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目的 探讨逆转录病毒介导的红色酵母D 氨基酸氧化酶 (DAAO)基因在K5 6 2细胞中的表达及其功能。方法 将DAAOcDNA克隆至逆转录病毒载体pLSN中 ,构建了载体pLDAAOSN ,经ΦNXA细胞包装后 ,用NIH3T3细胞测定病毒滴度 ,以重组逆转录病毒感染K5 6 2白血病细胞 ,G418筛选出抗性克隆 ,命名为KDAAO 。PCR、原位杂交分析外源基因整合和表达 ,并以不同浓度的D 丙氨酸 (D Ala)处理KDAAO 细胞。结果 重组逆转录病毒载体中含有完整的DAAO基因。包装细胞产生了高滴度病毒 (5 .2× 10 6cfu ml)。DAAO基因已整合至KDAAO细胞基因组中 ,并在mRNA水平表达。D Ala能明显杀伤KDAAO 细胞。结论 DAAO D Ala自杀基因系统可以进一步用于肿瘤的基因治疗研究。
Objective To investigate the expression and function of retroviral-mediated red yeast amino acid oxidase (DAAO) gene in K562 cells. Methods The DAAO cDNA was cloned into the retroviral vector pLSN and the vector pLDAAOSN was constructed. After being packed in ΦNXA cells, the titer of the virus was measured using NIH3T3 cells. The recombinant retrovirus was used to infect K562 leukemic cells and a resistant clone was selected by G418. Named KDAAO. PCR and in situ hybridization analysis of foreign gene integration and expression, and treatment of KDAAO cells with different concentrations of D-alanine (D Ala). Results The recombinant DAAO gene was contained in the recombinant retroviral vector. The packaging cells produced high titer virus (5.2 x 10 6 cfu ml). The DAAO gene has been integrated into the KDAAO cell genome and expressed at the mRNA level. D Ala can significantly kill KDAAO cells. Conclusion The DAAO D Ala suicide gene system can be further used in gene therapy research of tumors.