利用人的天然抗体抗肿瘤

目的构建含D-氨基酸氧化酶(DAAO)基因的重组逆转录病毒载体，初步观察DAAO基因的功能。方法利用重组DNA技术将DAAOcDNA亚克隆至逆转录病毒载体（pLDAAOSN）中，以磷酸钙沉淀法介导转染包装细胞ΦXNA，用NIH3T3细胞测定病毒滴度，将重组逆转录病毒感染K562白血病细胞，G418筛选出抗性克隆，命名为K

Construction andExpression of Recombinant Retroviral Vector Containing D-Amino Acid Oxidase cDNA

Objective: To construct retroviral vector pLDAAOSN and observethe function of DAAO gene. Methods: With recombinant DNA technology, DAAO cDNA was cloned into retroviral vector (pLDAAOSN). The vector was transfected into ΦXNA cells by CaPO4 method, and the DAAO cDNA was transferred by recombinant retroviral vector into leukemia cell line K562. The positive clones were obtained after G418 selection and termed KDAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in KDAAO. In order to observe the function of DAAO, KDAAO was treated with 50 mm/L D-Ala. Results: Results of plasmid pLDAAOSN digested with KpnⅠ and the sequence determination confirmed pLDAAOSN contains full-length DAAO cDNA. Infectious titer generated by the packaging cells was 5.2×106 CFU/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in KDAAO and expression of DAAO gene at mRNA level. Preliminary observation suggested that D-Ala could effectively kill KDAAO. Conclusion: Retroviral vector pLDAAOSN may be useful for futher study of gene therapy in cancer.