修饰hTERT核心启动子靶向转录FCY1的重组腺病毒的构建及鉴定
末端转移酶,,端粒，末端转移酶；启动区(遗传学)；雌激素反应元件；缺氧反应元件；靶向转录；卵巢肿瘤,0引言,1材料和方法,2结果
(第四军医大学西京医院：妇产科，检验科，骨科，陕西 西安 710033)Construction of transcriptional targeting replicationdefective recombinant adenoviruses containing FCY1 driven by modified hTERT core promoter
HUA Wei, XIN XiaoYan, SU MingQuan , LI LiWen
Department of Obstetrics & Gynecology, Department of Clinical Laboratories, Department of Orthopedics, Xijing Hospital, Fourth Military Medical University, Xian 710033 , China
【Abstract】AIM：To construct and identify transcriptional targeting replication-defective recombinant adenoviruses containing FCY1 driven by hTERT core promoter modified by estrogen response elements and hypoxiaresponsive elements. METHODS: The hTERT core promoter modified by tandem sequences of estrogen response elements and hypoxiaresponsive elements were constructed. The promoter sequence and the FCY1 gene fragment were subcloned into shuttle plasmid, then the shuttle plasmid and rescue plasmid were used to cotransfect the HEK 293 cells. Thus the recombinant adenoviruses were obtained. The viral DNA was identified by PCR reaction. After the amplification, the recombinant adenoviral titer was determined by endpoint dilution assay. RESULTS: The sequence of the modified hTERT core promoter was identified by DNA sequence analyses. Typical cytopathic effect (CPE) appeared in all the infected 293 cells within 710 days after cotransfection. PCR reactions showed that the viruses amplified the 122 bp targeted fragment. The recombinant adenoviral titer determined by endpoint dilution assay was 62×107 pfu·mL-1. CONCLUSION: Replicationdefective recombinant adenoviruses containing FCY1 driven by hTERT core promoter modified by tandem sequences of estrogen response elements and hypoxiaresponsive elements are successfully constructed and recombinant viruses of high titer have been obtained. ......
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