Construction and Expression of Recombinant Retroviral Vector Containing D-Amino Acid Oxidase cDNA / 中国肿瘤生物治疗杂志

ABSTRACT

Objective: To construct retroviral vector pLDAAOSN and observe the function of DAAO gene. Methods: With recombinant DNA technology, DAAO cDNA was cloned into retroviral vector (pLDAAOSN). The vector was transfected into ?XNA 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 K DAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in K DAAO. In order to observe the function of DAAO, K DAAO 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?10 6 CFU/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in K DAAO and expression of DAAO gene at mRNA level. Preliminary observation suggested that D-Ala could effectively kill K DAAO. Conclusion: Retroviral vector pLDAAOSN may be useful for futher study of gene therapy in cancer.