【摘要】 目的克隆正、反义人PIN1基因(hPIN1)及构建hPIN1基因正、反义真核表达载体。方法应用RT-PCR技术从人骨肉瘤细胞系MG-63细胞总RNA中成功扩增出990bp包含hPIN1基因编码区的cDNA序列,按正、反方向加上BamHⅠ及HindⅢ双酶切位点后形成正、反义hPIN1基因,然后连入含BamHⅠ及HindⅢ双酶切位点的pIRES2-EGFP表达质粒上,构建正、反义hPIN1基因的重组真核表达质粒phPIN1。结果扩增的cDNA经测秩序与基因文库完全一致;BamHⅠ及HindⅢ双酶切后,正、反义表达质粒形成5.3+0.99kb两条带,与理论计算值完全一致。结论成功克隆正、反义人PIN1基因及构建hPIN1基因的正、反义真核表达载体。更多还原

【Abstract】 Objective To clone and construct eukaryotic expressing vectors of sense and antisense human PIN1(hPIN1) genes.Methods Total RNA was extracted from MG-63 cells,then the hPIN1 cDNA was amplified by RT-PCR.At the same time the sense and antisense hPIN1 genes were formed by binding BamHⅠand HindⅢ in cis and trans-directions.At the end they were cloned into the eukaryotic expressing vector pIRES2-EGFP in cis and trans directions using DNA recombinant technology.The recombinant vectors were further identified by digestion of BamHⅠ and HindⅢ.Results Sequencing analysis revealed that the orientation of the ligations and the reading frame were correct.After digested by BamHⅠ and HindⅢ,two fragments of(5.3 kb) and(0.898 kb) respectively were formed in sense and antisense eukaryotic expressing vectors.Electrophoretic results were completely coincident with theoretical calculation.Conclusion Human PIN1 sense and antisense genes were successfully cloned and eukaryotic expressing vectors were successfully constructed.更多还原