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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′-end transcriptional regulatory sequence of human CD34 gene with specific regulatory activity. METHODS: Two pairs of primers were designed based on the 5′-end promoter regulatory sequence of the known CD34 antigen gene. A 6-1 bp CD34 antigen transcriptional control sequence (CD34TRS) was successfully cloned from chromosomal DNA by nested PCR. CD34TRS The fragment was inserted into the promoter reporter plasmid pEGFP1 to observe the regulatory expression of the recombinant plasmid pCD34EGFP in hematopoietic and nonhematopoietic cells. RESULTS : Restriction endonuclease digestion and DNA sequence analysis confirmed that the cloned CD34 promoter sequence was basically identical to the sequence reported in the literature and could specifically regulate the expression of EGFP gene in hematopoietic cell line K562. Conclusion : The cloned human CD34 differentiation antigen transcriptional regulatory sequence can specifically regulate the expression of eukaryotic genes. This study laid a foundation for the construction of hematopoietic stem cell-specific expression vectors.