D-氨基酸氧化酶基因重组逆转录病毒载体的构建及表达  

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

作　　者：翟勇平[1] 王健民[1] 周虹[1] 张雨生[1] 

机构地区：[1]第二军医大学附属长海医院血液科,上海200433

出　　处：《中国肿瘤生物治疗杂志》2001年第1期52-54,共3页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金重点项目!(3 9870 710 )

摘　　要：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 ,观察 5 0mm/LD 丙氨酸对KDAAO杀伤作用。结果 :经酶切鉴定和DNA测序证明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)。PCR、原位杂交分析证明DAAO基因已整合至KDAAO基因组中 ,并在mRNA水平表达。初步观察到D 丙氨酸能明显杀伤KDAAO。结论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 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 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.

关 键 词：逆转录病毒载体 DAAO基因 白血病细胞系 D-氨基酸氧化酶 

分 类 号：R733.7[医药卫生—肿瘤]