目的利用婴儿双歧杆菌对实体瘤低氧区的靶向效应,构建肿瘤厌氧靶向双自杀基因治疗系统pH2/CD和pH2/UPRT。方法用PCR的方法从质粒pGEX/CD和pGEX/UPRT中扩增出CD基因和UPRT基因,用内切酶BamHI和SalI酶切CD基因,UPRT基因和质粒pH2,分别连接后重组于大肠杆菌中。用电转化的方法将重组质粒转入婴儿双歧杆菌中。通过双酶切和基因测序的方法检验质粒pH2/CD和pH2/UPRT的正确性,RT-PCR检测该系统的mRNA表达。在黑色素瘤B16-F10细胞上检测该系统的体外肿瘤细胞杀伤效果。结果成功地将CD基因和UPRT基因转入了乳酸菌表达质粒pH2,在纯化后的质粒被双酶切后,CD基因被分割为6.9kb和1.3kb的两部分,UPRT基因被分割为6.9kb和620bp的两部分。CD基因和UPRT基因的测序结果表明序列与Genebank公布的序列一致。RT-PCR检测到CD和UPRTmRNA的明显表达。黑色素瘤细胞的低存活率证明pH2/CD,pH2/UPRT自杀基因治疗系统对黑色素瘤的强大杀伤作用。结论肿瘤厌氧靶向双自杀基因治疗系统pH2/CD,pH2/UPRT构建成功并显示出强大的杀伤肿瘤细胞的作用。 Objective To construct the anaerobic target dual suicide gene therapy system pH2 / CD and pH2 / UPRT for the targeted effect of Bifidobacterium infantis on the hypoxia region of solid tumors. Methods The CD gene and UPRT gene were amplified by PCR from plasmids pGEX / CD and pGEX / UPRT. The CD gene was digested with BamHI and SalI, and the UPRT gene and plasmid pH2 were ligated and recombined in E. coli . The method of electrotransformation was used to transfer the recombinant plasmid into Bifidobacterium infantis. The correctness of plasmid pH2 / CD and pH2 / UPRT was tested by double enzyme digestion and gene sequencing. The mRNA expression of this system was detected by RT-PCR. The in vitro tumor cell killing effect of this system was tested on melanoma B16-F10 cells. Results The CD gene and UPRT gene were successfully transferred into Lactobacillus expression plasmid pH2. After the purified plasmid was double-digested, the CD gene was divided into two parts of 6.9kb and 1.3kb. The UPRT gene was divided into 6.9kb and 620bp in two parts. Sequencing of the CD and UPRT genes showed that the sequence was identical to that published by Genebank. The expression of CD and UPRT mRNA was detected by RT-PCR. The low survival rate of melanoma cells demonstrates the powerful killing effect of the pH2 / CD, pH2 / UPRT suicide gene therapy system on melanoma. Conclusion The anaerobic target dual suicide gene therapy system pH2 / CD and pH2 / UPRT were successfully constructed and showed a powerful killing effect on tumor cells.