目的: 构建携带1/100、1/1 000减毒活性的突变减毒志贺样毒素Ⅰ(Shigalike toxin 1, Stx1) 编码序列的复制缺陷型腺病毒，并观察其抗人乳腺癌细胞裸鼠移植瘤的活性。方法：重叠PCR法构建毒性为原毒素毒性1/100、1/1000的突变减毒Stx1编码序列，TA克隆并测序后构建携带该编码序列的复制缺陷型腺病毒载体AdvStx1R170L。制备人乳腺癌T47D细胞移植瘤裸鼠模型，AdvStx1R170L肿瘤局部注射给药，评价其体内抑瘤能力。结果：经测序证实正确克隆了1/100、1/1 000减毒活性的突变减毒Stx1编码序列，成功构建携带该突变减毒Stx1编码序列的复制缺陷型腺病毒载体AdvStx1R170L。体内实验显示，AdvStx1R170L可以有效抑制裸鼠体内移植瘤的生长，与携带绿色荧光蛋白编码序列的腺病毒对照组、PBS对照组相比，差异有统计学意义（P<0.05）。结论：成功构建了携带1/1 000原毒素活性的突变减毒Stx1编码序列的重组复制缺陷型腺病毒载体，该病毒载体可以有效抑制裸鼠体内移植瘤的生长，且未见明显毒性作用

Objective:To construct recombinant replication defective adenoviral vectors encoding 1/100 and 1/1000 attenuated Shigalike toxinⅠ(Stx1) gene, and to study their therapeutic effects against breast cancer T47D cells in vivo.Methods: Genes encoding 1/100 or 1/1000 attenuated Stx1 were amplified by overlapping PCR and were cloned into T vectors. The inserted gene was verified by nucleotide sequencing. Replication defective adenoviral vector AdvStx1R170L containing 1/1000 attenuated Stx1 mutant gene was generated by AdMAX Adenoviral Vector System. Nude mouse models bearing human breast cancer T47D cells were established, and the tumor inhibitory effect of AdvStx1R170L was studied by intratumor injection of the recombinant adenovirus. Results: Vectors carrying 1/100 or 1/1000 attenuated Stx1 gene were successfully constructed and were verified by nucleotide sequencing. Recombinant replication defective adenoviral vector AdvStx1R170L containing 1/1000 attenuated Shigalike toxinⅠgene was constructed. In vivo study showed that AdvStx1R170L significantly inhibited the growth of implanted T47D tumor in the nude mice compared with AdvGFP and PBS group（P<0.05）. Conclusion: Recombinant replication defective adenoviral vector AdvStx1R170L encoding 1/1000 attenuated Shigalike toxin Ⅰgene has been successfully constructed, and it can effectively inhibit the growth of implanted T47D tumor in nude mice, without obvious toxicity.

天津市卫生局科技基金（No.05KY32）；天津医科大学附属肿瘤医院博士启动基金（No.07B02）