人干细胞因子(SCF)5’旁侧调控序列与其全长cDNA融合克隆的构建及鉴定

Construction and identification of the fusion clone of the SCF 5’flanking sequence and its full-length cDNA

目的 :为研究人干细胞因子 (SCF) 5’旁侧 1.42 kb区域内不同序列对全长 c DNA在真核细胞中表达的调控作用 ,构建了 SCF5’旁侧 1.42 kb的调控序列与其 1.2 kb的全长 c DNA融合克隆。方法 :将 PCR获得的 SCF5’旁侧 1.42kb的调控序列与 RT- PCR获得的其 1.2 kb的全长 c DNA克隆入 p GEM- T载体 ,筛选正确插入方向 ,利用合适的限制性内切酶从中切出三个片段依次亚克隆入 p UC19克隆载体中。结果 :获得了 SCF5’旁侧 1.42 kb的调控序列与其 1.2 kb的全长 c DNA并成功地构建了它们的融合克隆。结论 :T-载体在克隆添加了少量具有 3’→ 5’外切核酸酶的 PCR产物中仍然有效 ;在稍大片段的基因克隆操作中 ,利用分段亚克隆的方法 ,避开干扰另外的酶切位点 ,依次分段亚克隆更为可行。

Objective:To study the effect of different length DNA sequences of the 5’SCF 1.42 kb flanking sequence in the expression and regulation of full length cDNA in eukaryotic cells,a fused clone of the 5’SCF 1.42kb flanking sequence and its full length cDNA were constructed.Methods:A 1.42 kb flanking sequence and a 1.2 kb full length cDNA were achieved by PCR from human genomic DNA and by RT PCR from HepG2 mPNA,respectively,and then cloned into pGEM T cloning vector and identified.Both clones were digested with fitly restricted endonuclease and three DNA fragments,480 bp,980 bp,1.2 kb cDNA were harvested.Finally,these three DNA fragments were subcloned into pUC19 cloning vector in turn.Results:The fused clone of 5’SCF 1.42 kb flanking sequence and its full length cDNA were successfully constructed.Conclusion:To avoid the interruption of some restricted endonuclease sites,the way to cut larger fragments into smaller fragments is still useful and effective in the process of gene cloning.