肿瘤厌氧靶向双自杀基因治疗系统pH2／CD，pH2／UPRT的构建

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人婴儿双歧杆菌中。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。

Construction of tumor-targeting anaerobic double suicide gene therapy system pH2/CD and pH2/UPRT in Bifidobacterium Infantis

Objective To construct pH2/CD and pH2/UPRT double suicide gene therapy system in tumor-hypoxia-targeting vector Bifidobacterium Infantis.Methods CD gene and UPRT gene were amplified from pGEX/CD and pGEX/UPRT using PCR,and then the CD gene,UPRT gene and lactic acid bacteria expression plasmid pH2 were digested by restriction endonulease BamH I and Sal I,and constructed recombinant plasmids pH2/CD and pH2/UPRT in E.coli.The recombinant plasmids were then transfected into Bifidobacterium Infantis by electroporation.Identification of pH2/CD and pH2/UPRT was processed by double restriction endonulease digesting and sequencing.RT-PCR was used to examine the expression of CD and UPRT genes at RNA level.The killing effects for tumor cells by pH2/CD and pH2/UPRT suicide gene therapy system with 5-FC were done on Melanoma B16-F10 cells.Results The CD gene and UPRT gene were successfully recombined into lactic acid bacteria expression plasmid pH2.After double endonuclease digestion of plasmid purified from the positively transfected E coli,two fragments of 6.9 kb and 1.3 kb were found for CD gene and two fragments of 6.9 kb and 620 bp were found for UPRT gene.The sequencing of CD gene and UPRT gene proved consistent sequences with Genebank published data.A fragment of 1.3 kb for CD gene and fragment of 620 bp for UPRT gene was found in recombinant Bifidobacterium by RT-PCR.The survival rate of tumor cells treated by extracts from culture of recombinant Bifidobacterium with 5-FC showed a strong killing effects of pH2/CD and pH2/UPRT double suicide gene therapy system on Melanoma B16-F10 cells.Conclusion CD gene and UPRT gene were successfully inserted into pH2 and transfected into tumor-hypoxia-targeting vector Bifidobacterium Infantis.This double suicide gene therapy system showed a high efficiency for tumor cells killing.