| 摘要: |
| 【目的】制备抗肠致病性大肠杆菌（EPEC）的重组嗜酸乳酸杆菌，并初步探索其免疫效果。【方法】采用DNAStar软件选取EPEC E2348/69的eae全基因中抗原性较高的984 bp片段(编码328个氨基酸残基，命名为eae Int328)为目标序列，以EPEC E2348/69基因组为模板，PCR扩增eae Int328基因，构建重组表达载体pSIP409-eae Int328，电转化法获得相应的重组嗜酸乳酸杆菌，采用SppIP诱导重组菌表达，并通过SDS-PAGE和Western-blot鉴定重组蛋白。以BALB/c雌性小鼠为受试动物，灌胃重组嗜酸乳酸杆菌15 d后，再灌胃EPEC，第21天，取结肠PP结和肠内容物，15和21 d称体质量，同时设PBS（对照）、EPEC和pSIP409空载体转化嗜酸乳酸杆菌处理。采用流式细胞术检测结肠PP结中的CD11c+ DC、CD4+ T、CD19+ B细胞比例，采用ELISA检测肠内容物中的特异性sIgA水平。【结果】克隆得到了984 bp的eae Int328基因，构建了重组质粒pSIP409-eae Int328，制备了相应的重组嗜酸乳酸杆菌，用SppIP诱导表达后，重组嗜酸乳酸杆菌表达了分子质量约为36 ku的重组蛋白，该重组蛋白与eae单克隆抗体可发生特异性结合。与EPEC和pSIP409空载体转化嗜酸乳酸杆菌组相比，21 d时，pSIP409重组嗜酸乳酸杆菌组小鼠体质量显著增加，且与PBS组小鼠无显著差异。该重组嗜酸乳酸杆菌可显著提高BALB/c小鼠结肠PP结中的CD19+ B细胞比例，极显著提高结肠PP结中的CD11c+ DC和CD4+ T细胞比例，使肠道黏膜产生针对EPEC的特异性sIgA。【结论】pSIP409-eae Int328重组嗜酸乳酸杆菌通过CD11c+ DC的介导，促进CD4+ T细胞数量增加，进而诱导B细胞活化产生针对EPEC的特异性sIgA，进而对小鼠EPEC的急性感染发挥作用。 |
| 关键词: 肠致病性大肠杆菌 eae基因 重组嗜酸乳酸杆菌 |
| DOI： |
| 分类号: |
| 基金项目:吉林省科技发展计划项目(20090246，201101004) |
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| Detecting immune efficacy of recombinant Lactobacillus acidophilus expressing enteropathogenic Escherichia coli |
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HAO Feng-qi,LI Jing-mei,YANG Zhou-hong
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| Abstract: |
| 【Objective】This study prepared anti-EPEC recombinant Lactobacillus acidophilus and determined its immune efficacy.【Method】DNAStar software was used to select the 984 bp fragment (encoding 328 aa) with high antigen index from EPEC E2348/69.eae Int328 gene was amplified by PCR using genome DNA of EPEC E2348/69 as template,and was inserted into plasmid pSIP409 to construct plasmid pSIP409-eae Int328.Then recombinant Lactobacillus acidophilus was obtained using electro-transformation.The recombinant protein was expressed with induction of SppIP,and identified by SDS-PAGE and Western-blot.Fifteen days after intragastric administration of pSIP409-eae Int328 recombinant Lactobacillus acidophilus,EPEC was also administrated to BALB/c mice.On the 21st day,mice were killed,and CD11c+ DC,CD4+ T cell,and CD19+ B cells in colon Peyer’s patch and specific sIgA level in intestine were detected using Flow cytometry and ELISA,respectively.【Result】The eae Int328 gene with length of 984 bp was amplified,and the recombinant plasmid pSIP409-eae Int328 was constructed successfully.The pSIP409-eae Int328 recombinant Lactobacillus acidophilus was successfully prepared and the recombinant protein with 36 ku was expressed after SppIP induction and its capacity of specific binding to eae monoclonal antibody was confirmed.The pSIP409-eae Int328 recombinant Lactobacillus acidophilus increased the ratios of CD11c+ DC,CD4+ T cell,and CD19+ B cell in colon Peyer’ patch.The specific sIgA against EPEC in intestine was produced by stimulation of pSIP409 eae Int328 recombinant Lactobacillus acidophilus.【Conclusion】The pSIP409-eae Int328 recombinant Lactobacillus acidophilus promoted CD4+ T cell expansion through the mediation of CD11c+ DC,and further induced activation of B cell to produce sIgA against EPEC,which prevented mice from EPEC acute infection. |
| Key words: EPEC eae gene recombinant Lactobacillus acidophilus |
