沙门菌Ⅲ型分泌蛋白融合HBx抗原介导的T细胞免疫效果观察

T-cell immune response induced by Salmonella typeⅢsecretion system

目的检测沙门菌减毒株作为载体,Ⅲ型分泌系统(typeⅢsecretion system,TTSS)效应蛋白-肿瘤抗原融合蛋白激发机体产生T细胞免疫反应的能力。方法重组原核表达质粒,表达Ⅲ型分泌蛋白SspH2和肿瘤抗原HBx融合分子,转化鼠伤寒沙门菌减毒株SL3261,口饲免疫小鼠。通过检测免疫小鼠脾细胞乳酸脱氢酶(LDH)释放确定疫苗激发CTL作用;利用ELISPOT方法检测免疫小鼠脾淋巴细胞释放IFN-γ能力;腋下接种稳定表达HBx的B16细胞,观察免疫小鼠肿瘤的体积及重量,检测该疫苗的免疫保护效果。结果通过LDH释放试验发现,经细菌体内表达及投递SspH2-HBx融合蛋白诱发后,小鼠脾淋巴细胞具有特异的CTL反应活性;ELISPOT结果显示细菌表达、投递SspH2- HBx蛋白具有特异诱发淋巴细胞分泌IFN-γ活性;肿瘤接种试验进一步证实这种基于TIKS的肿瘤疫苗可以特异抑制肿瘤的生长。结论以沙门菌减毒株作为载体、Ⅲ型分泌蛋白SspH2作为底物融合肿瘤抗原HBx能够激发显著的T细胞免疫反应、产生一定的免疫保护作用;这种基于沙门菌TTSS的疫苗具有一定的开发、应用前景。

Objective To examine specific immune responses eliciting capability of attenuated Salmonella type Ⅲ secretion system（TrSS） delivered tumor antigen. Me, otis To construct a recombinant plasmid to express SspH2-HBx fusion protein, transform it into attenuated Salmonella typhimurium SL3261 strain and to immunize mice with this strain beating recombinant plasmid; then to observe the specific CTL response elicited by fusion vaccine through examination of LDH releasing level; by ELISPOT examine specific IFN-γsecreting capability of splenocytes isolated from immunized mice; inoculate immunized mice with B16 cells stably expressing HBx and observe the tumor volumes and weights to evaluate tumor preventive effect of HBx vaccine. Results LDH release assay showed that, splenocytes of mice immunized by SspH2-HBx fusion vaccine delivered through TrSS of SL3261, can specifically and significantly kill HBx beating B16 tumor cells. ELISPOT assay showed that such fusion vaccine specifically elicited spleen lymphocytes to secret IFN-γ. Finally, tumor challenging experiments further corroborated that the fusion vaccine markedly inhibited tumor growth. Conclusion SspH2-HBx fusion vaccine delivered by attenuated Salmonella TrSS can elicit significant T-cell immune responses and effective protection with potential clinical significance.