目的：构建大肠埃希菌胞嘧啶脱氨酶（E. coli cytosine deaminase, ECCD）基因突变体:D314A: （即ECCD基因开放阅读框第314位氨基酸由天冬氨酸突变为丙氨酸）并研究其抗肿瘤作用。方法：构建含ECCD基因的真核表达质粒pcDNA3.1CDwt，应用点突变技术将pcDNA3.1CD中ECCD基因开放阅读框第314位氨基酸的碱基由A突变为C，即构成pcDNA3.1CDD314A。用LipofectamineTM2000将ECCD基因或:D314A: 基因转入人结肠癌细胞系LoVo细胞，以G418筛选出稳定表达的阳性克隆LoVoCDwt及LoVoCDD314A。应用MTT法检测ECCD基因及:D314A: 基因对LoVo细胞的直接杀伤作用及旁观者效应。结果：成功构建ECCD基因突变体:D314A: 并经测序确认。LoVo细胞转染ECCD基因及:D314A: 基因后均能表达相应的mRNA，LovoCDD314A细胞对5FC的IC50为(85.13±0.60) mmol/L，显著低于LoVoCDwt细胞的（689.76±0.45）μmol/L，(P=0.000)；在旁观者试验中，当LoVoCDwt细胞和LoVoCDD314A细胞比例均为30%时，细胞存活率分别为(48.5±0.49)%与(17.3±0.40)%（P=0.000）。结论：ECCD基因突变体:D314A: 的抗LoVo细胞作用显著强于野生型ECCD基因，有望成为新的肿瘤基因治疗候选基因。

Objective:To construct a mutant :D314A: of Escherichia coli cytosine deaminase (ECCD, substitution of an alanine (A) for the aspartic acid (D) at position 314 of cytosine deaminase) and investigate its antitumor effect. :Methods: :Eukaryotic expression plasmid containing ECCD gene (pcDNA3.1CDwt) was constructed, and the mutant pcDNA3.1CDD314A plasmid, with aspartic acid (D) at position 314 of ECCD gene substituted by alanine (A) (ECCDD314A), was established by sitedirected mutation. ECCDwt and ECCDD314A were transfected into human colon cancer cell line LoVo via LipofectamineTM 2000, and positive LoVoCDwt and LoVoCDD314A cells stably expressing corresponding genes were selected by G418. The cytotoxicity and bystander effects of ECCD and ECCDD314A genes on LoVo cells were evaluated by MTT assay. :Results: :The mutant :D314A: was confirmed by sequence analysis. ECCD and ECCDD314A mRNA were expressed after transfected into LoVo cells. The IC50 of LovoCDD314A cells was (85.13±0.60) mmol/L, which was significantly lower than that of LoVoCDwt cells (\[689.76±0.45\] μmol/L, P=0.000). Bystander effect assay showed that, when at the ratio of 30%, the survival rates of LoVoCDwt cells and LovoCDD314A cells were (48.5±049)% and (17.3±0.40)% (P=0.000), respectively. :Conclusion：:Mutatant ECCD gene (ECCDD314A) has a significantly increased antitumor effect on LoVo cells compared with wild type EGCD gene, and it may become a new candidate gene for tumor gene therapy.

南京市科技局社会发展基金项目（No. 200605010）