CN110964747A - 胶质瘤细胞中高效特异表达的自杀基因腺病毒及构建方法 - Google Patents
胶质瘤细胞中高效特异表达的自杀基因腺病毒及构建方法 Download PDFInfo
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Abstract
一种胶质瘤细胞中高效特异表达的自杀基因腺病毒及构建方法，其特征在于，腺病毒通过下述方法构建，使用PCR方法克隆c‑fos基因启动子片段，将其连接到腺病毒表达载体上，在c‑fos启动子下游连接自杀基因及miR‑128互补序列，构建出腺病毒重组表达载体；将腺病毒重组表达载体转染293细胞，待细胞有出毒迹象时，收集病毒上清，利用病毒上清将病毒扩增至P3代，其后用腺病毒纯化试剂盒纯化病毒。本发明通过将miR‑128互补序列放在自杀基因的下游，正常细胞高表达miR‑128，而miR‑128互补序列可沉默miR‑128表达的目的，从而可对自杀基因在正常细胞中的表达发挥抑制效果，而胶质瘤细胞中miR‑128表达缺失，自杀基因可以在胶质瘤细胞中大量转录，从而能够进一步提高自杀基因在胶质瘤细胞中的靶向性，实现了自杀基因在胶质瘤细胞中高效且特性性的表达。
Description
【技术领域】
本发明涉及生物医学领域，特别涉及一种胶质瘤细胞中高效特异表达的自杀基因腺病毒及构建方法。
【背景技术】
神经系统的肿瘤中，胶质瘤的恶性程度是最高的，发病人群主要集中在30-40岁之间的成人及10-20岁之间的青少年。目前，临床对胶质瘤的标准治疗包括手术切除、放疗及化疗。在成人中，患者手术后联合放化、疗的中位生存期只有14.6个月；一部分儿童患者，3年生存率低于20％。脑胶质瘤预后差，严重危害人民生命安全。其浸润性生长特点，与正常脑组织无明显界限，并向周围正常脑组织呈指状浸润破坏，导致传统的治疗方法如手术切除、放疗、化疗等均很难将其根除。近年来，基因治疗被广泛应用于多种肿瘤的治疗研究，为脑胶质瘤的治疗带来曙光。
目前，自杀基因治疗策略已被广泛应用于肿瘤基因治疗研究，具有潜在的重大临床应用价值。单纯疱疹病毒胸苷激酶(herpes simplex virus-thymidine kinase,HSV-TK)基因是肿瘤基因治疗应用最广泛的自杀基因。单纯疱疹病毒胸苷激酶能将一些对哺乳动物细胞无毒性的核苷类似物转化成有毒性的磷酸化物，阻止细胞DNA的正常合成。当HSV-TK基因被导入肿瘤细胞后，联合应用更昔洛韦(ganciclovir,GCV)不仅能杀灭转染的肿瘤细胞，并且能通过旁观者效应杀伤周围没有转染的肿瘤细胞。迄今为止，采用HSV-TK基因转染联合GCV的“自杀基因”疗法已在卵巢癌、恶性黑色素瘤等人类实体细胞肿瘤治疗实验中取得了较好的效果。
自杀基因在肿瘤细胞中高效、特异性的表达对基因治疗的有效性和安全性有着极其重要的影响，也是目前基因治疗所面临的巨大挑战之一。开发和利用肿瘤特异性启动子为解决这一问题提供了一种可行的途径。近年来，已知的可作为肿瘤研究的特异性启动子主要有端粒酶(Tert)启动子、甲胎蛋白(AFP)启动子、癌胚抗原(CEA)启动子以及前列腺特异性抗原启动子等。
虽然目前基于这些肿瘤或组织特异性启动子的靶向基因治疗已经取得了一定的疗效，但由于肿瘤特异性启动子在正常细胞中仍然有少量的表达，限制了基因治疗的给药剂量，为了保障安全，不得不延长给药间隔时间，从而明显降低了基因治疗的疗效。另外，启动子也可能会因体内信号传导的复杂性而逐渐失去活性。因此，为了实现基因治疗早日进入临床试验，需继续寻求更安全、更有效的策略以及高效、靶向性的方法表达自杀基因，从而高效发挥抗胶质瘤的作用。
经过长期研究发现，c-fos启动子在脑胶质瘤中具有较高的转录活性，其相比于其他启动子，具有更好的肿瘤选择性。C-fos是编码核蛋白的一个重要的癌基因，定位于染色体14q23.4，其产物由380个氨基酸残基组成。C-fos基因具有独特的表达特性，表现为在细胞增殖周期的静止期(G0期)基本不表达，而多在细胞周期的(G0～G1)期表达。C-fos癌蛋白在肿瘤细胞核内发挥着重要的作用，可以与Jun蛋白形成异二聚体构成AP-1，Fos蛋白通过使Jun蛋白C末端的磷酸化从而活化AP-1的转录因子活性，进而刺激核内其他细胞增值有关基因的转录，促使细胞由G0期启动而进入细胞增殖周期，导致细胞癌变。研究已经表明，c-fos癌基因与肿瘤的发生发展和预后有关，在胃癌、胶质瘤、宫颈癌、皮肤鳞状细胞癌、子宫内膜癌等肿瘤中c-fos蛋白的表达均有显著增加。在我们前期的研究当中也发现了类似的现象，我们克隆了c-fos基因编码区上游600bp的启动子片段，插入在pGL4载体上，通过其他广泛研究的肿瘤特异性启动子，包括端粒酶(Tert)启动子，Survivin启动子、Cox2启动子、E2F1启动子比较发现：c-fos启动子驱动下的荧光素酶报告基因在胶质瘤细胞系中的转录水平显著高于上述启动子。进一步的动物实验发现，与没有肿瘤选择性的CMV启动子相比，c-fos启动子在正常组织转录活性较低，具有比较高的安全性。
虽然，c-fos启动子在正常组织转录活性较低，但其在正常细胞中(主要是脑胶质细胞，神经元细胞)仍有一定的转录活性，这样就不可避免的出现自杀基因在正常细胞表达，造成毒副作用。
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【发明内容】
本发明旨在解决上述问题，而提供一种在胶质瘤细胞中高效特异表达的自杀基因腺病毒，使得自杀基因可在胶质瘤细胞中高效表达，在正常细胞中不表达，从而避免自杀基因对正常细胞造成毒副作用，提高安全性。此外，还提供一种该腺病毒的构建方法。
为实现上述目的，本发明提供了一种胶质瘤细胞中高效特异表达的自杀基因腺病毒，其特征在于，该腺病毒基因组中包含有c-fos启动子、自杀基因和在正常细胞高表达并在胶质瘤细胞中表达确实的miR-128的互补序列，所述自杀基因位于所述c-fos启动子的下游，所述miR-128的互补序列位于所述自杀基因的下游。
进一步地，所述腺病毒通过下述方法构建：
使用PCR方法克隆c-fos基因启动子片段，将其连接到腺病毒表达载体上，在c-fos启动子下游连接自杀基因及miR-128的互补序列，构建出腺病毒重组表达载体；将腺病毒重组表达载体转染293细胞，待细胞有出毒迹象时，收集病毒上清，并将病毒扩增至P3代，其后用腺病毒纯化试剂盒纯化病毒，以获得所述腺病毒。
进一步地，所述自杀基因为单纯疱疹病毒胸苷激酶HSV-TK，所述腺病毒表达载体为pDC315。
进一步地，所述腺病毒纯化试剂盒选用ViraBind腺病毒纯化试剂盒。
进一步地，所述miR-128的互补序列通过以下方法确定：
使用PCR方法克隆不同片段长度的c-fos启动子片段，设计CMV增强子(IE)序列，优化启动子与多克隆位点(MCS)及表达框(ORF)的距离，设计出高效转录表达的表达框；
将表达框连接到pGL4表达载体上，克隆出pGL4-cfos重组表达载体；
通过miRBase数据库，设计若干条序列不同的miR-128的互补序列，并将其分别串联到pGL4-cfos重组表达载体的3’末端，构建出若干种序列不同的pGL4-cfos-miR128重组表达载体；
利用外源荧光素酶法检测各pGL4-cfos-miR128重组表达载体中的c-fos启动子在胶质瘤细胞和正常细胞中的转录水平，筛选出在胶质瘤细胞中具有较高转录水平而在正常细胞中具有较低转录水平的pGL4-cfos-miR128重组表达载体，将该pGL4-cfos-miR128重组表达载体所对应的miR-128的互补序列作为构建腺病毒重组表达载体所需的miR-128的互补序列。
此外，本发明还提供一种腺病毒的构建方法，其包括以下步骤：
使用PCR方法克隆c-fos基因启动子片段，将其连接到腺病毒表达载体Pdc315上；
在c-fos启动子下游连接自杀基因及miR-128的互补序列，构建出腺病毒重组表达载体；
将腺病毒重组表达载体转染293细胞，待细胞有出毒迹象时，收集病毒上清，并将病毒扩增至P3代，其后用腺病毒纯化试剂盒纯化病毒，以获得所述腺病毒。
进一步地，在c-fos启动子下游连接自杀基因及miR-128的互补序列，构建出腺病毒重组表达载体时，其还可将用于标记的绿色荧光蛋白基因GFP连接在c-fos启动子下游。
进一步地，所述miR-128的互补序列通过以下方法确定：
使用PCR方法克隆不同片段长度的c-fos启动子片段，设计CMV增强子(IE)序列，优化启动子与多克隆位点(MCS)及表达框(ORF)的距离，设计出高效转录表达的表达框；
将表达框连接到pGL4表达载体上，克隆出pGL4-cfos重组表达载体；
通过miRBase数据库，设计若干条序列不同的miR-128的互补序列，并将其分别串联到pGL4-cfos重组表达载体的3’末端，构建出若干种序列不同的pGL4-cfos-miR128重组表达载体；
利用外源荧光素酶法检测各pGL4-cfos-miR128重组表达载体中的c-fos启动子在胶质瘤细胞和正常细胞中的转录水平，筛选出在胶质瘤细胞中具有较高转录水平而在正常细胞中具有较低转录水平的pGL4-cfos-miR128重组表达载体，将该pGL4-cfos-miR128重组表达载体所对应的miR-128的互补序列作为构建腺病毒重组表达载体所需的miR-128的互补序列。
本发明的有益贡献在于，其有效解决了上述问题。本发明通过将miR-128互补序列放在自杀基因的下游，正常细胞高表达miR-128，而miR-128互补序列可沉默miR-128表达的目的，从而可对自杀基因在正常细胞中的表达发挥抑制效果，而胶质瘤细胞中miR-128表达缺失，自杀基因可以在胶质瘤细胞中大量转录，从而能够进一步提高自杀基因在胶质瘤细胞中的靶向性。本发明利用在胶质瘤中有特异性的c-fos启动子驱动自杀基因高水平的表达，同时将microRNA调节机制整合到转录靶向性载体中，相当于为预防自杀基因在非肿瘤组织中的表达上了一道"双保险"，从而实现了自杀基因在胶质瘤细胞中高效且特性性的表达。
【附图说明】
图1肿瘤特异性启动子C-fos、hTERT、Survivin、E2F1、Cox2的pGL4载体：pGL4-cfos、pGL4-TERT、pGL4-Survivin、pGL4-E2F1、pGL4-Cox2重组表达载体的示意图。
图2是pGL4-CMV、pGL4-Tert、pGL4-Survivin、pGL4-E2F1、pGL4-Cox2、pGL4-Vector、pGL4-cfos重组表达载体在胶质瘤细胞中的荧光素酶表达活性检测结果；其中，横坐标表征各重组表达载体，纵坐标表征荧光素酶的表达活性。
图3是pDC315-CFOS-TK-IRES-hrGFP和pDC315-CMV-TK-IRES-hrGFP分别感染胶质瘤细胞系U373的荧光图像。
图4是c-fos/HSV-TK/miR-128腺病毒和CMV/HSV-TK/miR-128腺病毒对U87、U251、U373及正常胶质细胞的杀伤实验结果，其中，横坐标为前体药物更昔洛韦GCV的浓度，纵坐标为细胞存活率。
图5是C-fos和CMV启动子在小鼠体内的转录活性比较分析
【具体实施方式】
下列实施例是对本发明的进一步解释和补充，对本发明不构成任何限制。
一、优化c-fos启动子，并验证c-fos启动子在胶质瘤细胞中的表达效率及特异性：
首先通过PCR方法克隆不同片段长度的c-fos基因启动子片段，设计CMV增强子(IE)序列，优化启动子与多克隆位点(MCS)和表达框(ORF)的距离，设计出高效转录表达的表达框。经测序验证后，将c-fos启动子片段插入到荧光素酶报告基因——pGL4表达载体上，克隆出pGL4-cfos重组表达载体。
作为对比，以相同或相似的方法分别构建出肿瘤特异性启动子CMV、Tert、Survivin、Cox2、E2F1、Vector的pGL4载体：pGL4-CMV重组表达载体、pGL4-Tert重组表达载体、pGL4-Survivin重组表达载体、pGL4-Cox2重组表达载体、pGL4-E2F1重组表达载体、pGL4-Vector重组表达载体(如图1所示)。
其后，将pGL4-CMV重组表达载体、pGL4-Tert重组表达载体、pGL4-Survivin重组表达载体、pGL4-E2F1重组表达载体、pGL4-Cox2重组表达载体、pGL4-Vector重组表达载体、pGL4-cfos重组表达载体分别转入胶质瘤细胞株U87、U251、U373中，并检测荧光素酶的表达活性。如图2所示，通过检测，我们发现c-fos启动子在胶质瘤细胞中的转录活性仅次于CMV启动子，高于Tert、Survivin、E2F1、Cox2、Vector等启动子。
二、c-fos启动子在小鼠体内的转录活性验证
两组小鼠分别以公知方法注射pGL4-CMV重组表达载体和pGL4-cfos重组表达载体。24h后分别取小鼠心、肝、脾、肺、肾进行荧光素酶活性检测，其检测结果如图5所示。
从图5中可以看出，c-fos启动子在小鼠主要脏器中的转录活性远低于CMV启动子。
三、确定microRNA-128互补序列
通过miRBase数据库，设计若干条miR-128的互补序列，将其分别串联到pGL4-cfos重组表达载体的3’末端，构建出若干种具有不同miR-128互补序列的pGL4-cfos-miR128重组表达载体；
利用外源荧光素酶法检测各pGL4-cfos-miR128重组表达载体中的c-fos启动子在胶质瘤细胞和正常细胞(胶质细胞、神经元细胞)中的转录水平，筛选出在胶质瘤细胞中具有较高转录水平而在正常细胞中具有较低转录水平的pGL4-cfos-miR128重组表达载体，以该pGL4-cfos-miR128重组表达载体所对应的miR-128的互补序列作为最优的miR-128互补序列。
四、c-fos/HSV-TK/miR-128自杀基因重组腺病毒载体的制备和病毒包装
使用PCR方法克隆c-fos基因启动子片段，将其连接到腺病毒表达载体pDC315上，在c-fos启动子下游连接自杀基因HSV-TK、miR-128互补序列和用于标记的绿色荧光蛋白基因GFP，构建出包含自杀基因HSV-TK的腺病毒重组表达载体pDC315-CFOS-TK-IRES-hrGFP；
将腺病毒重组表达载体pDC315-CFOS-TK-IRES-hrGFP转染HEK293细胞，待细胞有出毒迹象时，收集病毒上清。将病毒扩增至P3代，其后用ViraBind腺病毒纯化试剂盒纯化病毒，获得纯化浓缩的c-fos/HSV-TK/miR-128腺病毒。
五、c-fos/HSV-TK/miR-128自杀基因重组腺病毒的高效特性表达的验证
通过病毒扩增法使c-fos/HSV-TK/miR-128腺病毒的滴度达到1×10^10PFU/ML；
以不同感染复数(MOI：100,10，1)的c-fos/HSV-TK/miR-128腺病毒分别加入到胶质瘤细胞系U87、U251、U373及正常胶质细胞(购买自ScienCell,Carlsbad,CA)中，并分别在胶质瘤细胞系U87、U251、U373及正常胶质细胞中加入1000μM、100μM、10μM、1μM、0.1μM、0.01μM浓度梯度的前体药物更昔洛韦(ganciclovir，GCV)，分别在48h、72h、96h后通过CCK-8法检测U87、U251、U373及正常胶质细胞的数量，计算U87、U251、U373及正常胶质细胞的存活率(其检测结果如图4所示)。其中，图3示出了pDC315-CFOS-TK-IRES-hrGFP腺病毒感染胶质瘤细胞系U373的荧光图像。
由于实验验证了c-fos启动子在胶质瘤细胞中的转录活性仅次于CMV启动子，高于Tert、Survivin、E2F1、Cox2、Vector等启动子，因此，作为对照组，以同样的方法构建出包含CMV启动子的腺病毒重组表达载体——pDC315-CMV-TK-IRES-hrGFP，并对比研究其对胶质瘤细胞和正常胶质细胞的作用：
将腺病毒重组表达载体pDC315-CMV-TK-IRES-hrGFP转染293细胞，待细胞有出毒迹象时，收集病毒上清，利用病毒上清将病毒扩增至P3代，其后用ViraBind腺病毒纯化试剂盒纯化病毒，获得纯化浓缩的CMV/HSV-TK/miR-128腺病毒。
通过病毒扩增法使CMV/HSV-TK/miR-128腺病毒的滴度达到1×10^10PFU/ML；
以不同感染复数(MOI：100,10，1)的CMV/HSV-TK/miR-128腺病毒分别加入到胶质瘤细胞系U87、U251、U373及正常胶质细胞Astrocytes(购买自ScienCell,Carlsbad,CA)中，然后分别在胶质瘤细胞系U87、U251、U373及正常胶质细胞中加入1000μM、100μM、10μM、1μM、0.1μM、0.01μM浓度梯度的前体药物更昔洛韦(ganciclovir，GCV)，分别在48h、72h、96h后通过CCK-8法检测U87、U251、U373及正常胶质细胞的数量，计算U87、U251、U373及正常胶质细胞的存活率(检测结果见图4)。其中，图3示出了pDC315-CMV-TK-IRES-hrGFP腺病毒感染胶质瘤细胞系U373的荧光图像。
图4中，Ad-CMV代表CMV/HSV-TK/miR-128腺病毒，Ad-CFOS代表c-fos/HSV-TK/miR-128腺病毒。
根据图4可以看出，两种腺病毒都能抑制胶质瘤细胞，但是，CMV/HSV-TK/miR-128腺病毒对正常胶质细胞有杀伤作用，而c-fos/HSV-TK/miR-128腺病毒对正常胶质细胞无明显杀伤作用。
综上所述，c-fos/HSV-TK/miR-128腺病毒在胶质瘤细胞中可高效特异性的表达，其在正常细胞中转录活性低，其可提高自杀基因HSV-TK在胶质瘤细胞中的靶向性，并对自杀基因HSV-TK在正常细胞中的表达进行抑制，本发明的腺病毒对胶质瘤具有高度靶向特异性。
尽管通过以上实施例对本发明进行了揭示，但是本发明的范围并不局限于此，在不偏离本发明构思的条件下，以上各构件可用所属技术领域人员了解的相似或等同元件来替换。
Claims (8)
1.一种胶质瘤细胞中高效特异表达的自杀基因腺病毒，其特征在于，该腺病毒基因组中包含有c-fos启动子、自杀基因和在正常细胞高表达并在胶质瘤细胞中表达确实的miR-128的互补序列，所述自杀基因位于所述c-fos启动子的下游，所述miR-128的互补序列位于所述自杀基因的下游。
2.如权利要求1所述的胶质瘤细胞中高效特异表达的自杀基因腺病毒，其特征在于，所述腺病毒通过下述方法构建：
使用PCR方法克隆c-fos基因启动子片段，将其连接到腺病毒表达载体上，在c-fos启动子下游连接自杀基因及miR-128的互补序列，构建出腺病毒重组表达载体；将腺病毒重组表达载体转染293细胞，待细胞有出毒迹象时，收集病毒上清，并将病毒扩增至P3代，其后用腺病毒纯化试剂盒纯化病毒，以获得所述腺病毒。
3.如权利要求2所述的胶质瘤细胞中高效特异表达的自杀基因腺病毒，其特征在于，所述自杀基因为单纯疱疹病毒胸苷激酶HSV-TK，所述腺病毒表达载体为pDC315。
4.如权利要求3所述的胶质瘤细胞中高效特异表达的腺病毒，其特征在于，所述腺病毒纯化试剂盒选用ViraBind腺病毒纯化试剂盒。
5.如权利要求2所述的胶质瘤细胞中高效特异表达的自杀基因腺病毒，其特征在于，所述miR-128的互补序列通过以下方法确定：
使用PCR方法克隆不同片段长度的c-fos启动子片段，设计CMV增强子(IE)序列，优化启动子与多克隆位点(MCS)及表达框(ORF)的距离，设计出高效转录表达的表达框；
将表达框连接到pGL4表达载体上，克隆出pGL4-cfos重组表达载体；
通过miRBase数据库，设计若干条序列不同的miR-128的互补序列，并将其分别串联到pGL4-cfos重组表达载体的3’末端，构建出若干种序列不同的pGL4-cfos-miR128重组表达载体；
利用外源荧光素酶法检测各pGL4-cfos-miR128重组表达载体中的c-fos启动子在胶质瘤细胞和正常细胞中的转录水平，筛选出在胶质瘤细胞中具有较高转录水平而在正常细胞中具有较低转录水平的pGL4-cfos-miR128重组表达载体，将该pGL4-cfos-miR128重组表达载体所对应的miR-128的互补序列作为构建腺病毒重组表达载体所需的miR-128的互补序列。
6.一种腺病毒的构建方法，其特征在于，其包括以下步骤：
使用PCR方法克隆c-fos基因启动子片段，将其连接到腺病毒表达载体Pdc315上；
在c-fos启动子下游连接自杀基因及miR-128的互补序列，构建出腺病毒重组表达载体；
将腺病毒重组表达载体转染293细胞，待细胞有出毒迹象时，收集病毒上清，并将病毒扩增至P3代，其后用腺病毒纯化试剂盒纯化病毒，以获得所述腺病毒。
7.如权利要求6所述的腺病毒的构建方法，其特征在于，在c-fos启动子下游连接自杀基因及miR-128的互补序列，构建出腺病毒重组表达载体时，其还可将用于标记的绿色荧光蛋白基因GFP连接在c-fos启动子下游。
8.如权利要求6所述的腺病毒的构建方法，其特征在于，所述miR-128的互补序列通过以下方法确定：
使用PCR方法克隆不同片段长度的c-fos启动子片段，设计CMV增强子(IE)序列，优化启动子与多克隆位点(MCS)及表达框(ORF)的距离，设计出高效转录表达的表达框；
将表达框连接到pGL4表达载体上，克隆出pGL4-cfos重组表达载体；
通过miRBase数据库，设计若干条序列不同的miR-128的互补序列，并将其分别串联到pGL4-cfos重组表达载体的3’末端，构建出若干种序列不同的pGL4-cfos-miR128重组表达载体；
利用外源荧光素酶法检测各pGL4-cfos-miR128重组表达载体中的c-fos启动子在胶质瘤细胞和正常细胞中的转录水平，筛选出在胶质瘤细胞中具有较高转录水平而在正常细胞中具有较低转录水平的pGL4-cfos-miR128重组表达载体，将该pGL4-cfos-miR128重组表达载体所对应的miR-128的互补序列作为构建腺病毒重组表达载体所需的miR-128的互补序列。
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