人CD34分化抗原转录调控序列的克隆与调控表达

Cloning and Regulating Expression of Human CD34 TRS

目的 :克隆有特异调控活性的人CD34基因 5′端转录调控序列。方法 :根据已知的CD34抗原基因 5′ 端启动子调控序列 ,自行设计 2对引物 ,用巢式 PCR方法从染色体DNA中成功的克隆 6 6 1bp长度的CD34抗原转录调控序列 (CD34TRS) ,CD34TRS片段插入启动子报告质粒 pEGFP 1,观察重组质粒pCD34EGFP在造血系细胞和非造血系细胞中的调控表达作用。结果 :经限制性内切酶酶切鉴定及DNA序列分析证实 ,克隆的CD34启动子序列与文献报道的顺序基本一致 ,能特异调控EGFP基因在造血细胞系细胞K5 6 2中表达。结论 :所克隆的人CD34分化抗原转录调控序列有特异调控真核基因表达作用 ,本研究为构建造血干细胞特异性表达载体奠定了基础。

Objective: To clone the 5′ flanking region of the human CD34 gene containing transcriptional regulatory sequence (TRS). Methods: According to the registered 5′ flanking region of CD34 gene, two pairs of primers were designed and net PCR was used to amplify 661 bp long TRS of CD34 gene. The CD34 TRS fragment was cloned into reported plasmid pEGFP 1. The role of the regulating the specific expression of recombinant plasmid pCD34 EGFP in hematopoietic and non hematopoietic cells was observed. Results: Restrictive endonuclease identification and DNA sequencing proved that the CD34 promoter cloned was consistent with the sequence reported to a large extent. It could induce the EGFP gene to express in hematopoietic cell line K562 specifically,while has no effect on hepatocellular carcinoma cell HepG 2. Conclusion: The cloned CD34 gene TRS has the effect of regulating gene expression specifically. The study established the fundament for the construction of specific gene expression vector used in hematopoietic system cells.