以减毒沙门菌作为DNA疫苗载体的研究  

Attenuated Salmonella typhimurium as DNA delivery vehicle for DNA-mediated immunization

作　　者：何平[1] 陈洪[1] 刘晶星[1] 陈淑云[1] 胡宝瑜[1] 

机构地区：[1]上海第二医科大学微生物教研室,200025

出　　处：《中华实验和临床病毒学杂志》2004年第3期238-242,F002,共6页Chinese Journal of Experimental and Clinical Virology

基　　金：国家自然科学基金资助 ( 3 9970 691)

摘　　要：目的　以真核表达质粒pCMVβ为报告基因 ,研究用芳香族氨基酸合成缺陷的沙门菌SL72 0 7为载体以提高DNA疫苗免疫应答的可行性。方法　携带质粒pCMVβ的SL72 0 7体外感染小鼠腹腔巨噬细胞后 ,用X gal染色方法和RT PCR方法检测巨噬细胞内 β gal的表达。小鼠口服免疫SL72 0 7(pCMVβ)后 ,用RT PCR方法检测 β gal基因在淋巴组织中的转录产物 ;用ELISA方法检测体液免疫 ;用3H TdR掺入法检测脾淋巴细胞增殖 ;用JAM试验检测杀伤性T淋巴细胞反应。结果　SL72 0 7能有效地将质粒DNA传递到体外培养的巨噬细胞中并进行表达 ;小鼠口服携带有pCMVβ的SL72 0 7后 ,能在脾脏、派伊尔结、肠系膜淋巴结中检测到目的基因的转录 ,并可诱导小鼠产生特异性体液免疫和细胞免疫。与肌内注射pCMVβ相比较 ,口服SL72 0 7(pCMVβ)能更有效地诱导出细胞免疫应答。结论　减毒沙门菌SL72 0 7作为DNA疫苗的载体 ,可经口服途径进行免疫 ,并可将质粒直接传递给抗原递呈细胞 ,诱导出以细胞免疫为主的免疫应答。Objective To study whether the live attenuated AroA-auxotrophic mutant of Salmonella (S.) typhimurium (SL7207) could be used as DNA delivery vehicle to induce more efficient immune response by using the eukaryotic expression plasmid pCMVβ as report gene Methods Murine peritoneal macrophages were infected with SL7207(pCMVβ) in vitro, then the expression of the β-gal were detected by X-gal staining or RT-PCR After mice were orally immunized with SL7207(pCMVβ), the expression of β-gal in the lymphoid tissue were tested by RT-PCR, humoral responses were tested by ELISA, splenic lymphocyte proliferation were tested by 3 H-TdR incorporation and cytotoxic T lymphocyte reaction were tested by JAM test Results The results indicated that the plasmid pCMVβ could be delivered by SL7207 into the nucleus of the murine macrophages efficiently and expressed well in vitro; after mice received oral immunizations with attenuated Styphimurium SL7207 harboring plasmid pCMVβ mice, the expression of β-gal could be detected in the spleen, mesenteric lymph nodes and Peyer′s patches of the mice. Furthermore, the experiments demonstrated that specific humoral immune responses and cell-mediated immune responses were successfully induced in these immunized mice Compared with the naked DNA vaccination, SL7207(pCMVβ) oral immunization were more efficient in inducing cellular immune responses Conclusion Attenuated Salmonella typhimurium SL7207 could be used as DNA delivery vehicle for oral immunization, which have the ability to deliver the antigen-encoding DNA specifically to APC directly for inducing the specific immune response being dominant with cellular immune response

关 键 词：CMV 沙门菌 DNA疫苗 口服 减毒 小鼠 诱导 SL RT-PCR方法 ELISA方法 

分 类 号：R392[医药卫生—免疫学]