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目的:构建重组人BAG3(WT)与BAG3(ΔPXXP)的真核表达载体,建立稳定表达EGFP-BAG3(WT)、EGFP-BAG3(ΔPXXP)的人甲状腺癌FRO细胞系。方法:基因克隆方法构建pcDNA3-N-EGFP-BAG3(WT)和pcDNA3-N-EGFP-BAG3(ΔPXXP)的真核表达载体,应用脂质体介导的转染技术将重组质粒导入FRO细胞,用G418筛选稳定表达的细胞系。FRO细胞中EGFP-BAG3(WT)、EGFP-BAG3(ΔPXXP)的表达用倒置荧光显微镜方法检测。结果:分别成功构建pcDNA3-N-EGFP-BAG3(WT)和pcDNA3-N-EGFP-BAG3(ΔPXXP)重组质粒,获得转染并经G418反复筛选的稳定表达EGFP-BAG3(WT)、EGFP-BAG3(ΔPXXP)的FRO细胞系。FRO细胞均能表达绿色荧光,EGFP-BAG3(WT)主要分布在细胞质内,而EGFP-BAG3(ΔPXXP)在细胞质和细胞核内均有分布。结论:成功构建了BAG3(WT)与BAG3(ΔPXXP)真核表达载体。
OBJECTIVE: To construct eukaryotic expression vector of recombinant human BAG3 (WT) and BAG3 (ΔPXXP) and to establish a human thyroid carcinoma FRO cell line stably expressing EGFP-BAG3 (WT) and EGFP-BAG3 (ΔPXXP). METHODS: The eukaryotic expression vector pcDNA3-N-EGFP-BAG3 (WT) and pcDNA3-N-EGFP-BAG3 (ΔPXXP) was constructed by gene cloning method. The recombinant plasmid was introduced into FRO cells by liposome- Stably expressing cell lines were screened with G418. The expression of EGFP-BAG3 (WT), EGFP-BAG3 (ΔPXXP) in FRO cells was detected by inverted fluorescence microscopy. Results: The recombinant plasmids pcDNA3-N-EGFP-BAG3 (WT) and pcDNA3-N-EGFP-BAG3 (ΔPXXP) were successfully constructed and transfected and EGFP-BAG3 (ΔPXXP) FRO cell line. FRO cells were able to express green fluorescence, EGFP-BAG3 (WT) mainly distributed in the cytoplasm, and EGFP-BAG3 (ΔPXXP) in the cytoplasm and nucleus were distributed. Conclusion: Eukaryotic expression vectors of BAG3 (WT) and BAG3 (ΔPXXP) were successfully constructed.