p53基因真核表达质粒PLJM1-p53-GFP构建及其应用

Construction and application of mouse PLJM1-p53-GFP plasmid

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 PLJM1-p53-GFP plasmid of mouse p53 gene and study the role of p53 in the prostate cancer cells PC3.Methods：The open reading frame（ORF） of mouse p53 gene was amplified from mouse 3T3-L1 cells by RT-PCR,and inserted into the PLJM1-NRG1-GFP vector.The recombinant plasmid was transformed into DH5α competent E.coli.The positive clones were screened by PCR and the inserts were confirmed by DNA sequencing.The PLJM1-p53-GFP plasmid was transfected into the prostate cancer cells PC3.QPCR was used to detect the efficiency of the transfection.The invasive activity and proliferation of PC3 cells were measured after transfecting the PLJM1-p53-GFP plasmid.Results：DNA sequencing demonstrated that the recombinant plasmid PLJM1-p53-GFP was constructed successfully.The invasive activity and proliferation of PC3 cells were obviously decreased after transfection of the PLJM1-p53-GFP plasmid.Conclusion：The plasmid of PLJM1-p53-GFP of mouse p53 gene was successfully constructed.It can be used in the functional research for cancer.