(chǎn)物純化試劑盒、T4 DNA 連接酶購自Promega公司；引物合成由上海生工生物工程技術(shù)服務(wù)有限公司完成；核苷酸序列測(cè)定由上海英駿生物技術(shù)有限公司完成。

胎牛血清、DMEM培養(yǎng)基、轉(zhuǎn)染試劑Lipofectamine? Plus購自Life Technologies公司；anti-MTB CFP10、anti-MTB ESAT6、anti-MTB Ag85A 和anti-MTB Ag85B 多克隆抗體購自Abcam公司；HRP標(biāo)記的羊抗兔IgG購自北京中杉金橋生物技術(shù)有限公司；凱基全蛋白提取試劑盒及凱基BCA蛋白含量檢測(cè)試劑盒購自南京凱基生物科技發(fā)展有限公司；WesternBrightTMECL Western blotting檢測(cè)試劑盒購自Advansta公司。

1.3 目的基因的擴(kuò)增

根據(jù)已經(jīng)報(bào)道的CFP10、ESAT6、Ag85A、Ag85B抗原基因序列信息以及質(zhì)粒pIRES2-EGFP中IRES基因序列信息和質(zhì)粒pREP10中RSV啟動(dòng)子的序列信息，結(jié)合質(zhì)粒pcDNA3.1(+)多克隆位點(diǎn)限制性內(nèi)切酶信息，利用Primer Premier5軟件設(shè)計(jì)并由上海生工生物工程技術(shù)服務(wù)有限公司合成了7對(duì)引物，引物序列如表1所示。PCR產(chǎn)物經(jīng)1%瓊脂糖凝膠電泳檢測(cè)，割膠純化后進(jìn)行酶切和連接反應(yīng)。

1.4 重組質(zhì)粒pcDNA-CEAB的構(gòu)建

1.4.1 質(zhì)粒pcDNA-CFP10-ESAT6的構(gòu)建

用Afl Ⅱ和EcoRⅠ雙酶切CFP10 PCR產(chǎn)物，用EcoR Ⅰ和Not Ⅰ雙酶切ESAT6 PCR產(chǎn)物，用Afl Ⅱ和Not Ⅰ雙切質(zhì)粒pcDNA3.1(+)，三者用T4 DNA 連接酶4 ℃連接過夜，菌落PCR法挑取陽性克隆子，酶切并測(cè)序驗(yàn)證。

1.4.2 質(zhì)粒pcDNA-CFP10-ESAT6-BGHpA-RSV的構(gòu)建

EcoRⅠ和AscⅠ雙酶切ESAT6 PCR產(chǎn)物，AscⅠ和NotⅠ酶切BGH pA PCR產(chǎn)物，EcoRⅠ和NotⅠ雙酶切質(zhì)粒pcDNA3.1(+)，三者用T4 DNA連接酶進(jìn)行連接，構(gòu)建重組質(zhì)粒pcDNAESAT6-BGHpA。然后用NotⅠ和XhoⅠ雙酶切重組質(zhì)粒pcDNA-ESAT6-BGHpA和RSV PCR產(chǎn)物，將RSV啟動(dòng)子插入到質(zhì)粒pcDNA-ESAT6-BGHpA中，構(gòu)建重組質(zhì)粒pcDNA-ESAT6-BGHpA-RSV，最后，用AflⅡ和AscⅠ酶切質(zhì)粒pcDNA-CFP10-ESAT6，回收CFP10-ESAT6片段，同時(shí)用這兩種酶酶切質(zhì)粒pcDNA-ESAT6-BGHpA-RSV，切掉ESAT6基因，將CFP10-ESAT6整體插入到質(zhì)粒中，構(gòu)建質(zhì)粒pcDNA-CFP10-ESAT6-BGHpA-RSV(pcDNA-CEBR)，酶切并測(cè)序驗(yàn)證。

1.4.3 質(zhì)粒pcDNA-Ag85A-IRES-Ag85B的構(gòu)建

利用設(shè)計(jì)好的酶切位點(diǎn)，逐個(gè)將Ag85A、IRES和Ag85B基因插入到質(zhì)粒pcDNA3.1(+)中，構(gòu)建質(zhì)粒pcDNA-Ag85A-IERS-Ag85B(pcDNA-AIB)，菌落PCR法挑取陽性克隆子后直接測(cè)序驗(yàn)證。

1.4.4 質(zhì)粒pcDNA-CEAB的構(gòu)建

重組質(zhì)粒pcDNA-CEBR經(jīng)AflⅡ和NheⅠ雙酶切，同時(shí)用NheⅠ和XbaⅠ雙酶切質(zhì)粒pcDNA-AIB，AflⅡ和XbaⅠ雙酶切質(zhì)粒pcDNA3.1(+)，目的片段割膠回收后連接，構(gòu)建質(zhì)粒pcDNA-CEAB，酶切及測(cè)序驗(yàn)證。

1.5 HEK 293T細(xì)胞轉(zhuǎn)染及總蛋白提取

無內(nèi)毒素大提質(zhì)粒試劑盒大提質(zhì)粒pcDNA-CEAB。質(zhì)粒轉(zhuǎn)染前1天于60 mm細(xì)胞培養(yǎng)皿中接種不同濃度的HEK 293T細(xì)胞，次日選取80%–90%融合的細(xì)胞進(jìn)行轉(zhuǎn)染。每60 mm細(xì)胞培養(yǎng)皿內(nèi)細(xì)胞轉(zhuǎn)染20 μg質(zhì)粒pcDNA-CEAB，同時(shí)轉(zhuǎn)染質(zhì)粒pcDNA3.1(+)作為陰性對(duì)照，轉(zhuǎn)染攜帶GFP的質(zhì)粒pIRES2-EGFP作為陽性對(duì)照。轉(zhuǎn)染采用Lipofectamine? Plus試劑盒進(jìn)行，具體轉(zhuǎn)染操作參照試劑盒說明書進(jìn)行。

轉(zhuǎn)染48 h后提取轉(zhuǎn)染了質(zhì)粒pcDNA3.1(+)和重組質(zhì)粒pcDNA-CEAB的細(xì)胞總蛋白，采用凱基蛋白定量試劑盒進(jìn)行定量后，加5×SDS-PAGE蛋白上樣緩沖液，沸水中煮沸5 min后分裝，-20 ℃凍存?zhèn)溆谩?/p>

表1 目的基因PCR擴(kuò)增引物Table 1 Primer sequences for the target genes

1.6 Western blotting分析

蛋白經(jīng)15% SDS-PAGE電泳后(20 μg/泳道)轉(zhuǎn)印至硝酸纖維素膜 (NC膜) 上，封閉液 (含5%脫脂牛奶的TBST) 封閉2 h后，加一抗 (1∶1 000封閉液稀釋) 室溫孵育2 h，TBST洗膜3次，每次10 min，然后加入二抗 (1∶5 000封閉液稀釋)，室溫孵育2 h，TBST洗膜2次后PBS洗膜1次，每次10 min。用ECL試劑顯色，暗室中用X光膠片曝光。

2 結(jié)果與分析

2.1 目的基因的擴(kuò)增結(jié)果

以MTB毒株 H37Rv基因組DNA為模板成功擴(kuò)增了4種抗原基因，以質(zhì)粒pIRES2-EGFP為模板成功地?cái)U(kuò)增IRES序列，以質(zhì)粒pREP10為模板成功地?cái)U(kuò)增了RSV啟動(dòng)子序列，以pcDNA3.1(+)為模板成功地?cái)U(kuò)增了BGH pA 序列。PCR擴(kuò)增結(jié)果如圖1所示。

圖1 目的基因的PCR擴(kuò)增結(jié)果Fig. 1 Amplification of the target genes. M: Trans2kTMplus DNA marker; 1: PCR products of CFP10; 2: PCR products of ESAT6; 3: PCR products of Ag85A; 4: PCR products of Ag85B; 5: PCR products of IRES; 6: PCR products of BGH pA; 7: PCR products of RSV.

2.2 重組質(zhì)粒的酶切鑒定結(jié)果

2.2.1 pcDNA-CFP10-ESAT6的酶切鑒定結(jié)果

為了對(duì)質(zhì)粒pcDNA-CFP10-ESAT6進(jìn)行酶切鑒定，首先用NheⅠ和AscⅠ雙酶切該質(zhì)粒，理論上能將CFP10和ESAT6融合基因整體從質(zhì)粒上酶切下來，此外還用EcoRⅠ和AscⅠ進(jìn)行雙酶切，看能否將ESAT6和(Gly4Ser)3 linker從質(zhì)粒上切下來，酶切結(jié)果 (圖2) 證實(shí)，質(zhì)粒pcDNA-CFP10-ESAT6構(gòu)建正確。

圖2 重組質(zhì)粒pcDNA-CFP10-ESAT6的酶切電泳結(jié)果Fig. 2 Agarose gel electrophoresis of the recombiant plasmid pcDNA-CFP10-ESAT6 by restriction endonuclease digestion. M: Trans2kTMDNA marker; 1: pcDNA3.1(+) digested with Xho I; 2: pcDNA-CFP10-ESAT6 digested with Nhe Iand Asc I; 3: pcDNACFP10-ESAT6 digested with EcoR I and Asc I.

2.2.2 pcDNA-CEBR的酶切鑒定結(jié)果

由于插入片段CFP10-ESAT6-BGHpA-RSV兩端各有一個(gè)NheⅠ酶切位點(diǎn)，故采用NheⅠ對(duì)該質(zhì)粒進(jìn)行單酶切驗(yàn)證。另外，還用EcoRⅠ和NotⅠ對(duì)質(zhì)粒進(jìn)行酶切，理論上應(yīng)該能將(Gly4Ser)3 linker、ESAT6和BGH pA 整體從質(zhì)粒上切下來，酶切片段大小與理論值相符，說明質(zhì)粒構(gòu)建正確，酶切結(jié)果如圖3所示。

圖3 重組質(zhì)粒pcDNA-CEBR的酶切電泳結(jié)果Fig. 3 Agarose gel electrophoresis of the recombiant plasmid pcDNA-CEBR by restriction endonuclease digestion. M: Trans8k DNA marker; 1: pcDNA3.1(+) digested with Xho I; 2: pcDNA-CEBR digested with Nhe I; 3: pcDNA-CEBR digested with EcoR I and Not I.

圖4 重組質(zhì)粒pcDNA-CEAB的結(jié)構(gòu)模式圖Fig. 4 Ideograph of the recombinant plasmid pcDNA-CEAB.

2.2.3 pcDNA-CEAB的酶切鑒定結(jié)果

質(zhì)粒pcDNA-CEAB (圖4) 構(gòu)建完成后，首先用PmeⅠ 進(jìn)行酶切驗(yàn)證，看能否將CFP10、ESAT6、BGH pA、RSV、Ag85A、IRES和Ag85B序列整體切下來。另外，考慮到CFP10、Ag85A和Ag85B基因內(nèi)部各有一個(gè)XhoⅠ酶切位點(diǎn)，故用XhoⅠ對(duì)質(zhì)粒pcDNA-CEAB 繼續(xù)進(jìn)行酶切驗(yàn)證，酶切結(jié)果如圖5所示，各酶切片段大小與理論值相符。

2.3 Western blotting分析結(jié)果

質(zhì)粒轉(zhuǎn)染HEK 293T細(xì)胞48 h后裂解細(xì)胞，提取細(xì)胞總蛋白進(jìn)行SDS-PAGE分析，電泳結(jié)果如圖6所示。電泳后蛋白轉(zhuǎn)印至NC膜上進(jìn)行Western blotting分析。結(jié)果顯示，在約26 kDa位置處出現(xiàn)CFP10和ESAT6特異性抗體識(shí)別條帶，在約 32 kDa和34 kDa位置處出現(xiàn)Ag85A抗體和Ag85B抗體識(shí)別條帶，表明所有蛋白均可以被相應(yīng)的特異性抗體所識(shí)別，結(jié)果如圖7所示。

3 討論

CFP10、ESAT6、Ag85A和Ag85B是MTB的主要免疫優(yōu)勢(shì)抗原，目前已被廣泛用于TB疫苗的研究中[10-13]。其中，CFP10和ESAT6是從MTB早期培養(yǎng)濾液中分離到的兩種低分子量分泌蛋白，它們都由結(jié)核分枝桿菌基因組RD1區(qū)編碼，同時(shí)翻譯表達(dá)[14]。1996年，Mahairas等研究發(fā)現(xiàn)，編碼CFP10和ESAT6基因的RD1區(qū)只存在于致病性MTB菌株中，而在BCG和非致病性MTB基因組中缺失[15]。這一研究發(fā)現(xiàn)使得CFP10和ESAT6基因在疫苗研究領(lǐng)域受到廣泛關(guān)注，而BCG免疫效果不佳，可能與其在長期傳代過程中喪失一些免疫優(yōu)勢(shì)抗原有一定關(guān)系。Ag85A和Ag85B是MTB在生長過程中分泌到細(xì)胞外的兩種主要的分泌性蛋白，是從Ag85復(fù)合物中分離到的兩種蛋白，它們的分子量大小分別為31 kDa和30 kDa[16]，是MTB的兩種重要的免疫保護(hù)性抗原，它們既能誘導(dǎo)體液免疫應(yīng)答，又能誘導(dǎo)特異性Th1型細(xì)胞免疫應(yīng)答，具有良好的免疫保護(hù)作用[17-18]。疫苗研究中，這兩種抗原基因也得到廣泛的應(yīng)用。因此，本研究選取這4種抗原基因構(gòu)建真核共表達(dá)載體，并證實(shí)它們可以在真核細(xì)胞HEK 293T中表達(dá)。

圖5 重組質(zhì)粒pcDNA-CEAB酶切電泳結(jié)果Fig. 5 Agarose gel electrophoresis of the recombiant plasmid pcDNA-CEAB by restriction endonuclease digestion. M: Trans8K DNA marker; 1: pcDNA3.1(+) digested with XhoⅠ; 2: pcDNA-CEAB digested with PmeⅠ; 3: pcDNA-CEAB digested with XhoⅠ.

圖6 質(zhì)粒pcDNA3.1(+)和pcDNA-CEAB轉(zhuǎn)染HEK 293T細(xì)胞后目的蛋白表達(dá)情況SDS-PAGE分析Fig. 6 SDS-PAGE analysis of cell lysates from HEK 293T cells transfected with pcDNA3.1 (+) and pcDNA-CEAB. M: Blue PlusTMII protein marker; 1: cell lysates from HEK 293T cells transfected with empty vector pcDNA3.1 (+); 2: cell lysates from HEK 293T cells transfected with the recombinant plasmid pcDNA-CEAB.

圖7 質(zhì)粒轉(zhuǎn)染HEK 293T細(xì)胞后分別用anti-CFP10(A)、anti-ESAT6(B)、anti-Ag85A(C) 和 anti-Ag85B(D)多克隆抗體對(duì)基因在HEK 293T細(xì)胞中的表達(dá)情況進(jìn)行Western blotting分析Fig. 7 Western blotting analysis of cell lysates using anti-CFP10(A), anti-ESAT6(B), anti-Ag85A(C) and anti-Ag85B(D) antibody, respectively. M: Blue PlusTMII protein marker; 1: cell lysates from HEK 293T cells transfected with empty vector pcDNA3.1 (+); 2: cell lysates from HEK 293T cells transfected with recombinant plasmid pcDNA-CEAB.

CFP10和ESAT6抗原的分子量較小，分子量太小對(duì)抗原免疫原性存在一定的影響。對(duì)于這兩種蛋白，多數(shù)研究者采用融合蛋白的形式進(jìn)行表達(dá)。因此，我們也采用基因融合技術(shù)，將二者以蛋白linker連接起來，以融合蛋白的形式進(jìn)行表達(dá)。在蛋白linker的選擇方面，我們選擇目前已被廣泛使用的(Gly4Ser)3蛋白linker[19-21]。而對(duì)Ag85A和Ag85B蛋白而言，其分子量大小適中，適合單獨(dú)表達(dá)，研究也證實(shí)它們具有誘導(dǎo)機(jī)體產(chǎn)生特異性Th1型細(xì)胞免疫應(yīng)答的能力[22-24]。為了實(shí)現(xiàn)這兩種蛋白的獨(dú)立表達(dá)，我們利用IRES序列將兩者相連。目前，來源于腦心肌炎病毒(EMCV)的IRES元件已被廣泛使用于帶有報(bào)道基因的真核表達(dá)載體中，它能招募核糖體啟動(dòng)其下游基因的翻譯表達(dá)[25]。實(shí)踐證明，通過引入IRES序列，我們最終成功地利用一個(gè)RSV啟動(dòng)子，實(shí)現(xiàn)了對(duì)Ag85A和Ag85B蛋白的獨(dú)立表達(dá)。

總之，本研究成功地構(gòu)建了雙啟動(dòng)子共表達(dá)載體pcDNA-CEAB，并利用(Gly4Ser)3蛋白linker實(shí)現(xiàn)了CFP10和ESAT6的融合表達(dá)，利用IRES序列實(shí)現(xiàn)了Ag85A和Ag85B蛋白的獨(dú)立表達(dá)。最終，在同一個(gè)載體中共表達(dá)了MTB四種優(yōu)勢(shì)抗原基因，并且在HEK 293T 細(xì)胞中對(duì)它們的體外表達(dá)進(jìn)行了驗(yàn)證，為進(jìn)一步研究這幾種抗原的免疫原性和免疫效果奠定了基礎(chǔ)。另外，本研究構(gòu)建的載體系統(tǒng)本身已被證明是一個(gè)良好的多基因共表達(dá)系統(tǒng)，也可以用作研究其他基因共表達(dá)的工具，尤其是在多價(jià)疫苗的研制中可能發(fā)揮一定的作用。

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(本文責(zé)編 郝麗芳)

Construction and expression of a eukaryotic vector co-expressing immunodominant antigens of CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis

Wu Li, Guangcun Deng, Xiaoming Liu, and Yujiong Wang
Key Laboratory of Ministry of Education for Conservation and Utilization of Special Biological Resources in the Western, Ningxia University, Yinchuan 750021, Ningxia, China

CFP10, ESAT6, Antigen 85A (Ag85A) and antigen 85B (Ag85B) are the key immunodominant antigens of Mycobacterium tuberculosis. In order to construct a eukaryotic vector able to co-express the four genes in one vector, we amplified the target gene fragments encoding the CFP10, ESAT6, Ag85A and Ag85B antigens and inserted them into the multicloning site of the shuttle plasmid vector pcDNA3.1 (+), of which the CFP10 and ESAT6 encoding genes were in frame fused with a linker encoding (Gly4Ser)3 residue, before the fused gene was inserted downstream of CMV promoter with a bovine growth hormone poly A(BGH pA) sequence at the 3’-end; Ag85A and Ag85B encoding genes were fused with a separation of internal ribosome entry site (IRES) sequence before the fused gene cassette was inserted downstream of RSV promoter with a BGH pA sequence at the 3’-end. The final plasmid containing all four genes was confirmed by sequence analysis and designated as pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB). In order to verify the ability of this construct to express target proteins, we then transfected the recombinant plasmid into Human embryonic kidney (HEK) 293T cells and harvested the cell lysates, and the cell lysates were then separated by SDS-PAGE and subjected to Western blot analysis 48 h after transfection. All four of the target proteins were detected in the cell lysates against the respective specific antibodies, suggesting that we have successfully constructed a eukaryotic vector co-expressing the four immunodominant antigens of Mycobacterium tuberculosis, which lay a foundation for the further study of the immunogenicity and protective activity of the four antigens.

Mycobacterium tuberculosis, immunodominant antigen, co-expression, Western blotting

April 24, 2013; Accepted: August 12, 2013

Yujiong Wang. Tel/Fax: +86-951-2062033; E-mail: wyj@nxu.edu.cn

李武, 鄧光存, 劉曉明, 等. 結(jié)核分枝桿菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表達(dá)載體的構(gòu)建與表達(dá). 生物工程學(xué)報(bào), 2014, 30(2): 265–273.

Li W, Deng GC, Liu XM, et al. Construction and expression of a eukaryotic vector co-expressing immunodominant antigens of CFP10, ESAT6, Ag85A and Ag85B of Mycobacterium tuberculosis. Chin J Biotech, 2014, 30(2): 265–273.

Supported by: National Basic Research Program of China (973 Program) (Nos. 2012CB126301, 2012CB518801), National Natural Science Foundation of China (No. 31160515), Research Fund for the Doctoral Program of Higher Education of China (No. 20126401110001), Key Technologies Research and Development Program of China (No. 2012BAD12B07-4).

國家重點(diǎn)基礎(chǔ)研究發(fā)展計(jì)劃 (973計(jì)劃) (Nos. 2012CB126301，2012CB518801)，國家自然科學(xué)基金 (No. 31160515)，高等學(xué)校博士學(xué)科點(diǎn)專項(xiàng)科研基金 (No. 20126401110001)，國家科技支撐計(jì)劃項(xiàng)目 (No. 2012BAD12B07-4) 資助。

時(shí)間：2013-09-12 網(wǎng)絡(luò)出版地址：http://www.cnki.net/kcms/detail/11.1998.Q.20130912.0214.005.html