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为研究P~(53),mdm2基因在肿瘤细胞中的表达与调控作用,将克隆于质粒载体中的P~(53),mdm2和neo基因分别用双酶切得到P~(53),mdm2,cDNA和neo基因片段,采用地高辛随机引物法标记探针,用于转染含P~(53)和mdm2基因的肺腺癌GLC-82细胞中DNA和RNA斑点杂交,以及肿瘤细胞中P~(53)和mdm2基因的检测。结果显示,以neo基因为探针的DNA斑点杂交中转染P~(53)和mdm2的细胞出现阳性信号,GLC-82亲本细胞为阴性,表明转染成功。以P~(53),mdm2,cDNA为探针的RNA斑点杂交中转染P~(53)和mdm2的细胞出现阳性信号,提示P~(53)和mdm2在细胞中获得表达。地高辛标记探针具有简单、快速、特异性强、灵敏度高、无污染等特点,有推广应用价值。
In order to study the expression and regulation of P53 in mdm2 gene in tumor cells, the P53, mdm2 and neo genes cloned in the plasmid vector were digested with double enzymes respectively to obtain P53 and mdm2. , cDNA and neo gene fragments were labeled with digoxigenin random primers for DNA and RNA dot blot hybridization in lung adenocarcinoma GLC-82 cells containing P53 and mdm2 genes, and in tumor cells. Detection of P53 and mdm2 genes. The results showed that cells transfected with P53 and mdm2 with a neo gene as a probe showed a positive signal, and GLC-82 parental cells were negative, indicating successful transfection. Positive signals were detected in cells transfected with P53 and mdm2 by RNA blot hybridization with P53, mdm2, and cDNA as probes, suggesting that P53 and mdm2 were expressed in cells. The digoxigenin labeled probe has the characteristics of simplicity, rapidity, specificity, high sensitivity, no pollution, etc. It has the value of popularization and application.