【摘要】 目的:电泳并真核细胞鉴定pcDNA3.0-PTEN真核表达质粒。方法:将含有pcDNA3.0-PTEN真核表达质粒的EcoliJM109细胞挑少许于LB液体培养基上,振荡过夜,离心,碱裂解法裂解细菌,参照UNIQ-10柱式质粒抽提试剂盒操作步骤抽提质粒,电泳鉴定,将获得的pcDNA3.0-PTEN真核表达质粒采用脂质体转染法转入COS-7细胞株,传代培养细胞株,West-Blot鉴定PTEN蛋白质的表达。结果:实验得到的pcDNA3.0-PTEN真核表达质粒大小约为6.0kb,质粒浓度为0.080μg/μl。该质粒目的基因在COS-7细胞株表达产物经West-Blot鉴定证实为目的基因表达产物。结论:pcDNA3.0-PTEN真核表达质粒内含有所需目的基因,该真核表达系统能在真核细胞株COS-7表达所需目的基因产物,可以应用该质粒对PTEN基因缺失或杂合性缺失的肿瘤细胞进行试验性治疗。更多还原

【Abstract】 Objective: Identify the eukaryotic expression plasmid pcDNA3.0-PTEN by electrophoresis and eukaryotic expression. Methods: Develop the E coli JM109 germs 24 hours, which have pcDNA3.0-PTEN eukaryotic expression plasmid, on LB liquid substrate; centrifugation, schizolysis E coli JM109 germs by alkaline lysis proceure. Take out the plasmid by UNIQ-10, identify the plasmid by electrophoresis. Take the plasmid into COS-7 cell line by liposome transfection, develop it, then identify the PTEN protein by West-Blot. Results: The size of pcDNA3.0-PTEN plasmid got by the experiment is 6.0kb. Identical by references. The protein expressed in COS-7 cell line identify by Western-blot is PTEN. Conclusion: pcDNA3.0-PTEN contain the target gene. It can express itself protein in COS-7 cell line. We may use this plasmid for gene therapy on loss or loss of heterozyosity tumors.更多还原