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本研究提取大肠杆菌JM109菌株的全基因组,克隆获得CD基因,将CD基因和IRES-EGFP基因构建到pEE12.4表达载体上,形成pEE12.4IE-CD真核表达载体。用该表达载体转染人肝癌细胞后,以不同浓度的5-氟胞嘧啶(5-flucytosine,5-FC)处理细胞,采用噻唑蓝(MTT)法测定CD/5-FC系统对人肝癌细胞HepG2的杀伤效果。当前导药物5-FC的浓度分别为10、20和30 mmol.L 1时,CD/5-FC系统对HepG2细胞的杀伤效率分别为18.07%、42.98%和62.20%,与对照组相比较差异具有统计学意义。急性毒性实验结果表明,小鼠尾静脉注射不同剂量5-FC,组间剂量比为1∶0.5,采用改良寇氏法测定5-FC对小鼠的LD50为507 mg.kg 1,95%可信限为374～695 mg.kg 1。再以LD50实验中最大LD0剂量为基准,组间剂量差值为10 mg.kg 1,测得最大安全剂量为200 mg.kg 1,并对体重、体征指标进行跟踪观察。这些检测结果可为动物实验奠定基础。将CD基因连于新城疫病毒(NDV)rClone30载体中并建立肿瘤动物模型,抑制肿瘤实验结果表明,重组新城疫病毒rClone30-CD/5-FC系统具有较高的体内抗肿瘤活性。综上所述,克隆获得的CD基因具有生物学活性,重组rClone30-CD/5-FC系统是一种潜在的治疗肿瘤的方法。
In this study, we extracted the whole genome of Escherichia coli JM109 strain and cloned the CD gene. The CD gene and IRES-EGFP gene were constructed into the pEE12.4 expression vector to form the eukaryotic expression vector pEE12.4IE-CD. After transfecting human hepatocellular carcinoma cells with this expression vector, the cells were treated with 5-flucytosine (5-FC) at various concentrations. The effect of CD / 5-FC on human hepatocellular carcinoma cells HepG2 killing effect. The results showed that the killing efficiency of CD / 5-FC system on HepG2 cells was 18.07%, 42.98% and 62.20% when the concentration of 5-FC was 10, 20 and 30 mmol.L 1, respectively. Compared with the control group has statistical significane. The results of acute toxicity test showed that mice were injected different doses of 5-FC into the tail vein, the dose ratio was 1: 0.5. The LD50 of 5-FC was 507 mg.kg and 1, 85% The credit limit is 374 ~ 695 mg.kg 1. Based on the maximum dose of LD0 in the LD50 experiment, the difference in dose between the two groups was 10 mg.kg1, and the maximum safe dose was 200 mg.kg1. The body weight and body mass index were tracked. These test results can lay the foundation for animal experiments. The CD gene was ligated into rClone30 vector of Newcastle disease virus (NDV) and tumor model was established to inhibit the tumor. The experimental results showed that rClone30-CD / 5-FC system has high in vivo anti-tumor activity. In conclusion, the CD gene obtained by cloning is biologically active, and the recombinant rClone30-CD / 5-FC system is a potential tumor treatment method.