中国农业科学 ›› 2009, Vol. 42 ›› Issue (11): 4085-4092 .doi: 10.3864/j.issn.0578-1752.2009.11.041

• 兽医 • 上一篇    下一篇

表达产肠毒素性大肠杆菌F41重组干酪乳杆菌免疫小鼠的保护性分析

刘建奎,魏春华,侯喜林,余丽芸,王桂华

  

  1. (黑龙江八一农垦大学动物科技学院)
  • 收稿日期:2008-11-10 修回日期:2009-02-20 出版日期:2009-11-10 发布日期:2009-11-10
  • 通讯作者: 余丽芸

Protection Against ETEC F41 After Intranasal Immunization with Recombinant Lactobacillus casei

LIU Jian-kui, WEI Chun-hua, HOU Xi-lin, YU Li-yun, WANG Gui-hua   

  1. (黑龙江八一农垦大学动物科技学院)
  • Received:2008-11-10 Revised:2009-02-20 Online:2009-11-10 Published:2009-11-10
  • Contact: YU Li-yun

摘要:

【目的】利用干酪乳酸菌作为抗原传递系统来刺激机体产生黏膜免疫反应,从而研制有效的黏膜疫苗预防ETEC F41的感染。【方法】重组菌在MRS培养基中进行表达,经SDS-PAGE、Western blot检测目的蛋白的表达,间接免疫荧光分析及流式细胞术检测外源蛋白展示到菌体表面。将重组菌及空质粒菌株分别滴鼻接种SPF级Balb/c小鼠,采集血液样品测定小鼠产生抗F41的特异性IgG,收集小鼠肺部冲洗液、肠道冲洗液、阴道冲洗液及粪便样品测定小鼠产生抗F41的特异性sIgA,并对小鼠进行攻毒保护性试验。【结果】重组干酪乳杆菌pLA-F41/L.casei免疫小鼠能够产生明显的抗F41的sIgA和IgG抗体水平,主动免疫组保护率在85%以上,对照组则全部死亡,被动免疫组新生幼鼠的保护率达80%,对照组保护率仅为5%。【结论】细胞表面锚定ETEC F41菌毛蛋白的重组干酪乳杆菌pLA-F41/L.casei通过滴鼻途径免疫能够有效预防F41型ETEC的感染。

关键词: ETEC F41, 干酪乳酸菌, 滴鼻免疫, 保护率

Abstract:

【Objective】 The use of live Lactobacillus casei as an antigen delivery system elicit mucosal immunity and thus represents a promising strategy for mucosal vaccination to prevent ETEC F41 infection. 【Method】 The recombinant fusion proteins were detected by SDS-PAGE and Western blot. Surface localization of the fusion protein was verified by immunofluorescence microscopy and flow cytometry. Intranasal immunization of SPF Balb/c mice was performed with recombinant strain harboring pLA-F41/L. casei and pLA/L. casei. Specific anti-F41 IgG antibody in the serum and specific anti-F41 secret immunoglobulin A (sIgA) antibody in the lung, intestines, vigna fluid and feces of mice were detected by indirect ELISA after intranasal immunization. The mice intranasaly immunized with pLA-F41/L. casei and pLA/L. casei were challenged with standard-type ETEC F41(C83919). 【Result】 Mice immunized with pLA-F41/L.casei could produce remarkable anti-F41 antibody level. More than 80% of mice survived after challenged with C83919 (2×103LD50). All the mice immunized with a control L. casei harboring pLA vector were died. Eighty-five percent of the pups survived after challenged with C83919, but only a 5% survival rate for pups that were either immunized with a control L. casei harboring pLA vector or unimmunized. 【Conclusion】 These results indicate that mucosal immunization with recombinant L. casei expressing ETEC F41 fimbriae protein on its surface provides an effective means for eliciting protective immune response against the ETEC F41.

Key words: ETEC F41, Lactobacillus casei, intranasal immunization, protection