目的：构建含有TIP30与人IFN-γ 基因的真核共表达质粒的减毒伤寒沙门氏菌，观察以减毒伤寒沙门氏菌为载体的，联合TIP30与IFN-γ 的基因治疗对腺样囊性癌的疗效。方法：PCR扩增TIP30和人IFN-γ 基因，分别插入真核表达载体pCI-neo，构建成重组表达质粒pCI-TIP30、pCI-IFN和TIP30、IFN-γ 基因共表达质粒pCI-TIP30/IFN。分别将重组表达质粒转化到减毒鼠伤寒沙门氏菌SL7207，体外感染小鼠巨噬细胞株，用免疫印迹和ELISA对表达产物进行鉴定，将重组菌口服给腺样囊性癌荷瘤裸鼠，观察其疗效。结果：免疫印迹试验表明质粒pCI-TIP30和pCI-TIP30/IFN转化菌感染的小鼠巨噬细胞能表达TIP30蛋白，ELISA检测则显示pCI-IFN和pCI-TIP30/IFN转化菌感染的小鼠巨噬细胞能分泌性表达人IFN-γ ，减毒沙门氏菌本身和3种表达质粒转化的重组沙门氏菌对腺样囊性癌均有治疗作用，且TIP30与IFN-γ 具有明显的协同作用。结论：成功构建了分别携有真核表达质粒pCI-TIP30、pCI-IFN、pCI-TIP30/IFN的减毒鼠伤寒沙门氏菌，对腺样囊性癌荷瘤裸鼠有显著的治疗作用。

Objective:To construct attenuated salmonella typhimurium haboring an eukaryotic co-expression plasmid encoding TIP30 and human IFN-γ gene and observing its effect on the growth of adenoid cystic carcinoma. Methods:The TIP30 and human IFN-γ genes were amplified by PCR and inserted into eukaryotic expression vector pCI-neofor the construction of expression plasmids pCI-TIP30 and pCI-IFN, respectively. A co-expression plasmid pCI-TIP30/IFN was constructed by linking TIP30 and human IFN-γ gene using the sequence of internal ribosome binding sequence (IRES). Three recombinant expression plasmids were transformed into an attenuated AroA -autotrophic mutant of salmonella typhimurium SL7207, the resultant bacteria were used to infected murine macrophage in vitro and the expressed products were detected by Western blot and ELISA, respectively. Tumor growth was observed by oral administration of the recombinant salmonella typhimuriums to the nude mouse with adenoid cystic carcinoma.Results:Murine macrophage infected with recombinant salmonella transformed with both plasmids pCI-TIP30 and pCI-TIP30/IFN could express TIP30 protein, and murine macrophage infected with recombinant salmonella transformed with pCI-IFN or pCI-TIP30/IFN could secret human IFN-γ in the culture supernatant. Attenuated salmonella typhimurium and three constructed recombinant salmonella typhimuriums all had evident inhibition onthe tumor growth in nude mouse with adenoid cystic carcinoma.Conclusion: The recombinant attenuated salmonella typhimuriums haboring plasmid pCI-TIP30, plasmidpCI-IFN and co-expressing plasmidpCI-TIP30/IFN were successfully constructed, which could inhibit the growth of adenoid cystic carcinoma in nude mouse.

R730.5

国家自然科学基金资助(30171055)