携带flk1的重组减毒沙门氏菌的构建及其抗肿瘤血管生成的研究

目的:观察携带鼠血管内皮生长因子受体-2(vascular endothelial growth factor receptor-2,flk1)的重组减毒鼠伤寒沙门氏菌诱导的flk1特异性免疫应答及抗肿瘤血管生成作用.方法:构建真核表达载体pcDNA3.1-flk1,并将其转化到减毒鼠伤寒沙门氏菌SL7207中,体外感染小鼠巨噬细胞,用Western印迹对表达产物进行鉴定,将重组菌口服免疫小鼠,分析免疫后小鼠体内的特异性CTL应答.采用藻酸盐微囊实验观察其对体内肿瘤血管生成的影响.结果:免疫印迹试验表明携带pcD-NA3.1-flk1表达载体的重组菌感染的小鼠巨噬细胞能表达flk1蛋白,免疫小鼠脾淋巴细胞产生针对flk1的特异性CTL活性.重组疫苗菌的免疫能够明显抑制小鼠体内肿瘤血管的形成.结论:携带flk1的重组减毒沙门氏菌经口服免疫,诱导小鼠产生抗flk1的特异性免疫反应,且能明显产生抗肿瘤血管生成作用.

Construction of Attenuated Salmonella Typhimurium Carrying flk1 Expression Vector and Priliminary Investigation of Its Inhibition of Tumor Angiogenesis

Objective: To investigate the specific anti-flk1 immunity and the anti-angiogenesis effect of attenuated Salmonella typhimurium strain carrying flk1 expression vector encoding flkl cDNA. Methods: Plasmid pcDNA3.1-flk1 was constructed and transformed into live attenuated Salmonella typhimurium strain SL7207. The recombinant bacteria were used to infect murine macrophage in vitro and the expressed products were detected by Western blot. C57BL/6J mice were immunized with recombinant bacteria encoding flk1, and CTL activity of the splenocytes derived from the immunized mice was measured by MTT assay. Alginate bead assay was designed to measure in vivo angiogenesis induced by tumor cells. Result: Murine macrophage infected with recombinant S. typhimurium transformed with plasmid pcDNA3.1-flk1 could express flkl protein. A strong CTL activity against flkl was found in pcDNA3.1-flk1 immunized animals. The recombinant S. typhimurium vaccination inhibited tumor-induced angiogenesis in vivo. Conclusion: Attenuated Salmonella typhimurium strain encoding flk1 cDNA can elicit specific cellular immune response and inhibit tumor-induced angiogenesis in vivo.