作　　者： ; ; ; ;

机构地区： 暨南大学医学院血液病研究所

出　　处： 《暨南大学学报（自然科学与医学版）》 2008年第2期121-124,共4页

摘　　要： 目的:构建胰腺十二指肠同源框蛋白1(pancreatic and duodenal homeobox factor1,Pdx1)转录因子真核表达质粒,并检测其在真核细胞中的表达能力及其生物活性。方法:以人胚胎胰腺组织基因组的mRNA为模板经RT-PCR扩增获得Pdx1基因编码序列,克隆到真核表达载体pEGFP-N1的多克隆位点中,构建pEGFP-N1/Pdx1真核表达质粒,并转染L02细胞,用RT-PCR、免疫组化、间接荧光法和Western Blot检测目的基因表达情况。结果:酶切与测序结果表明目的基因序列克隆正确;RT-PCR检测目的基因Pdx1 mRNA在靶细胞L02中表达;免疫组化和间接荧光结果证实Pdx1主要存在于细胞核内;Western blot检测到细胞核内Pdx1目的蛋白。结论:完成人Pdx1基因克隆,成功构建了目的基因Pdx1真核表达载体,并在L02细胞中获得有效表达。 Aim： To construct recombinant eukaryotic expression vector of Pdxl transcription factor and detect its expression in eukaryocyte. Methods： We obtained the Pdxl gene exon by using human embryo pancreas tissue mRNA as the template, and cloned it into multiple cloning sites of eukaryotic expression vector pEGFP-N1 to construct pEGFP-N1/Pdxl eukaryotie expression plasmid and the constructed plasmid was transfected L02 ceil. The expression in transfected ceils was detected by RT-PCR immunocytochemistry, indirect fluorescence assay and Western Blot respectively. Results： Restriction endonucleas and sequence analysis verified that the fragment cloned in pEGFP-N1 vector was Pdxl cDNA. In the supematant of L02 ceils transfected by the pEGFP-N1/Pdxl plasmid. RT-PCR verified that Pdxl mRNA was positive in L02 ceils, the expression of PDX1 protein in the nuclears of L02 ceils was detected by indirect fluorescence assay and immunohistochemistry, and about 48KDa protein was detected by Western blot in the cellular nucleus of L02 ceils. Conclusion： Eukaryotic expression plasmid of Pdxl gene （pEGFP-N1/Pdxl） is successfully constructed and expressed effectively in L02 ceils.

关 键 词： 胰腺十二指肠同源框蛋白 质粒 转录因子 内切酶

领　　域： [生物学]