正、反义hTERT基因真核表达载体的构建与鉴定  

Construction and identification of eukaryotic expression vector for sense and antisense human telomerase reverse transcriptase

作　　者：孙来保[1] 李成荣[1] 文剑明[2] 张萌[2] 

机构地区：[1]深圳市儿童医院,广东深圳518026 [2]中山大学医学院病理教研室,广东广州510089

出　　处：《第一军医大学学报》2003年第9期899-902,共4页Journal of First Military Medical University

基　　金：深圳市卫生科技计划基金资助项目(199805021)~~

摘　　要：目的构建正、反义hTERT基因真核表达载体,为进一步研究端粒酶的活性调节与恶性肿瘤基因治疗作准备。方法根据已发表在Genebank的hTERT cDNA序列,设计并合成了1对两端带特定限制性酶切位点引物,提取子宫颈癌HeLa细胞株总RNA,进行RT-PCR,并将扩增产物TA首先克隆至pGEMR-T载体,然后双酶切pGEM-T载体回收目的片段再克隆至真核表达质粒pcDNA3.1(±)中,并对重组体进行酶切鉴定和测序分析。结果PCR扩增片段与预期结果相符,双酶切证实构建的正、反义hTERT基因真核表达载体克隆成功,插入片段测序结果与Genebank的hTERT cDNA的部分序列完全一致。结论成功地构建了正、反义hTERT基因真核表达载体,为下一步研究端粒酶的活性调节与恶性肿瘤基因治疗打下了实验基础。Objective To construct eukaryotic expression vectors for sense and antisense human telomerase reverse transcriptase (hTERT), to facilitate further study of the regulation of telomerase activity and gene therapy of malignant tumors. Methods According to the published hTERT cDNA sequence in Genbank, a pair of primers containing the sites for given restrictive endonuclease at both ends were designed and synthesized. Reverse transcriptional PCR (RT-PCR) of the total RNA extracted from HeLa cell line was performed, the product of which was cloned into pGEM-T vector by using TA cloning and then subcloned into a highly efficient eukaryotic expression vector pcDNA3.1 (±). The recombinants were finally sequenced and identified by restrictive endonuclease digestion. Results A 210-bp DNA fragment was amplified by PCR as expected. The sense and antisense hTERT eukaryotic expression vector were successfully constructed and identified by double restrictive endonuclease digestion. Sequence analysis of the inserted target fragment revealed the same sequence as that of partial hTERT cDNA published in Genbank. Conclusion The sense and antisense hTERT eukaryotic expression vector has been successfully constructed.

关 键 词：恶性肿瘤 HTERT基因 真核表达载体 构建 鉴定 基因治疗 

分 类 号：R73-36[医药卫生—肿瘤]