摘要
目的:运用分子生物学技术扩增CD、TK基因,进行其DNA序列分析,并构建真核表达质粒pIRES-CD及pIRES-TK,将其共同转染ACC-2细胞,为进一步研究双自杀基因对ACC-2细胞杀伤作用奠定基础。方法:根据CD和TK基因DNA的序列分别设计、合成引物,构建克隆载体pMD18-T-CD和pMD18-T-TK,并进行DNA序列分析;构建以内部核糖体进入位点(IRES)相连的CD和TK基因的真核表达质粒pIRES-CD及pIRES-TK,用电穿孔法以真核表达质粒共同转染ACC-2细胞,用RT-PCR检测CD和TK的表达。结果:克隆DNA片段和GeneBank上报道的CD、TK序列基本一致,且经酶切鉴定,CD、TK基因成功插入真核表达质粒IRES,RT-PCR检测表明,以真核表达质粒pIRES-CD及pIRES-TK共同转染的ACC-2细胞能表达CD和TK。结论:成功扩增了CD、TK的DNA片段,并进行了序列分析;成功构建CD和TK基因的重组真核表达质粒pIRES-CD及pIRES-TK,将其转染ACC-2细胞后能分泌性表达CD和TK,为肿瘤基因治疗的研究提供一定途径。
Objective： To amplify the CD and TK gene fragment and analyze the DNA sequence, and then to construct eukaryotic expression plasmids pIRES-CD and pIRES-TK and determine their expression in ACC-2 cells. Methods： The primers of CD and TK were designed and composed at the base of the CD and TK＇s DNA sequence. CD and TK genes were cloned by PCR and then the eukaryotic expression plasmids pIRES-CD and pIRES-TK were constructed and transfected into ACC-2 cells by electroporation. The transfection and transcription of CD and TK were successfully demonstrated by RT-PCR. Results： The cloned CD and TK gene segments accorded with those reported in the GeneBank. The recombination expression vector contained CD and TK genes by enzyme restriction. RT-PCR analysis demonstrated that CD and TK genes effectively expressed in ACC-2 cells. Condusion： The CD and TK genes are successfully amplified and the sequence is analyzed. The pIRES-CD and pIRES-TK expression plasmids are successfully constructed and expressed in ACC-2 cells, which offers a pathway for research on gene therapy of tumors.
出处
《山东大学学报（医学版）》
CAS
北大核心
2007年第2期117-123,共7页
Journal of Shandong University:Health Sciences
基金
山东省自然科学基金资助课题(Z2003C03)
