CAJ | 학술논문

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cDNA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cDNA融合克隆。方法：将PCR获得的SCF5’旁侧1．42 kb的调控序列与RT-PCR获得的其1．2 kb的全长cDNA克隆入pGEM-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pUC19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cDNA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。
목적：위연구인간세포인자（SCF）5’방측1.42 kb구역내불동서렬대전장cDNA재진핵세포중표체적조공작용，구건료SCF5’방측1．42 kb적조공서렬여기1．2 kb적전장cDNA융합극륭。방법：장PCR획득적SCF5’방측1．42 kb적조공서렬여RT-PCR획득적기1．2 kb적전장cDNA극륭입pGEM-T재체，사선정학삽입방향，이용합괄적한제성내절매종중절출삼개편단의차아극륭입pUC19극륭재체중。결과：획득료SCF5’방측 1．42 kb적조공서렬여기1．2 kb적전장cDNA병성공지구건료타문적융합극륭。결론：T-재체재극륭첨가료소량구유3’→5’외절핵산매적PCR산물중잉연유효；재초대편단적기인극륭조작중，이용분단아극륭적방법，피개간우령외적매절위점，의차분단아극륭경위가행。
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