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目的:构建小鼠p53基因真核表达质粒PLJM1-p53-GFP,研究其对前列腺癌细胞株PC3侵袭、增殖能力的影响。方法:从小鼠3T3-L1细胞提取总RNA,经RT-PCR获得cDNA,扩增p53基因,经酶切纯化后的产物与双酶切后的PLJM1-NRG1-GFP慢病毒载体连接,得到PLJM1-p53-GFP慢病毒载体并转化感受态细菌DH5α,通过PCR筛选阳性克隆,抽提质粒并测序鉴定。将PLJM1-p53-GFP质粒瞬时转染到PC3细胞中,提取RNA,定量PCR鉴定p53高表达水平,采用细胞侵袭实验观察高表达p53的前列腺癌细胞株PC3的侵袭能力,采用流式细胞及划痕试验研究p53对前列腺癌细胞株PC3增殖活性的影响。结果:测序证实,目的基因p53插入完全正确且无任何突变;高表达p53的PC3细胞的侵袭能力、增殖能力明显下降。结论:成功构建小鼠p53基因真核表达质粒PLJM1-p53-GFP,为深入研究肿瘤等相关疾病提供了有效的工具。
OBJECTIVE: To construct a mouse p53 gene eukaryotic expression plasmid PLJM1-p53-GFP and study its effect on the invasion and proliferation of prostate cancer cell line PC3. METHODS: The total RNA was extracted from mouse 3T3-L1 cells, and the cDNA was amplified by RT-PCR. The p53 gene was amplified and ligated with PLJM1-NRG1-GFP lentiviral vector. The PLJM1- p53-GFP lentivirus vector and transformed into competent bacteria DH5α, positive clones were screened by PCR, plasmid was extracted and identified by sequencing. The plasmids of PLJM1-p53-GFP were transiently transfected into PC3 cells, RNA was extracted and the high expression level of p53 was identified by quantitative PCR. The invasiveness of prostate cancer cell line PC3 with high expression of p53 was observed by cell invasion assay. The Effect of p53 on the Proliferation of Prostate Cancer Cell Line PC3. Results: Sequencing confirmed that p53 gene was inserted correctly and without any mutation. The invasiveness and proliferation of PC3 cells with high expression of p53 were significantly decreased. Conclusion: The mouse p53 gene eukaryotic expression plasmid PLJM1-p53-GFP was successfully constructed, which provided an effective tool for further study of tumor-related diseases.