携带双启动子DNA疫苗载体的减毒沙门菌在体内定植及动态变化  

Colonization and kinetics of attenuated Salmonella-based Dualpromoter DNA vaccine of HPV16 by inoculating through oral, intranasal and transcutaneous pathways

作　　者：卞继峰[1] 于修平[1] 耿昭[1] 卢翌[1] 胡海燕[1] 王晓明[1] 

机构地区：[1]山东大学医学院分子生物学实验室,山东济南250012

出　　处：《山东大学学报（医学版）》2004年第5期531-533,539,共4页Journal of Shandong University:Health Sciences

基　　金：国家自然科学基金(30200246;30170045);山东省卫生厅青年基金穴2001CAZCAB3雪;山东大学青年基金资助课题。

摘　　要：目的:探讨双启动子DNA疫苗载体在减毒沙门菌中的稳定性和重组减毒沙门菌在小鼠体内的定植及动态变化。方法:将人乳头瘤病毒双启动子DNA疫苗载体pCN鄄16L1E7转化减毒沙门菌S.SL3261熏经口服、鼻饲和皮肤途径免疫小鼠,在免疫后的第3、4、5、6周取小鼠粘膜相关淋巴组织,电镜观察细菌在组织中的定植;组织匀浆,细菌培养计数确定细菌的增殖;从细菌中提取质粒酶切鉴定以确定质粒的稳定性。结果:重组减毒沙门菌S.SL3261能在小鼠脾脏、肝脏和粘膜相关淋巴组织中定植,电镜可检测到多个细菌定植灶;重组减毒沙门菌可有效进入机体细胞并有限增殖达6周,在第4～5周菌落数量达到高峰,然后逐渐减少以至最后被清除。pCN鄄16L1E7可在减毒沙门菌中稳定存在,载体的产量和酶切图谱没有明显变化。结论:双启动子DNA疫苗载体pCN鄄16L1E7与减毒沙门菌有较好的相容性,能够在小鼠体内定植及有限增殖。口服、鼻饲和皮肤粘膜接种途径均可有效地将减毒沙门菌携带的DNA疫苗载体导入粘膜相关淋巴组织。Objective: To evaluate the stability and colonization of the Salmonella-based Dual-promoter DNA vaccine in vivo. Methods: Dual-promoter DNA vaccine of human Paillomavirus type 16 contained in vivo-inducible bacterial promoter nirB and HCMVie promoter. AroA-mutant Salmonella SL3261 strain was used as the carrier of the DNA vaccine, and BALB/c mice were inoculated through oral, intranasal and transcutaneous pathways with anaerobically-induced SL3261/pCN-16L1E7. Mice were sacrificed from weeks 3 to 6. The liver, spleen, cervical lymphoid nodes, mediastinal lymph nodes and Peyer's Patch were harvested and homogenized with PBS buffer, spread onto the LB plates with ampicillin (100 μg/ml) and cultivated overnight. Then the number of the colonies was counted. Several colonies were randomly picked up from each plate and cultivated, and then plasmid was extracted. Colonization of the salmonella strain in Peyer's Patch was confirmed by electron microscope. Results: Salmonella SL3261/pCN-16L1E7 DNA vaccine was stable in vivo for up to 6 weeks, and entered and replicated in mucosa-associated lymph organs. Replication kinetics reached peak at around weeks 4 and 5, then recombinant Salmonella declined quickly. These three different pathways were all effective, and no significant difference was found, except the nasal pathway was more toxic than the others. The transcutaneous pathway was very easy to operate and accept by patients. Conclusion: These data indicate Dual-promoter DNA vaccine pCMVnir and pCN-16L1E7 is stable in salmonella cells in vivo for up to 6 weeks. High expression of the antigens from the nirB promoter does not result in the instability of the pCMVnir and recombinant bacteria. The transcutaneous pathway is a prosperous vaccine delivery strategy for protein, DNA and recombinant bacterial vaccines.

关 键 词：乳头状瘤病毒 人 疫苗 DNA 沙门菌属 

分 类 号：R373.9[医药卫生—病原生物学] Q782[医药卫生—基础医学]