知识与财富的链接
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 CDwt,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 CDD314A。用LipofectamineTM2000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 CDwt及LoVo CDD314A。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 CDD314A细胞对5 FC的IC50为(85.13±0.60) mmol/L,显著低于LoVo CDwt细胞的（689.76±0.45）μmol/L,(P=0.000);在旁观者试验中,当LoVo CDwt细胞和LoVo CDD314A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 e-valuated 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 in-creased antitumor effect on LoVo cells compared with wild type EG-CD gene, and it may become a new candidate gene for tumor gene therapy.