杂合启动子HREAF的构建及其肝癌特异性分析

本研究将在几乎所有肝癌细胞中均有高特异性的杂合启动子HREAF用于构建肝癌特异性溶瘤腺病毒。根据献报道的缺氧应答元件(HRE)和人甲胎蛋白(AFP)核心启动子(AF0.3)基因序列，设计并合成2对引物，采用PCR方法从肝癌细胞基因组DNA中扩增获得大小分别约为560bp的HRE DNA片段和310bp的AF0.3DNA片段，连接到pGEM—T Easy载体进行测序，证明获得了HRE和AF0.3的基因片段。将HRE和AF0．3的PCR产物分别进行PstI和SspI酶切并末端补平后直接连接，将此约700bp的连接产物克隆到pGEM—T Easy载体进行测序，证明杂合启动子HREAF构建成功。将HREAF亚克隆至腺病毒穿梭载体pShuttle并在其后克隆入受其调控的腺病毒早期基因E1，构建成肝癌特异性溶瘤腺病毒穿梭载体pShuttle—HREAF—E1，经PCR及酶切鉴定。将pShuttle—HREAF—E1转染肺癌细胞株及AFP产量不等的不同人肝癌细胞株，经Ela的RT—PCR检测证实杂合启动子HREAF可调控目的基因在AFP产量不等的不同人肝癌细胞系中特异性高表达。杂合启动子HREAF及腺病毒穿梭载体pShuttle—HREAF—E1的构建为进一步研制肝癌特异性重组溶瘤腺病毒及其体内外肝癌特异杀伤作用的研究打下了基础。

Construction of hybrid promoter HREAF and the study of its specificity to hepatocellular carcinoma

In this study, hybrid promoter HREAF, which has high specificity to nearly all hepatocellular carcinoma, was developed to construct a hepatocellular carcinoma selectively oncolytic Adenovirus. Two pair primers was designed and synthesized according to reported gene sequences of the hypoxia responsive element(HER) and human a-fetoprotein(AFP) core promoter(AF0.3). And two DNA fragments with the size of 560bp(HRE) and 310bp(AF0.3) were successfully amplified by PCR from hepatocelluar carcinoma genome DNA. The target genes ligated into pGEM-T easy vector were sequenced and proved that gene fragments of HRE and AF0.3 have been acquired. PCR products of HRE and AF0.3 were separately digested by PstI or SspI, and directly ligated in vitro after smoothing the ends. This ligated products cloned into pGEM-T easy vector were sequenced and proved the successful construction of the hybrid promoter HREAF. A hepatocellular carcinoma selectively oncolytic adenovirus shuttle plasmid pShuttle-HREAF-E1 was constructed by subcloned HREAF into adenovirus shuttle vector and then it followed with cloned adenovirus early gene E1, to be under the control of HREAF. And this plasmid was identified by PCR method and restriction endonucleases digestion. The specificity of HREAF to hepatocellular carcinoma was verified by RT-PCR of E1a after transfected the pShuttle-HREAF-E1 into the hepatocellular carcinoma cells with different amount of AFP products and lung cancer cells. The construction of HREAF and pShuttle-HREAF-E1 established the ground for the next study of construction of hepatocellular carcinoma selectively oncolytic adenovirus and its treatment of hepatocellular carcinoma in vitro and in vivo.