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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。
Objective: Electrophoresis and eukaryotic cell identification of pcDNA3.0-PTEN eukaryotic expression plasmid. Methods: EcoliJM109 cells containing pcDNA3.0-PTEN eukaryotic expression plasmid were picked on LB liquid medium. After shaking overnight, the bacteria were lysed by centrifugation and alkaline lysis. According to the operating procedure of UNIQ-10 column plasmid extraction kit The plasmid pcDNA3.0-PTEN was transfected into COS-7 cells by lipofection method. The cell lines were subcultured and the expression of PTEN protein was identified by West-Blot. Results: The eukaryotic expression plasmid pcDNA3.0-PTEN was about 6.0 kb in size and the plasmid concentration was 0.080 μg / μl. The expression product of the target gene of the plasmid in COS-7 cell line was confirmed by West-Blot identification as the target gene expression product. CONCLUSION: The pcDNA3.0-PTEN eukaryotic expression plasmid contains the desired gene. The eukaryotic expression system can express the desired gene product in the eukaryotic cell line COS-7. The eukaryotic expression system can be used for the deletion or hybridization of the PTEN gene Lack of tumor cells for experimental treatment.