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目的 :克隆人端粒酶逆转录酶 (humantelomerasereversetranscriptasehTERT)基因第 16外显子 ,用于后续研究。方法 :使用PfuDNA聚合酶以PCR法从人外周血白细胞DNA中扩增出包括hTERT第 16外显子的891bpDNA片段 ,连接于 pGEM -Teasyvector ,酶切筛选重组克隆 ,测序证实序列的正确性。结果 :PCR产物电泳证实有符合大小的条带 ,连接及转菌后酶切筛选出的重组克隆经测序证实序列正确性可达 99.9%。结论 :本研究成功克隆了hTERT第 16外显子 ,可用于 3’UTR对hTERT表达调节研究及以hTERT为靶的肿瘤基因治疗研究。
Objective: To clone human exon 16 of human telomerase reverse transcriptase (hTERT) gene for further study. METHODS: The 891 bp DNA fragment including exon 16 of hTERT was amplified by PCR from human peripheral blood leukocytes using Pfu DNA polymerase and ligated to pGEM-Teleasyctor. The recombinant clones were screened by restriction enzyme digestion and sequenced. Results: PCR products were confirmed by electrophoresis. The recombinant clones that were selected by ligation and transfection were proved to be 99.9% correct by sequencing. Conclusion: The hTERT exon 16 was successfully cloned and could be used to study the regulation of hTERT expression by 3’UTR and tumor gene therapy targeting hTERT.