【摘要】 目的　探讨表达人乳头状瘤病毒 6b型 (HPV6b)L1 E7嵌合蛋白的减毒沙门菌诱导小鼠粘膜及系统免疫反应的可行性。方法　对重组减毒沙门菌S .BRD5 0 9 pTETnir15 6bL1E7体外厌氧诱导其表达 ,Westernblot方法鉴定表达的L1 E7蛋白。此重组沙门菌经滴鼻和口服联合粘膜免疫BALB c小鼠 ,采用ELISA方法及足垫肿胀试验分别检测免疫小鼠血清及阴道冲洗液中特异性抗体和DTH反应。结果　Westernblot分析显示 ,重组沙门菌在预计相对分子质量 (Mr) 5 6× 10 3 处有特异染色带 ,提示此重组沙门菌能表达特异的HPV6bL1 E7蛋白。ELISA检测结果表明实验组小鼠阴道冲洗液中HPV6bL1特异性sIgA显著升高 ,和对照组比较两者差异有显著性 (P <0 .0 1) ,但小鼠血清中HPV6bL1IgG抗体无明显变化 ,实验组和对照组两者差异无显著性。DTH结果显示 :与对照组比较 ,实验组小鼠接受HPV6bL1 E7抗原刺激后产生明显阳性反应 ,提示重组沙门菌免疫小鼠产生了针对HPV6bL1 E7抗原的特异性细胞免疫反应。结论　构建的重组减毒沙门菌S .BRD5 0 9 pTETnir15 6bL1E7可激发有效的粘膜免疫及细胞免疫反应 ,为进一步研制新型HPV粘膜疫苗奠定了实验基础。更多还原

【Abstract】 Objective To assess the ability of the attenuated Salmonella typhimurium strain expressing human papillomavirus type 6b(HPV 6b) L1-E7 chimeric protein in inducing the specific mucosal and systemic immune responses against HPV. Methods A recombinant Salmonella strain S.BRD509/pTETnir15-6bL1E7 was constructed and the anaerobic expression of HPV6b L1-E7 protein under the control of nirB promoter was confirmed by Western blot in vitro. BALB/c mice were immunized by intranasal and oral rout with the Salmonella vaccine. Serum and vaginal washes were collected following second immunization and assessed for specific L1 antibody by ELISA. HPV6b L1-E7 specific DTH response was evaluated 2 weeks after the last immunization. Results Western blot showed an additional protein with molecular relative mass of 56×103 which could specifically react with both L1 and E7 antisera. Results of animal experiment indicated that the L1-specific IgA titer in vaginal washes from mice immunized by S.BRD509/pTETnir15-6bL1E7 was significantly higher than that in control group. However, IgG in the sera was low and there was no difference in both experiment and control groups. A positive 48 h DTH reaction to HPV6b L1-E7 protein was detected among mice immunized with S.BRD509/pTETnir15-6bL1E7 but not with the blank vector. Conclusion It is concluded that the attenuated Salmonella typhimurium strains S.BRD509/pTETnir15-6bL1E7 could elicit potent mucosal and cell-mediated immune responses, which play an important role in preventing and treating HPV6b infection and relative diseases. [更多还原