hPARP-1基因cDNA翻译起始区域的T载体克隆

The translational initiation region of hPARP-1 gene cDNA cloning by T vector

目的 克隆及构建含限制性内切酶位点BamHⅠ、SacⅠ的人聚腺苷二磷酸核糖聚合酶 1(hPARP 1)基因cDNA翻译起始区域的pGEM T S载体 ,为以后的亚克隆和毒理学研究提供实验材料。方法 采用RT PCR方法 ,用正常人胚肺成纤维细胞 (HLF)抽提的总RNA逆转录成cDNA ,再以cDNA为模板 ,扩增hPARP 1基因片段 ,并直接与T载体连接后转化大肠埃希菌DH5α ,用蓝 (白 )斑试验筛选出阳性克隆 ,抽提重组质粒并进行PCR及酶切鉴定 ,再行序列分析。结果 经RT PCR获得 5 0 7bp含限制性内切酶位点的阳性产物 ,T载体克隆、PCR及酶切鉴定和序列分析后证实 ,克隆片段与Genbank中该基因的序列同源性为 99 9%。结论 该实验成功地构建了含hPARP 1基因cDNA翻译起始区域的T载体克隆 。

Objective To clone and construct the pGEM-T-S vector containing the translational initiation region of poly(ADP-ribose)polymerase(hPARP-1) gene cDNA with two recongnition sites for the enzymes BamH Ⅰ and Sac Ⅰ,then compare its sequence with Genbank and provide experimental material for subclone and toxicological study.Methods Following to total RNA being extracted from human embryo lung fibroblast and RT-PCR being carried out,T vector was linked with PCR products.After E.coil DH5α was transformed with recombitants and screened with blue/white blot test for positive clones from which plasmide were then extracted,the recombinants were identified by PCR, restriction analysis and sequencing.Results 507 base pairs fragment contraining restriction sites was obtained through RT-PCR.After colning by T vector and being identified by restriction analysis and sequening,it was confirmed that the object fragment had 99^9% homology with hPARP-1 sequence in Genbank.Conclusion The T vector clone with the translational initiation region of hPARP-1 gene cDNA has been constructed successfully.This clone can be used in research subclone and PARP-1 gene deficiency cell.