分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达
1中国人民解放军第四军医大学基础部病理学教研室 陕西省西安市 710033
2中国人民解放军第四军医大学唐都医院骨科 陕西省西安市 710038
程虹，女，1967-06-24生，安徽省宣州市人，汉族. 1996年获第四军医大学病理学博士, 主要从事肝癌的免疫基因治疗研究.
项目负责人 程虹, 710033, 陕西省西安市长乐西路169号，中国人民解放军第四军医大学基础部病理学教研室.
Department of Pathology, Faculty of Preclinical Medicine, Fourth Military Medical University, Xi＇an 710033, Shaanxi Province, China
Correspondence to:Hong Cheng, Department of Pathology, Fourth Military Medical University, Xi＇an 710033,Shaanxi Province, China
Tel. 0086-29-3577167(H), 3374541 Ext.324(O)
Email. chenghong@fmmu.edu.cn
Received 2001-02-06 Accepted 2001-02-12
Construction and expression of anti-HCC immunotoxin of sFv-TNF-α and GFP fusion proteins
Hong Cheng, Yan-Fang Liu, Hui-Zhong Zhang, Wan-An Shen and Su-Zhen Zhang
Abstract
AIM To study the construction and expression of anti-HCC single-chain bifunctional antibody (sFv-TNF-α) and GFP fusion proteins.
METHODS A single-chain antibody (sFv) gene comprising the linked heavy and light chain variable regions derived from a monoclonal antibody with high specifity and affinity binding to hepatocellular carcinoma (HCC) were first added leader sequence by polymerase chain reaction (PCR) and then fused inframe with human tumor necrosis factor-α (TNF-α) gene. The recombinant sFv-TNF-α gene was cloned into an expression vector pEGFP-N3 containing the report gene of GFP, and the recombinant vector was first identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. To investigate whether mammalian cells could produce fusion protein, NIH3T3 cells were transfected with the plasmid pEGFP-ST by Calcium Phosphate method.
RESULTS The recombinant vector was identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. Bright green fluorescence in the cytoplasm of the transfected NIH3T3 cells was observed under the fluorescence microscope 20 hours after transfection of the recombinant vector.
CONCLUSION The gene of single-chain bifunctional antibody was constructed. The fusion proteins of anti-HCC single-chain bifunctional antibody and GFP were successfully expressed in NIH3T3 cells. It is suggested that mammalian cells can express antibody-cytokine fusion proteins, which may be theoretically necessary for our next step of study in a gene therapy.
Subject headings hepatocellular carcinoma; single-chain antibody; tumor necrosis factor-α; green fluorescent protein; gene expression
Cheng H, Liu YF, Zhang HZ, Shen WA, Zhang SZ. Construction and expression of anti-HCC immunotoxin of sFv-TNF-α and GFP fusion proteins. Shijie Huaren Xiaohua Zazhi, 2001;9(6):640-644
摘要
目的 分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达.
方法 采用PCR方法在抗肝癌sFv的5＇端引入引导序列使其能够在真核细胞中表达并分泌，在其下游连接人TNF-α基因，构建分泌型抗肝癌单链双功能抗体基因，并将该基因克隆入带有GFP报告基因的真核表达载体，用磷酸钙共沉淀法转染小鼠成纤维细胞NIH3T3进行瞬时表达.
结果 DNA序列分析证实在sFv 的5＇端引入正确的60bp引导肽序列, 酶切鉴定证明成功构建了分泌型抗肝癌单链双功能抗体融合GFP真核表达载体，并在NIH3T3细胞中成功地表达了融合荧光蛋白.
结论 成功构建并表达了分泌型抗肝癌单链双功能抗体融合GFP基因，为肝癌免疫基因治疗的进一步研究奠定了基础.
主题词 肝细胞癌;单链抗体(sFv); 肿瘤坏死因子(TNF-α); 绿色荧光蛋白(GFP); 基因表达
程虹, 刘彦仿, 张惠中, 沈万安, 张素珍. 分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达.
世界华人消化杂志，2001；9(6)：640-644
0 引言
肝细胞肝癌(hepatocellular carcinoma, HCC)恶性程度极高，在我国有着较高发病率和死亡率[1-4]，迄今尚无理想的治疗手段[5-14]. 随着现代分子生物学和免疫学的发展，基因工程抗体的诞生为肿瘤的诊断和治疗带来了希望[15-17]，尤其是单链抗体的应用成为肿瘤免疫基因治疗的热点[18,19]. 我室曾成功构建并高效原核表达了抗肝癌sFv，体外免疫细胞化学实验表明，其对肝癌细胞具有明确的反应[20,21]. 我们用PCR方法在该sFv 的5＇端引入引导序列使其能够在真核细胞中表达并分泌，在其下游连接人肿瘤坏死因子(tumour necrosis factor-α, TNF-α)基因，构建分泌型抗肝癌单链双功能抗体基因，并将该基因克隆入带有绿色荧光蛋白(green fluorescent protein，GFP)报告基因的真核表达载体，在小鼠成纤维细胞NIH3T3中瞬时表达sFv-TNFα-GFP融合蛋白，以便灵敏、直观、有效地观察目的基因在真核细胞中的表达，为肝癌免疫基因治疗的进一步研究奠定了基础.
1 材料和方法
1.1 材料 pEGFP-N3质粒(内含GFP基因)由赵利军博士惠赠，抗肝癌sFv基因为本室构建，TNF-α基因由孙志伟博士惠赠. 大肠杆菌JM109，pGEM-T载体和磷酸钙转染试剂盒购自Promega公司；限制性内切酶、T4DNA连接酶、X-gal及IPTG购自Gibco公司；PCR引物由上海生物工程公司合成.
1.2 方法
1.2.1 PCR引物的设计与合成 根据本实验室克隆抗肝癌sFv的cDNA序列，Oligo软件优化设计出上、下游引物，在sFv的5＇端引入酶切位点EcoRⅠ和引导肽基因序列，3＇端引入酶切位点SalⅠ. 由于引导肽基因序列较长，故设计了一对上游引物，通过两轮PCR反应来引入完整引导序列. 根据人TNFα的cDNA序列，Oligo软件优化设计出上、下游引物，两端分别引入SalⅠ和SacⅡ位点. 所有引物均由上海生物工程有限公司合成，序列如下：
SFv下游引物: sFv-1: 5＇CCGTCGACACGT-TTGATCTCGACCTTGG3＇
SFv上游引物: sFv-2: 5＇GTTTTAAAAGGT-GTCCAGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCGG3＇
sFv-3: 5＇CTGAATT-CATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGCCAGTG3＇
引导肽基因序列：ATGAACTTCGGGCT-CAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGCCAGTGT 引自Peter Buckel,1987 (Gene,1987; 51:13-19).
TNFα下游引物: TNFα-1: 5＇TCCCCGCGGCAG-GGCAATGACCCCAAAGTAG3＇
TNFα上游引物: TNFα-2: 5＇CCGTCGACGTCA-GATCATCTTCTCGAAC3＇
1.2.2 sFv和TNF-α基因片段的扩增 以质粒pGEX-sFv为模板，用引物sFv-1和sFv-2进行第1轮PCR反应，15g·L-1琼脂糖凝胶电泳分离PCR产物，低熔点琼脂糖凝胶回收760bp大小的条带，WizardTM Minicolumn 纯化(按试剂盒提供方法进行). 再以纯化的第1轮PCR产物为模板，sFv-1和sFv-3为引物进行第2轮PCR反应，循环结束后，同样方法分离、回收、纯化PCR产物中790bp大小的条带，-20℃保存备用. 以PET-TNF-α为模板，用引物TNFα-1和TNFα-2进行PCR扩增. 15g·L-1琼脂糖凝胶电泳分离PCR产物，低熔点琼脂糖凝胶回收470bp大小的条带，WizardTM Minicolumn纯化. 循环参数为：94℃ 60s，58℃ 60s，72℃ 90s，共30个循环，72℃保温10min.
1.2.3 sFv和TNF-α基因片段的克隆[22]和鉴定 sFv和TNF-α的PCR产物纯化后分别连入pGEM-T载体，连接反应体系为：PCR产物5μL，pGEM-T载体1μL, 10×连接反应缓冲液1μL，T4DNA连接酶1μL, 16℃连接反应16h. 连接产物分别命名为pGEM-sFv和pGEM-TNFα. 参照《分子克隆操作指南》制备JM109感受态宿主菌，冰浴条件下分别加入上述连接产物，冰浴30min， 42℃热休克90s后，迅速冰浴2min,加LB培养液800μL，37℃保温1h，涂于氨苄青霉素抗性的X-gal/LB平板进行蓝白筛选，37℃培养过夜. 分别随机挑取上述连接产物转化的10个白色菌落，扩大培养，提取质粒并分别用pGEM-T载体中外源基因插入位点两侧sacⅡ，pstⅠ双酶切鉴定，酶切产物于15g·L-1琼脂糖凝胶电泳，EB染色，紫外射仪上观察结果.择酶切鉴定符合的克隆作为阳性克隆，Qiagen20 TIP纯化质粒(按Qiagen公司试剂盒说明操作)，用通用引物Dye Primer(M13RP1)(上游：AACAGCTATGACCATG，下游：TGACCGGCAGCAAAATG)进行双向测序.
1.2.4 双功能抗体基因与GFP基因融合表达载体的构建和鉴定 pEGFP-N3质粒用SalⅠ和SacⅡ双酶切，15g·L-1琼脂糖凝胶电泳，低熔点琼脂糖凝胶回收并纯化4.7kb大小的线性化载体片段，pGEM-TNF质粒用SalⅠ和SacⅡ双酶切，15g·L-1琼脂糖凝胶电泳，低熔点琼脂糖凝胶回收470bp大小的TNF-α片段，回收TNF-α片段. 将纯化的TNF-α酶切片段5μL与线性化pEGFP-N3载体片段1μL于16℃连接反应16h，连接产物命名为pEGFP-TNF. 转化JM109感受态宿主菌，涂于卡那霉素抗性的LB平板上，37℃培养过夜. 随机挑取12个单菌落，扩大培养，小量提取质粒，用SalⅠ和SacⅡ双酶切鉴定，15g·L-1琼脂糖凝胶电泳，EB染色，紫外透射仪上观察结果. 选择酶切鉴定的pEGFP-TNF阳性克隆扩大培养，提取质粒，用EcoRⅠ和SalⅠ双酶切并回收5.2kb大小的线性化载体片段，pGEM-sFv用EcoRⅠ和SalⅠ双酶切并回收790bp大小的sFv片段，取载体片段1μL和sFv酶切片段5μL进行连接反应，连接产物命名为pEGFP-ST, 即为单链双功能抗体和GFP融合表达的真核表达载体. 将上述连接产物转化 JM109感受态宿主菌，涂于卡那霉素抗性的LB平板上，37℃培养过夜. 随机挑取12个单菌落，扩大培养，小量提取质粒，用EcoRⅠ和SacⅡ切割sFv-TNF-α片段及SalⅠ和SacⅡ切割TNF-α片段，分别进行双酶切鉴定，15g·L-1琼脂糖凝胶电泳，EB染色，紫外透射仪上观察结果.
1.2.5 重组双功能抗体-GFP融合蛋白的真核表达活化重组pEGFP-ST宿主菌JM109单个菌落，接种于3mL LB培养液中培养过夜. Qiagen20 Tip纯化质粒，溶于30μL无菌水中，-20℃保存备用. 于转染前1d，用2.5g·L-1蛋白酶消化小鼠成纤维细胞NIH3T3，将6×105细胞接种于90mm培养皿中. 转染前4h换10mL含100mL·L-1小牛血清的DMEM培养液，置37℃，50mL·L-1 CO2孵箱培养. 严格按照磷酸钙转染试剂盒说明，将20μg纯化重组pEGFP-ST质粒DNA溶于无菌水中，总体积为438μL，加入2mol·L-1 CaCl2 62μL混匀，缓慢滴入2×HBS 0.5mL中. DNA-磷酸钙共沉淀物置室温30min，逐滴加至接种NIH3T3细胞的培养皿中，置37℃，50mL·L-1 CO2孵箱培养12h，换完全DMEM培养液继续培养12h～24h. 转染后20h，于倒置荧光显微镜下观察活细胞中GFP融合蛋白的表达.
2 结果
2.1 sFv和TNF-α基因的克隆和鉴定 纯化的sFv和TNF-α PCR产物分别与pGEM-T载体连接，转化感受态JM109大肠杆菌，均长出数10个克隆，分别随机挑取10个白色菌落，用pGEM-T载体中外源基因插入位点两侧的sacⅡ，pstⅠ双酶切，pGEM-sFv质粒切下590bp左右和200bp左右的2个片段(sFv基因序列中含有一个pstⅠ酶切位点，符合插入片段sFv的大小)，pGEM-TNF-α质粒切下470bp左右的片段(符合插入片段TNF-α的大小). 将酶切鉴定过的阳性克隆测序，证实sFv的基因序列与已知序列一致，其5＇端引入EcoRⅠ酶切位点及正确的60bp引导肽序列, 3＇端引入SalⅠ酶切位点；TNF-α基因序列与文献报道一致，5＇端引入SalⅠ酶切位点，3＇端引入SacⅡ酶切
位点.
2.2 重组融合蛋白表达载体pEGFP-ST的构建和酶切鉴定 TNF-α片段与线性化的pEGFP-N3载体片段连接，连接产物转化JM109感受态菌，涂卡那霉素抗性平板培养长出数10个克隆，随机挑取的12个克隆均可用SalⅠ和SacⅡ双酶切下470bp左右的片段，符合插入片段TNF-α的大小. sFv片段与相同酶切的pEGFP-TNF-α载体大片段连接，连接产物转化JM109感受态菌，涂卡那霉素抗性平板培养长出数10个克隆， 随机挑取的12个克隆均可用EcoRⅠ和SalⅠ双酶切下790bp左右的片段，符合插入片段sFv的大小，进一步用EcoRⅠ和SacⅡ及SalⅠ和SacⅡ分别双酶切鉴定pEGFP-ST，分别切下1260bp左右及470bp左右的片段，符合sFv-TNF-α及TNF-α片段大小(图1)，证明插入正确，从而获得重组融合蛋白表达载体pEGFP-ST.
图1 pEGFP-ST质粒的酶切鉴定结果.1. SalⅠ酶切空载体pEGFP-N3; 2. EcoRⅠ和SacⅡ酶切pEGFP-ST质粒(1260bp); 3. SalⅠ和SacⅡ酶切pEGFP-ST质粒(470bp); 4. EcoRⅠ和SalⅠ酶切pEGFP-ST质粒(790bp); 5. PCR markers
2.3 细胞内融合蛋白表达 转染后20h倒置荧光显微镜下即可观察到经pEGFP-ST转染的部分细胞发出较强的绿色荧光，散在或成对出现，阳性细胞约为50%. 连续观察发光细胞逐渐增多，约72h达最高，有灶性发光细胞出现(图2).
图2 融合荧光蛋白在小鼠成纤维细胞NIH3T3内瞬时表达结果. ×200
3 讨论
双功能抗体是近几年发展起来的一类基因工程抗体，将抗体基因与一种或几种具有其他生物活性的效应分子(如毒素分子，细胞因子等)基因相连并融合表达，这种融合表达的蛋白不仅具有与抗原特异性结合活性，而且具有效应分子的特殊功能. 双功能基因工程抗体的代表是重组单链免疫毒素[23,24]，它是将特异的sFv基因与毒素基因片段相连构建为重组基因,以表达sFv与毒素的融合蛋白. 1989年Chaudhary et al[25]先将抗卵巢癌的sFv与去除受体结合区的假单孢杆菌外毒素(PE40)基因融合表达单链免疫毒素，使荷瘤裸鼠体内的移植瘤完全消失[26]. 目前已有多种单链免疫毒素基因治疗计划进入临床试验，均采用PE40或切除PE40中部分氨基酸残基及定点突变几个氨基酸残基而获得的毒性更强的PE38和PE38KDEL活性片段，作为双功能抗体中的效应分子[27-29]. 但由于毒素均来源于细菌，且分子质量通常较大，具有较强的免疫原性，因而在临床应用中受到一定的限制[30,31].
肿瘤的细胞因子基因治疗方法近年来倍受关注 ，将细胞因子基因转染至肿瘤细胞并注入体内，使肿瘤局部细胞因子浓度增高，有效地激发和加强局部及全身的免疫功能，避免因外源输入大量细胞因子而带来的一系列毒副作用. 自1989年Rosenberg[32]首次用逆转录病毒载体将TNF-α基因转入人TIL细胞，细胞因子基因治疗开始应用于临床. 常用的细胞因子有IL-2[32,33]、TNF-α[34-39]、IFN[33,40]等. 以细胞因子为效应分子而构建双功能抗体的报道较少[41]，将细胞因子与抗体基因连接并融合表达，融合蛋白除了仍保留肿瘤靶向性外，尚具有细胞因子的功能，可使细胞因子特异性杀伤肿瘤细胞[42-44]，增强抗肿瘤活性，并降低其毒副作用[45]. 1991年Hoogenboom et al[46]首次构建了抗体-TNF-α融合蛋白，将人TNF-α基因融合于肿瘤特异性人-鼠嵌合抗体CH2或CH3下游，并在骨髓瘤细胞中表达，对肿瘤细胞具有杀伤活性，但因分子质量大仍可被人体的免疫系统识别并产生人抗鼠抗体(human anti-mouse antibody，HAMA)，因而限制了它的重复使用[47,48]. 单链抗体是由Fv抗体衍生而来的一类无Fc段的小分子抗体，将抗体重链可变区和轻链可变区通过一段连接肽(linker)连接而成[49,50]，具有天然抗体的亲合力，而分子质量只有完整抗体的六分之一. 其最大特点是分子质量小、穿透力强、容易进入实体瘤周围的血液循环[51]，体内应用具有较大的分布容积和较高的组织分布比例[52,53]，是构建双功能抗体、双特异性抗体等多种新功能抗体分子的理想元件[54]. 同时由于免疫原性低，人体可以重复使用，目前尚未见人体试用产生HAMA报道，因而具有巨大的临床应用潜力[55-58].
我室自1982年先后制备出10余株鼠源性抗肝癌单克隆抗体,并对其中特异性较强、亲和性较高的2株进行了基因工程改造[59-61]，成功构建并高效原核表达了抗肝癌单链抗体. 在此基础上，我们尝试构建分子质量尽可能小，免疫原性尽可能弱的分泌型双功能抗体. 在sFv的5＇端加上引导序列，3＇端连上TNF-α基因，构成分泌型抗肝癌单链双功能抗体基因. 首先采用PCR方法将人工合成的抗体分泌信号肽序列加在抗肝癌sFv基因的N末端，再将sFv基因的C末端与人TNF-α基因连接构成分泌型单链双功能抗体基因，并将该重组基因克隆至带有GFP报告基因的真核表达载体中，观察重组基因在真核细胞中的瞬时表达.
GFP来源于水母Aequorea victoria，由238个氨基酸组成，分子质量27ku. 与其他发光性报告分子不同，GFP不需要其他蛋白质、底物或共同因子的参与，紫外线激发即可产生明亮、稳定的绿色荧光，但只有完整的GFP才能激发出绿色荧光[62]. GFP无种系依赖性，且对宿主细胞无毒性，目前已作为一种新型的报告分子广泛应用于生命科学的众多领域，尤其在基因表达和定位等方面更具优越性[63,64]. 我们所用pEGFP-N3载体中EGFP为一种突变型GFP，其基因编码区含有190多个沉默碱基突变，具有更适合于在人类细胞中表达的开放读框，而且其mRNA翻译效率提高，GFP表达增强，所产生的荧光比野生型GFP增强35倍，因而更适用于检测各类细胞中GFP融合蛋白的表达情况. 我们将构建好的融合GFP表达载体通过磷酸钙介导转染小鼠成纤维细胞NIH3T3，20h后即可观察到转染的部分细胞发出较强的绿色荧光，散在或成对出现，阳性细胞约为50%，72h左右达最高，有灶性发光细胞出现.
我们在NIH3T3细胞中成功地表达了融合荧光蛋白，证实构建的分泌型抗肝癌单链双功能抗体基因可以在真核细胞中有效地表达，为肝癌的免疫基因治疗研究提供了一种灵敏、直观、有效的方法，为今后进一步的研究奠定了基础.
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2中国人民解放军第四军医大学唐都医院骨科 陕西省西安市 710038
程虹，女，1967-06-24生，安徽省宣州市人，汉族. 1996年获第四军医大学病理学博士, 主要从事肝癌的免疫基因治疗研究.
项目负责人 程虹, 710033, 陕西省西安市长乐西路169号，中国人民解放军第四军医大学基础部病理学教研室.
Department of Pathology, Faculty of Preclinical Medicine, Fourth Military Medical University, Xi＇an 710033, Shaanxi Province, China
Correspondence to:Hong Cheng, Department of Pathology, Fourth Military Medical University, Xi＇an 710033,Shaanxi Province, China
Tel. 0086-29-3577167(H), 3374541 Ext.324(O)
Email. chenghong@fmmu.edu.cn
Received 2001-02-06 Accepted 2001-02-12
Construction and expression of anti-HCC immunotoxin of sFv-TNF-α and GFP fusion proteins
Hong Cheng, Yan-Fang Liu, Hui-Zhong Zhang, Wan-An Shen and Su-Zhen Zhang
Abstract
AIM To study the construction and expression of anti-HCC single-chain bifunctional antibody (sFv-TNF-α) and GFP fusion proteins.
METHODS A single-chain antibody (sFv) gene comprising the linked heavy and light chain variable regions derived from a monoclonal antibody with high specifity and affinity binding to hepatocellular carcinoma (HCC) were first added leader sequence by polymerase chain reaction (PCR) and then fused inframe with human tumor necrosis factor-α (TNF-α) gene. The recombinant sFv-TNF-α gene was cloned into an expression vector pEGFP-N3 containing the report gene of GFP, and the recombinant vector was first identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. To investigate whether mammalian cells could produce fusion protein, NIH3T3 cells were transfected with the plasmid pEGFP-ST by Calcium Phosphate method.
RESULTS The recombinant vector was identified with enzyme digestions for the correct insertion and further confirmed by DNA sequence analysis. Bright green fluorescence in the cytoplasm of the transfected NIH3T3 cells was observed under the fluorescence microscope 20 hours after transfection of the recombinant vector.
CONCLUSION The gene of single-chain bifunctional antibody was constructed. The fusion proteins of anti-HCC single-chain bifunctional antibody and GFP were successfully expressed in NIH3T3 cells. It is suggested that mammalian cells can express antibody-cytokine fusion proteins, which may be theoretically necessary for our next step of study in a gene therapy.
Subject headings hepatocellular carcinoma; single-chain antibody; tumor necrosis factor-α; green fluorescent protein; gene expression
Cheng H, Liu YF, Zhang HZ, Shen WA, Zhang SZ. Construction and expression of anti-HCC immunotoxin of sFv-TNF-α and GFP fusion proteins. Shijie Huaren Xiaohua Zazhi, 2001;9(6):640-644
摘要
目的 分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达.
方法 采用PCR方法在抗肝癌sFv的5＇端引入引导序列使其能够在真核细胞中表达并分泌，在其下游连接人TNF-α基因，构建分泌型抗肝癌单链双功能抗体基因，并将该基因克隆入带有GFP报告基因的真核表达载体，用磷酸钙共沉淀法转染小鼠成纤维细胞NIH3T3进行瞬时表达.
结果 DNA序列分析证实在sFv 的5＇端引入正确的60bp引导肽序列, 酶切鉴定证明成功构建了分泌型抗肝癌单链双功能抗体融合GFP真核表达载体，并在NIH3T3细胞中成功地表达了融合荧光蛋白.
结论 成功构建并表达了分泌型抗肝癌单链双功能抗体融合GFP基因，为肝癌免疫基因治疗的进一步研究奠定了基础.
主题词 肝细胞癌;单链抗体(sFv); 肿瘤坏死因子(TNF-α); 绿色荧光蛋白(GFP); 基因表达
程虹, 刘彦仿, 张惠中, 沈万安, 张素珍. 分泌型抗肝癌单链双功能抗体融合GFP真核表达载体的构建及表达.
世界华人消化杂志，2001；9(6)：640-644
0 引言
肝细胞肝癌(hepatocellular carcinoma, HCC)恶性程度极高，在我国有着较高发病率和死亡率[1-4]，迄今尚无理想的治疗手段[5-14]. 随着现代分子生物学和免疫学的发展，基因工程抗体的诞生为肿瘤的诊断和治疗带来了希望[15-17]，尤其是单链抗体的应用成为肿瘤免疫基因治疗的热点[18,19]. 我室曾成功构建并高效原核表达了抗肝癌sFv，体外免疫细胞化学实验表明，其对肝癌细胞具有明确的反应[20,21]. 我们用PCR方法在该sFv 的5＇端引入引导序列使其能够在真核细胞中表达并分泌，在其下游连接人肿瘤坏死因子(tumour necrosis factor-α, TNF-α)基因，构建分泌型抗肝癌单链双功能抗体基因，并将该基因克隆入带有绿色荧光蛋白(green fluorescent protein，GFP)报告基因的真核表达载体，在小鼠成纤维细胞NIH3T3中瞬时表达sFv-TNFα-GFP融合蛋白，以便灵敏、直观、有效地观察目的基因在真核细胞中的表达，为肝癌免疫基因治疗的进一步研究奠定了基础.
1 材料和方法
1.1 材料 pEGFP-N3质粒(内含GFP基因)由赵利军博士惠赠，抗肝癌sFv基因为本室构建，TNF-α基因由孙志伟博士惠赠. 大肠杆菌JM109，pGEM-T载体和磷酸钙转染试剂盒购自Promega公司；限制性内切酶、T4DNA连接酶、X-gal及IPTG购自Gibco公司；PCR引物由上海生物工程公司合成.
1.2 方法
1.2.1 PCR引物的设计与合成 根据本实验室克隆抗肝癌sFv的cDNA序列，Oligo软件优化设计出上、下游引物，在sFv的5＇端引入酶切位点EcoRⅠ和引导肽基因序列，3＇端引入酶切位点SalⅠ. 由于引导肽基因序列较长，故设计了一对上游引物，通过两轮PCR反应来引入完整引导序列. 根据人TNFα的cDNA序列，Oligo软件优化设计出上、下游引物，两端分别引入SalⅠ和SacⅡ位点. 所有引物均由上海生物工程有限公司合成，序列如下：
SFv下游引物: sFv-1: 5＇CCGTCGACACGT-TTGATCTCGACCTTGG3＇
SFv上游引物: sFv-2: 5＇GTTTTAAAAGGT-GTCCAGTGTCCAGTGTGAGGTGCAGCTGGTGGAGTCGG3＇
sFv-3: 5＇CTGAATT-CATGAACTTCGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGCCAGTG3＇
引导肽基因序列：ATGAACTTCGGGCT-CAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGCCAGTGT 引自Peter Buckel,1987 (Gene,1987; 51:13-19).
TNFα下游引物: TNFα-1: 5＇TCCCCGCGGCAG-GGCAATGACCCCAAAGTAG3＇
TNFα上游引物: TNFα-2: 5＇CCGTCGACGTCA-GATCATCTTCTCGAAC3＇
1.2.2 sFv和TNF-α基因片段的扩增 以质粒pGEX-sFv为模板，用引物sFv-1和sFv-2进行第1轮PCR反应，15g·L-1琼脂糖凝胶电泳分离PCR产物，低熔点琼脂糖凝胶回收760bp大小的条带，WizardTM Minicolumn 纯化(按试剂盒提供方法进行). 再以纯化的第1轮PCR产物为模板，sFv-1和sFv-3为引物进行第2轮PCR反应，循环结束后，同样方法分离、回收、纯化PCR产物中790bp大小的条带，-20℃保存备用. 以PET-TNF-α为模板，用引物TNFα-1和TNFα-2进行PCR扩增. 15g·L-1琼脂糖凝胶电泳分离PCR产物，低熔点琼脂糖凝胶回收470bp大小的条带，WizardTM Minicolumn纯化. 循环参数为：94℃ 60s，58℃ 60s，72℃ 90s，共30个循环，72℃保温10min.
1.2.3 sFv和TNF-α基因片段的克隆[22]和鉴定 sFv和TNF-α的PCR产物纯化后分别连入pGEM-T载体，连接反应体系为：PCR产物5μL，pGEM-T载体1μL, 10×连接反应缓冲液1μL，T4DNA连接酶1μL, 16℃连接反应16h. 连接产物分别命名为pGEM-sFv和pGEM-TNFα. 参照《分子克隆操作指南》制备JM109感受态宿主菌，冰浴条件下分别加入上述连接产物，冰浴30min， 42℃热休克90s后，迅速冰浴2min,加LB培养液800μL，37℃保温1h，涂于氨苄青霉素抗性的X-gal/LB平板进行蓝白筛选，37℃培养过夜. 分别随机挑取上述连接产物转化的10个白色菌落，扩大培养，提取质粒并分别用pGEM-T载体中外源基因插入位点两侧sacⅡ，pstⅠ双酶切鉴定，酶切产物于15g·L-1琼脂糖凝胶电泳，EB染色，紫外射仪上观察结果.择酶切鉴定符合的克隆作为阳性克隆，Qiagen20 TIP纯化质粒(按Qiagen公司试剂盒说明操作)，用通用引物Dye Primer(M13RP1)(上游：AACAGCTATGACCATG，下游：TGACCGGCAGCAAAATG)进行双向测序.
1.2.4 双功能抗体基因与GFP基因融合表达载体的构建和鉴定 pEGFP-N3质粒用SalⅠ和SacⅡ双酶切，15g·L-1琼脂糖凝胶电泳，低熔点琼脂糖凝胶回收并纯化4.7kb大小的线性化载体片段，pGEM-TNF质粒用SalⅠ和SacⅡ双酶切，15g·L-1琼脂糖凝胶电泳，低熔点琼脂糖凝胶回收470bp大小的TNF-α片段，回收TNF-α片段. 将纯化的TNF-α酶切片段5μL与线性化pEGFP-N3载体片段1μL于16℃连接反应16h，连接产物命名为pEGFP-TNF. 转化JM109感受态宿主菌，涂于卡那霉素抗性的LB平板上，37℃培养过夜. 随机挑取12个单菌落，扩大培养，小量提取质粒，用SalⅠ和SacⅡ双酶切鉴定，15g·L-1琼脂糖凝胶电泳，EB染色，紫外透射仪上观察结果. 选择酶切鉴定的pEGFP-TNF阳性克隆扩大培养，提取质粒，用EcoRⅠ和SalⅠ双酶切并回收5.2kb大小的线性化载体片段，pGEM-sFv用EcoRⅠ和SalⅠ双酶切并回收790bp大小的sFv片段，取载体片段1μL和sFv酶切片段5μL进行连接反应，连接产物命名为pEGFP-ST, 即为单链双功能抗体和GFP融合表达的真核表达载体. 将上述连接产物转化 JM109感受态宿主菌，涂于卡那霉素抗性的LB平板上，37℃培养过夜. 随机挑取12个单菌落，扩大培养，小量提取质粒，用EcoRⅠ和SacⅡ切割sFv-TNF-α片段及SalⅠ和SacⅡ切割TNF-α片段，分别进行双酶切鉴定，15g·L-1琼脂糖凝胶电泳，EB染色，紫外透射仪上观察结果.
1.2.5 重组双功能抗体-GFP融合蛋白的真核表达活化重组pEGFP-ST宿主菌JM109单个菌落，接种于3mL LB培养液中培养过夜. Qiagen20 Tip纯化质粒，溶于30μL无菌水中，-20℃保存备用. 于转染前1d，用2.5g·L-1蛋白酶消化小鼠成纤维细胞NIH3T3，将6×105细胞接种于90mm培养皿中. 转染前4h换10mL含100mL·L-1小牛血清的DMEM培养液，置37℃，50mL·L-1 CO2孵箱培养. 严格按照磷酸钙转染试剂盒说明，将20μg纯化重组pEGFP-ST质粒DNA溶于无菌水中，总体积为438μL，加入2mol·L-1 CaCl2 62μL混匀，缓慢滴入2×HBS 0.5mL中. DNA-磷酸钙共沉淀物置室温30min，逐滴加至接种NIH3T3细胞的培养皿中，置37℃，50mL·L-1 CO2孵箱培养12h，换完全DMEM培养液继续培养12h～24h. 转染后20h，于倒置荧光显微镜下观察活细胞中GFP融合蛋白的表达.
2 结果
2.1 sFv和TNF-α基因的克隆和鉴定 纯化的sFv和TNF-α PCR产物分别与pGEM-T载体连接，转化感受态JM109大肠杆菌，均长出数10个克隆，分别随机挑取10个白色菌落，用pGEM-T载体中外源基因插入位点两侧的sacⅡ，pstⅠ双酶切，pGEM-sFv质粒切下590bp左右和200bp左右的2个片段(sFv基因序列中含有一个pstⅠ酶切位点，符合插入片段sFv的大小)，pGEM-TNF-α质粒切下470bp左右的片段(符合插入片段TNF-α的大小). 将酶切鉴定过的阳性克隆测序，证实sFv的基因序列与已知序列一致，其5＇端引入EcoRⅠ酶切位点及正确的60bp引导肽序列, 3＇端引入SalⅠ酶切位点；TNF-α基因序列与文献报道一致，5＇端引入SalⅠ酶切位点，3＇端引入SacⅡ酶切
位点.
2.2 重组融合蛋白表达载体pEGFP-ST的构建和酶切鉴定 TNF-α片段与线性化的pEGFP-N3载体片段连接，连接产物转化JM109感受态菌，涂卡那霉素抗性平板培养长出数10个克隆，随机挑取的12个克隆均可用SalⅠ和SacⅡ双酶切下470bp左右的片段，符合插入片段TNF-α的大小. sFv片段与相同酶切的pEGFP-TNF-α载体大片段连接，连接产物转化JM109感受态菌，涂卡那霉素抗性平板培养长出数10个克隆， 随机挑取的12个克隆均可用EcoRⅠ和SalⅠ双酶切下790bp左右的片段，符合插入片段sFv的大小，进一步用EcoRⅠ和SacⅡ及SalⅠ和SacⅡ分别双酶切鉴定pEGFP-ST，分别切下1260bp左右及470bp左右的片段，符合sFv-TNF-α及TNF-α片段大小(图1)，证明插入正确，从而获得重组融合蛋白表达载体pEGFP-ST.
图1 pEGFP-ST质粒的酶切鉴定结果.1. SalⅠ酶切空载体pEGFP-N3; 2. EcoRⅠ和SacⅡ酶切pEGFP-ST质粒(1260bp); 3. SalⅠ和SacⅡ酶切pEGFP-ST质粒(470bp); 4. EcoRⅠ和SalⅠ酶切pEGFP-ST质粒(790bp); 5. PCR markers
2.3 细胞内融合蛋白表达 转染后20h倒置荧光显微镜下即可观察到经pEGFP-ST转染的部分细胞发出较强的绿色荧光，散在或成对出现，阳性细胞约为50%. 连续观察发光细胞逐渐增多，约72h达最高，有灶性发光细胞出现(图2).
图2 融合荧光蛋白在小鼠成纤维细胞NIH3T3内瞬时表达结果. ×200
3 讨论
双功能抗体是近几年发展起来的一类基因工程抗体，将抗体基因与一种或几种具有其他生物活性的效应分子(如毒素分子，细胞因子等)基因相连并融合表达，这种融合表达的蛋白不仅具有与抗原特异性结合活性，而且具有效应分子的特殊功能. 双功能基因工程抗体的代表是重组单链免疫毒素[23,24]，它是将特异的sFv基因与毒素基因片段相连构建为重组基因,以表达sFv与毒素的融合蛋白. 1989年Chaudhary et al[25]先将抗卵巢癌的sFv与去除受体结合区的假单孢杆菌外毒素(PE40)基因融合表达单链免疫毒素，使荷瘤裸鼠体内的移植瘤完全消失[26]. 目前已有多种单链免疫毒素基因治疗计划进入临床试验，均采用PE40或切除PE40中部分氨基酸残基及定点突变几个氨基酸残基而获得的毒性更强的PE38和PE38KDEL活性片段，作为双功能抗体中的效应分子[27-29]. 但由于毒素均来源于细菌，且分子质量通常较大，具有较强的免疫原性，因而在临床应用中受到一定的限制[30,31].
肿瘤的细胞因子基因治疗方法近年来倍受关注 ，将细胞因子基因转染至肿瘤细胞并注入体内，使肿瘤局部细胞因子浓度增高，有效地激发和加强局部及全身的免疫功能，避免因外源输入大量细胞因子而带来的一系列毒副作用. 自1989年Rosenberg[32]首次用逆转录病毒载体将TNF-α基因转入人TIL细胞，细胞因子基因治疗开始应用于临床. 常用的细胞因子有IL-2[32,33]、TNF-α[34-39]、IFN[33,40]等. 以细胞因子为效应分子而构建双功能抗体的报道较少[41]，将细胞因子与抗体基因连接并融合表达，融合蛋白除了仍保留肿瘤靶向性外，尚具有细胞因子的功能，可使细胞因子特异性杀伤肿瘤细胞[42-44]，增强抗肿瘤活性，并降低其毒副作用[45]. 1991年Hoogenboom et al[46]首次构建了抗体-TNF-α融合蛋白，将人TNF-α基因融合于肿瘤特异性人-鼠嵌合抗体CH2或CH3下游，并在骨髓瘤细胞中表达，对肿瘤细胞具有杀伤活性，但因分子质量大仍可被人体的免疫系统识别并产生人抗鼠抗体(human anti-mouse antibody，HAMA)，因而限制了它的重复使用[47,48]. 单链抗体是由Fv抗体衍生而来的一类无Fc段的小分子抗体，将抗体重链可变区和轻链可变区通过一段连接肽(linker)连接而成[49,50]，具有天然抗体的亲合力，而分子质量只有完整抗体的六分之一. 其最大特点是分子质量小、穿透力强、容易进入实体瘤周围的血液循环[51]，体内应用具有较大的分布容积和较高的组织分布比例[52,53]，是构建双功能抗体、双特异性抗体等多种新功能抗体分子的理想元件[54]. 同时由于免疫原性低，人体可以重复使用，目前尚未见人体试用产生HAMA报道，因而具有巨大的临床应用潜力[55-58].
我室自1982年先后制备出10余株鼠源性抗肝癌单克隆抗体,并对其中特异性较强、亲和性较高的2株进行了基因工程改造[59-61]，成功构建并高效原核表达了抗肝癌单链抗体. 在此基础上，我们尝试构建分子质量尽可能小，免疫原性尽可能弱的分泌型双功能抗体. 在sFv的5＇端加上引导序列，3＇端连上TNF-α基因，构成分泌型抗肝癌单链双功能抗体基因. 首先采用PCR方法将人工合成的抗体分泌信号肽序列加在抗肝癌sFv基因的N末端，再将sFv基因的C末端与人TNF-α基因连接构成分泌型单链双功能抗体基因，并将该重组基因克隆至带有GFP报告基因的真核表达载体中，观察重组基因在真核细胞中的瞬时表达.
GFP来源于水母Aequorea victoria，由238个氨基酸组成，分子质量27ku. 与其他发光性报告分子不同，GFP不需要其他蛋白质、底物或共同因子的参与，紫外线激发即可产生明亮、稳定的绿色荧光，但只有完整的GFP才能激发出绿色荧光[62]. GFP无种系依赖性，且对宿主细胞无毒性，目前已作为一种新型的报告分子广泛应用于生命科学的众多领域，尤其在基因表达和定位等方面更具优越性[63,64]. 我们所用pEGFP-N3载体中EGFP为一种突变型GFP，其基因编码区含有190多个沉默碱基突变，具有更适合于在人类细胞中表达的开放读框，而且其mRNA翻译效率提高，GFP表达增强，所产生的荧光比野生型GFP增强35倍，因而更适用于检测各类细胞中GFP融合蛋白的表达情况. 我们将构建好的融合GFP表达载体通过磷酸钙介导转染小鼠成纤维细胞NIH3T3，20h后即可观察到转染的部分细胞发出较强的绿色荧光，散在或成对出现，阳性细胞约为50%，72h左右达最高，有灶性发光细胞出现.
我们在NIH3T3细胞中成功地表达了融合荧光蛋白，证实构建的分泌型抗肝癌单链双功能抗体基因可以在真核细胞中有效地表达，为肝癌的免疫基因治疗研究提供了一种灵敏、直观、有效的方法，为今后进一步的研究奠定了基础.
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