小鼠口服弓形虫DNA混合疫苗的免疫保护反应  

Oral Mixed DNA Vaccine Protects Mice from Infection ofToxoplasma gondii

作　　者：丛华[1] 古钦民[1] 周怀瑜[1] 郭岚[1] 杨婷婷[1] 何深一[1] 李瑛[1] 赵群力[1] 

机构地区：[1]山东大学医学院病原生物研究所寄生虫学教研室,济南250012

出　　处：《中国寄生虫学与寄生虫病杂志》2005年第3期159-162,共4页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：山东省自然科学基金(No.Y99C02);山东省可持续发展科技十大示范工程(山东[1998]28号)~~

摘　　要：目的观察携带弓形虫表面抗原复合基因重组沙门氏菌经口免疫BALB/c小鼠所诱导的免疫反应。方法构建弓形虫主要表面抗原SAG1、SAG2复合基因真核表达质粒,将其转入减毒鼠伤寒沙门氏菌BRD509(BRD509/pSAG1/SAG2),经口服免疫BALB/c小鼠,以携带空载体质粒的减毒沙门氏菌BRD509及生理盐水(NS)组为对照,分别于每次免疫前和免疫后断尾取血,并于末次免疫后第4周取小鼠脾脏测定T淋巴细胞增殖活性、天然杀伤细胞(NK细胞)活性和T细胞亚群;ELISA法测定IgG抗体及血清中的细胞因子干扰素(IFN-γ),白介素-4(IL-4)。与末次免疫后4周,每只小鼠腹腔注射0.1ml(105)弓形虫RH株速殖子攻击,观察小鼠存活时间。结果免疫组小鼠的IgG抗体水平明显提高,滴度为1∶100;NK细胞杀伤活性和T细胞增殖活性也明显增强,其中NK细胞杀伤活性为85%±7%,T细胞增殖指数为2.83,与对照组相比差异有统计学意义(P<0.05)。免疫鼠攻击感染后平均存活时间比对照组长5d。结论弓形虫口服DNA混合疫苗可诱导小鼠产生保护性免疫。Objective To examine the immune response in BALB/c mice induced by oral mixed Toxoplasma gondii DNA vaccine delivered by live attenuated Salmonella typhimurium. Methods Gene fragments SAG1 and SAG2 were amplified from the genomic DNA of T. gondii RH strain by PCR and were subcloned into pcDNA3.1(±) eukaryotic expression vector. The positive recombinant plasmid was transformed into aroA- and aroD-attenuated Salmonella typhimurium BRD509 (BRD509/pSAG1/SAG2). After screened by PCR, restrictive enzyme digestion and sequencing, recombinant Salmonella strain was used to immunize BALB/c mice by i.g. route, three times at 2-week interval. Humoral and cellular responses were assayed using ELISA for determining antibody, IFN-γ and IL-4. MTT assay was used to detect the proliferation activity of T lymphocytes and the activity of NK killers. FCM was also used to sort the lymphocyte subsets of spleen cells. All mice were then infected with highly virulent RH tachyzoites of T. gondii intraperitoneally. Results Significant increase of specific IgG level was observed in immunized mice with a titer of 1∶100. Proliferation activity of specific NK cells and T lymphocytes was highly enhanced in BRD509/ pSAG1/SAG2 vaccinated mice: the killing activity of NK cells was 85%±7%, the proliferation SI of T lymphocytes was 2.83, which resulted in a 5 days longer survival time than mice in control group after challenge infection. Conclusion The oral mixed DNA vaccine delivered by attenuated Salmonella typhimurium shows an immunoprotection against T. gondii in mice.

关 键 词：弓形虫 减毒沙门氏菌BRD509 SAG1/SAG2 DNA混合疫苗 

分 类 号：R382.5[医药卫生—医学寄生虫学] R392.1[医药卫生—基础医学]