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破伤风毒素C片段基因的克隆及在大肠杆菌中的高效表达

期刊名称：中国预防兽医学报，2007，29(6)：439-447
时间：0
分类：S852.43[农业科学—基础兽医学;农业科学—兽医学;农业科学—畜牧兽医]
作者机构：[1]扬州大学江苏省人兽共患病学重点实验室,江苏扬州225009, [2]扬州大学测试中心,江苏扬州225009
相关基金：国家杰出青年科学基金（No.30425031）、全国优秀博士学位论文作者专项资金（FANEDD200358）和教育部青年教师奖（175）资助
相关项目：兽医传染病学

关键词：减毒沙门菌, ELISPOT, CD8+T细胞表位, 黏膜免疫, attenuated Salmonella, ELISPOT, CD8^＋ T cell epitope, mucosal immunity

中文摘要：

以融合表达绿色荧光蛋白（Green fluorescent protein,GFP）和OVA、LCMV NP CD8＋T细胞表位的重组减毒沙门菌SL7207（ptG2F）为免疫原,经口服途径接种BALB/c小鼠,每2周加强免疫1次,分别于第2次、第3次、第4次、第5次免疫后,以ELISPOT法分别测定免疫小鼠派伊尔氏结细胞中特异性针对OVA和LCMVNP CD8＋T细胞表位的IFN-γ分泌细胞和IL-4分泌细胞.同时,运用ELISA分别检测血清、肠黏膜中的GFP特异性抗体,分析了口服重组减毒沙门菌诱导的特异性免疫应答动态规律.实验表明SL7207（ptG2F）口服免疫BALB/c小鼠后,诱导产生了LCMV NP CD8＋T细胞表位（H-2d）特异的细胞免疫应答.在第2次免疫后,即可检测到特异性IFN-γ分泌细胞和IL-4分泌细胞在第3次免疫后,IFN-γ分泌细胞数量明显高于IL-4分泌细胞数.然而,在第4、5次免疫后,IFN-γ分泌细胞数和IL-4分泌细胞数相当,未见明显变化.试验中未能检测到针对OVA CD8＋T细胞表位（H-2b）的细胞免疫应答.运用ELISA方法检测到SL7207（ptG2F）、SL7207（ptGFP）免疫组GFP特异性IgG和IgA抗体,与对照组相比较,差异显著（P＜0.05）.结果表明重组减毒沙门菌口服免疫可以有效运送外源抗原至宿主免疫系统,并诱导产生特异性细胞免疫应答和黏膜免疫应答.

英文摘要：

The immune responses induced by recombinant salmonella after oral immunization was investigated using a recombinant bacteria expressing GFP fused with OVA and LCMV NP CD8^＋ T cell epitope. The GFP fusion vector was constructed and transformed into the attenuated strain SL7207 of salmonella typhimurium to produce recombinant bacteria SL7207 （ptG2F）. BALB/c mice were orally immunized with SL7207 （ptG2F） in two weeks intervals. IFN-γ, and IL-4 secreting cells were detected by ELISPOT assay in murine peyer＇s patches cells after the second, third, fourth and fifth immunization, respectively. Antibodies against GFP were determined by ELISA in both serum and intestinal mucosa. The results showed that recombinant bacteria SL7207 （ptG2F） induced various cellular immune responses specific for LCMV NP CD8^＋ T epitope at different time point of immunization. IFN-γ and IL-4 secreting cells were detected after second immunization. The fi＇equency of IFN-γ secreting cells increased significantly over that of IL-4 secreting cells after third immunization. However after the fourth immunization, both IFN-γ and IL-4 secreting cells was detected at similar frequency. No cellular immune responses were detected against OVA CD8^＋ T cell epitope throughout the experiment. The levels of IgG and IgA antibodies detected by ELISA were significantly higher in immunized group than that of the control group （p〈0.05）. These results suggested that recombinant attenuated Salmonella could effectively deliver extraneous antigen to immune system and induce both cellular immune responses and mucosal responses through mucosal administration.

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