结核分枝杆菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表达载体的构建与表达

李武，邓光存，刘晓明，王玉炯宁夏大学 西部特色生物资源保护与利用教育部重点实验室，宁夏 银川 750021

医学与免疫生物技术

结核分枝杆菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表达载体的构建与表达

李武，邓光存，刘晓明，王玉炯
宁夏大学 西部特色生物资源保护与利用教育部重点实验室，宁夏 银川 750021

CFP10、ESAT6、Ag85A和Ag85B是结核分枝杆菌的主要免疫优势抗原。为了构建一种可同时表达这4种抗原的真核多基因共表达载体pcDNA-CFP10-ESAT6-Ag85A-Ag85B (pcDNA-CEAB)，并利用HEK 293T细胞对其体外表达进行检测。采用酶切、连接的方法，将CFP10和ESAT6编码基因以 (Gly4Ser) 3蛋白Linker连接，插入至质粒pcDNA3.1(+)多克隆位点CMV启动子与加尾信号BGH pA之间，使两者融合表达，将Ag85A和Ag85B编码基因以内部核糖体进入位点 (Internal ribosome entry site, IRES) 序列连接，并赋予RSV启动子和加尾信号BGH pA，使两者在RSV启动子作用下独立表达。重组质粒经酶切及测序验证后转染至HEK 293T细胞中进行体外表达实验，48 h后提取总蛋白，利用CFP10、ESAT6、Ag85A和Ag85B特异性抗体进行Western blotting检测。结果显示多基因共表达载体pcDNA-CEAB在真核细胞HEK 293T中得到表达，且CFP10、ESAT6、Ag85A和Ag85B抗原能被相应的特异性抗体所识别，表明质粒pcDNA-CEAB构建正确，这为进一步研究其免疫原性和免疫保护效果奠定了基础。

结核分枝杆菌，优势抗原，共表达，蛋白免疫印迹

结核病 (Tuberculosis, TB) 是由结核分枝杆菌 (Mycobacterium tuberculosis, MTB) 引起的慢性致死性人兽共患性传染病。据世界卫生组织估计，全世界大约有1/3的人群感染MTB。近年来，随着多重耐药性菌株的不断出现，MTB与艾滋病共感染等情况更加剧了全球结核病负担[1-2]。我国是世界上22个结核病高负担国家之一，结核病人数仅次于印度。对TB的防治目前仍然没有有效的手段，卡介苗 (Bacillus Calmette-Guerin, BCG) 虽然在全世界范围内广泛使用，但其免疫保护率在不同地区或人群中差异极大，从0–80%不等[3-4]，而且研究证实其保护时间不超过15年，加强免疫的效果也不太明显[5]。因此，关于TB预防疫苗及其免疫机理的研究一直是个热点研究领域。但是，到目前为止，仍然没有取得理论及实践的突破。

近年来，随着CFP10、ESAT6、Ag85A、Ag85B等一些结核分枝杆菌优势抗原基因被克隆，这些基因随之被用于DNA疫苗、重组BCG疫苗、纳米疫苗等多种形式的结核病预防疫苗的研究中，且被证实具有很好的免疫保护效果[6-9]。但是，现有的研究中，多数研究者将这些基因串联起来进行融合表达。考虑到多基因融合表达可能对抗原的免疫原性产生影响。因此，本研究以质粒pcDNA3.1(+)为基础，引入双启动子和IRES序列，构建共表达MTB免疫保护性抗原CFP10、ESAT6、Ag85A和Ag85B的重组质粒pcDNA-CEAB，最终实现在一个表达载体中共表达4种MTB优势抗原的目的。其中，Ag85A和Ag85B独立表达，CFP10和ESAT6以融合蛋白的形式进行表达，以期为进一步研究它们的免疫原性和免疫保护效果奠定基础。

1 材料与方法

1.1 菌株、质粒及细胞

MTB毒株H37Rv 基因组DNA，HEK 293T细胞，质粒 pcDNA3.1(+)、pREP10、pIRES2-EGFP等均由宁夏大学西部特色生物资源保护与利用教育部重点实验室保存；Trans5α感受态细胞购自北京全式金生物技术有限公司。

1.2 试剂

EasyPfu DNA聚合酶、DNA分子量marker、蛋白marker等购自北京全式金生物技术有限公司；限制性内切酶 AflⅡ、BamHⅠ、EcoRⅠ、NotⅠ、AscⅠ、XhoⅠ、NheⅠ、XbaⅠ、BstXⅠ、PmeⅠ等购自Fermentas公司；质粒小量提取试剂盒购自深圳市艾思进生物科技有限公司；无内毒素质粒大提试剂盒购自天根生化科技有限公司；PCR产物纯化试剂盒、T4 DNA 连接酶购自Promega公司；引物合成由上海生工生物工程技术服务有限公司完成；核苷酸序列测定由上海英骏生物技术有限公司完成。

胎牛血清、DMEM培养基、转染试剂Lipofectamine™ Plus购自Life Technologies公司；anti-MTB CFP10、anti-MTB ESAT6、anti-MTB Ag85A 和anti-MTB Ag85B 多克隆抗体购自Abcam公司；HRP标记的羊抗兔IgG购自北京中杉金桥生物技术有限公司；凯基全蛋白提取试剂盒及凯基BCA蛋白含量检测试剂盒购自南京凯基生物科技发展有限公司；WesternBrightTMECL Western blotting检测试剂盒购自Advansta公司。

1.3 目的基因的扩增

根据已经报道的CFP10、ESAT6、Ag85A、Ag85B抗原基因序列信息以及质粒pIRES2-EGFP中IRES基因序列信息和质粒pREP10中RSV启动子的序列信息，结合质粒pcDNA3.1(+)多克隆位点限制性内切酶信息，利用Primer Premier5软件设计并由上海生工生物工程技术服务有限公司合成了7对引物，引物序列如表1所示。PCR产物经1%琼脂糖凝胶电泳检测，割胶纯化后进行酶切和连接反应。

1.4 重组质粒pcDNA-CEAB的构建

1.4.1 质粒pcDNA-CFP10-ESAT6的构建

用Afl Ⅱ和EcoRⅠ双酶切CFP10 PCR产物，用EcoR Ⅰ和Not Ⅰ双酶切ESAT6 PCR产物，用Afl Ⅱ和Not Ⅰ双切质粒pcDNA3.1(+)，三者用T4 DNA 连接酶4 ℃连接过夜，菌落PCR法挑取阳性克隆子，酶切并测序验证。

1.4.2 质粒pcDNA-CFP10-ESAT6-BGHpA-RSV的构建

EcoRⅠ和AscⅠ双酶切ESAT6 PCR产物，AscⅠ和NotⅠ酶切BGH pA PCR产物，EcoRⅠ和NotⅠ双酶切质粒pcDNA3.1(+)，三者用T4 DNA连接酶进行连接，构建重组质粒pcDNAESAT6-BGHpA。然后用NotⅠ和XhoⅠ双酶切重组质粒pcDNA-ESAT6-BGHpA和RSV PCR产物，将RSV启动子插入到质粒pcDNA-ESAT6-BGHpA中，构建重组质粒pcDNA-ESAT6-BGHpA-RSV，最后，用AflⅡ和AscⅠ酶切质粒pcDNA-CFP10-ESAT6，回收CFP10-ESAT6片段，同时用这两种酶酶切质粒pcDNA-ESAT6-BGHpA-RSV，切掉ESAT6基因，将CFP10-ESAT6整体插入到质粒中，构建质粒pcDNA-CFP10-ESAT6-BGHpA-RSV(pcDNA-CEBR)，酶切并测序验证。

1.4.3 质粒pcDNA-Ag85A-IRES-Ag85B的构建

利用设计好的酶切位点，逐个将Ag85A、IRES和Ag85B基因插入到质粒pcDNA3.1(+)中，构建质粒pcDNA-Ag85A-IERS-Ag85B(pcDNA-AIB)，菌落PCR法挑取阳性克隆子后直接测序验证。

1.4.4 质粒pcDNA-CEAB的构建

重组质粒pcDNA-CEBR经AflⅡ和NheⅠ双酶切，同时用NheⅠ和XbaⅠ双酶切质粒pcDNA-AIB，AflⅡ和XbaⅠ双酶切质粒pcDNA3.1(+)，目的片段割胶回收后连接，构建质粒pcDNA-CEAB，酶切及测序验证。

1.5 HEK 293T细胞转染及总蛋白提取

无内毒素大提质粒试剂盒大提质粒pcDNA-CEAB。质粒转染前1天于60 mm细胞培养皿中接种不同浓度的HEK 293T细胞，次日选取80%–90%融合的细胞进行转染。每60 mm细胞培养皿内细胞转染20 μg质粒pcDNA-CEAB，同时转染质粒pcDNA3.1(+)作为阴性对照，转染携带GFP的质粒pIRES2-EGFP作为阳性对照。转染采用Lipofectamine™ Plus试剂盒进行，具体转染操作参照试剂盒说明书进行。

转染48 h后提取转染了质粒pcDNA3.1(+)和重组质粒pcDNA-CEAB的细胞总蛋白，采用凯基蛋白定量试剂盒进行定量后，加5×SDS-PAGE蛋白上样缓冲液，沸水中煮沸5 min后分装，-20 ℃冻存备用。

表1 目的基因PCR扩增引物Table 1 Primer sequences for the target genes

1.6 Western blotting分析

蛋白经15% SDS-PAGE电泳后(20 μg/泳道)转印至硝酸纤维素膜 (NC膜) 上，封闭液 (含5%脱脂牛奶的TBST) 封闭2 h后，加一抗 (1∶1 000封闭液稀释) 室温孵育2 h，TBST洗膜3次，每次10 min，然后加入二抗 (1∶5 000封闭液稀释)，室温孵育2 h，TBST洗膜2次后PBS洗膜1次，每次10 min。用ECL试剂显色，暗室中用X光胶片曝光。

2 结果与分析

2.1 目的基因的扩增结果

以MTB毒株 H37Rv基因组DNA为模板成功扩增了4种抗原基因，以质粒pIRES2-EGFP为模板成功地扩增IRES序列，以质粒pREP10为模板成功地扩增了RSV启动子序列，以pcDNA3.1(+)为模板成功地扩增了BGH pA 序列。PCR扩增结果如图1所示。

图1 目的基因的PCR扩增结果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 重组质粒的酶切鉴定结果

2.2.1 pcDNA-CFP10-ESAT6的酶切鉴定结果

为了对质粒pcDNA-CFP10-ESAT6进行酶切鉴定，首先用NheⅠ和AscⅠ双酶切该质粒，理论上能将CFP10和ESAT6融合基因整体从质粒上酶切下来，此外还用EcoRⅠ和AscⅠ进行双酶切，看能否将ESAT6和(Gly4Ser)3 linker从质粒上切下来，酶切结果 (图2) 证实，质粒pcDNA-CFP10-ESAT6构建正确。

图2 重组质粒pcDNA-CFP10-ESAT6的酶切电泳结果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的酶切鉴定结果

由于插入片段CFP10-ESAT6-BGHpA-RSV两端各有一个NheⅠ酶切位点，故采用NheⅠ对该质粒进行单酶切验证。另外，还用EcoRⅠ和NotⅠ对质粒进行酶切，理论上应该能将(Gly4Ser)3 linker、ESAT6和BGH pA 整体从质粒上切下来，酶切片段大小与理论值相符，说明质粒构建正确，酶切结果如图3所示。

图3 重组质粒pcDNA-CEBR的酶切电泳结果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.

图4 重组质粒pcDNA-CEAB的结构模式图Fig. 4 Ideograph of the recombinant plasmid pcDNA-CEAB.

2.2.3 pcDNA-CEAB的酶切鉴定结果

质粒pcDNA-CEAB (图4) 构建完成后，首先用PmeⅠ 进行酶切验证，看能否将CFP10、ESAT6、BGH pA、RSV、Ag85A、IRES和Ag85B序列整体切下来。另外，考虑到CFP10、Ag85A和Ag85B基因内部各有一个XhoⅠ酶切位点，故用XhoⅠ对质粒pcDNA-CEAB 继续进行酶切验证，酶切结果如图5所示，各酶切片段大小与理论值相符。

2.3 Western blotting分析结果

质粒转染HEK 293T细胞48 h后裂解细胞，提取细胞总蛋白进行SDS-PAGE分析，电泳结果如图6所示。电泳后蛋白转印至NC膜上进行Western blotting分析。结果显示，在约26 kDa位置处出现CFP10和ESAT6特异性抗体识别条带，在约 32 kDa和34 kDa位置处出现Ag85A抗体和Ag85B抗体识别条带，表明所有蛋白均可以被相应的特异性抗体所识别，结果如图7所示。

3 讨论

CFP10、ESAT6、Ag85A和Ag85B是MTB的主要免疫优势抗原，目前已被广泛用于TB疫苗的研究中[10-13]。其中，CFP10和ESAT6是从MTB早期培养滤液中分离到的两种低分子量分泌蛋白，它们都由结核分枝杆菌基因组RD1区编码，同时翻译表达[14]。1996年，Mahairas等研究发现，编码CFP10和ESAT6基因的RD1区只存在于致病性MTB菌株中，而在BCG和非致病性MTB基因组中缺失[15]。这一研究发现使得CFP10和ESAT6基因在疫苗研究领域受到广泛关注，而BCG免疫效果不佳，可能与其在长期传代过程中丧失一些免疫优势抗原有一定关系。Ag85A和Ag85B是MTB在生长过程中分泌到细胞外的两种主要的分泌性蛋白，是从Ag85复合物中分离到的两种蛋白，它们的分子量大小分别为31 kDa和30 kDa[16]，是MTB的两种重要的免疫保护性抗原，它们既能诱导体液免疫应答，又能诱导特异性Th1型细胞免疫应答，具有良好的免疫保护作用[17-18]。疫苗研究中，这两种抗原基因也得到广泛的应用。因此，本研究选取这4种抗原基因构建真核共表达载体，并证实它们可以在真核细胞HEK 293T中表达。

图5 重组质粒pcDNA-CEAB酶切电泳结果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.

图6 质粒pcDNA3.1(+)和pcDNA-CEAB转染HEK 293T细胞后目的蛋白表达情况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.

图7 质粒转染HEK 293T细胞后分别用anti-CFP10(A)、anti-ESAT6(B)、anti-Ag85A(C) 和 anti-Ag85B(D)多克隆抗体对基因在HEK 293T细胞中的表达情况进行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抗原的分子量较小，分子量太小对抗原免疫原性存在一定的影响。对于这两种蛋白，多数研究者采用融合蛋白的形式进行表达。因此，我们也采用基因融合技术，将二者以蛋白linker连接起来，以融合蛋白的形式进行表达。在蛋白linker的选择方面，我们选择目前已被广泛使用的(Gly4Ser)3蛋白linker[19-21]。而对Ag85A和Ag85B蛋白而言，其分子量大小适中，适合单独表达，研究也证实它们具有诱导机体产生特异性Th1型细胞免疫应答的能力[22-24]。为了实现这两种蛋白的独立表达，我们利用IRES序列将两者相连。目前，来源于脑心肌炎病毒(EMCV)的IRES元件已被广泛使用于带有报道基因的真核表达载体中，它能招募核糖体启动其下游基因的翻译表达[25]。实践证明，通过引入IRES序列，我们最终成功地利用一个RSV启动子，实现了对Ag85A和Ag85B蛋白的独立表达。

总之，本研究成功地构建了双启动子共表达载体pcDNA-CEAB，并利用(Gly4Ser)3蛋白linker实现了CFP10和ESAT6的融合表达，利用IRES序列实现了Ag85A和Ag85B蛋白的独立表达。最终，在同一个载体中共表达了MTB四种优势抗原基因，并且在HEK 293T 细胞中对它们的体外表达进行了验证，为进一步研究这几种抗原的免疫原性和免疫效果奠定了基础。另外，本研究构建的载体系统本身已被证明是一个良好的多基因共表达系统，也可以用作研究其他基因共表达的工具，尤其是在多价疫苗的研制中可能发挥一定的作用。

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(本文责编 郝丽芳)

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, 邓光存, 刘晓明, 等. 结核分枝杆菌CFP10、ESAT6、Ag85A和Ag85B抗原真核共表达载体的构建与表达. 生物工程学报, 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).

国家重点基础研究发展计划 (973计划) (Nos. 2012CB126301，2012CB518801)，国家自然科学基金 (No. 31160515)，高等学校博士学科点专项科研基金 (No. 20126401110001)，国家科技支撑计划项目 (No. 2012BAD12B07-4) 资助。

时间：2013-09-12 网络出版地址：http://www.cnki.net/kcms/detail/11.1998.Q.20130912.0214.005.html