非致病性大肠杆菌鞭毛蛋白对O型口蹄疫病毒的佐剂效果

doi:10.3864/j.issn.0578-1752.2017.09.017

摘要/Abstract

摘要： 【目的】口蹄疫（foot and mouth disease, FMD）的传染性强，传播广，给世界经济和社会发展带来巨大危害，目前疫苗接种仍是主要而有效的防控手段。以增强口蹄疫疫苗免疫效力为目的，基于国内外对鞭毛蛋白佐剂的深入了解与研究，对非致病性大肠杆菌鞭毛进行修饰，研究鞭毛蛋白（flagellin, F）以及有无LTMT佐剂不同重组鞭毛蛋白对口蹄疫病毒(foot and mouth disease virse, FMDV)的佐剂作用。【方法】通过体外表达获得pCold-F0、pCold-F0-LTMT、pCold-F0NC、pCold-F0NC-LTMT 4种不同重组鞭毛蛋白，用间接ELISA方法检测pCold-F0-LTMT和pCold-F0NC-LTMT蛋白的生物学活性，按照5 μg/只小鼠蛋白量与FMDV灭活抗原混合制备疫苗，同时设置添加或者不添加ISA 206佐剂组，皮下接种Balb/c小鼠，共免疫2次，每次间隔2周。分别于免疫前和免疫后 14、21、28、35和45 d采血并采集小鼠粪便，45 d后取小肠后段，采用阻断ELISA方法检测血清IgG抗体滴度，用间接ELISA方法检测小鼠粪便和肠洗液中特异的分泌性IgA（secretory IgA, SIgA）抗体滴度，以评估免疫效力。【结果】SDS-PAGE 和Western blot分析表明，4种不同重组鞭毛蛋白成功表达。并用GM l-ELISA方法验证了pCold-F0-LTMT和pCold-F0NC-LTMT的生物学活性，融合蛋白F0-LTMT和F0NC-LTMT保留了与GM l结合能力。动物试验结果表明，疫苗经皮下注射接种后，单独口蹄疫抗原组没有产生明显的抗体水平，而鞭毛蛋白佐剂组比单独的口蹄疫抗原组，IgG滴度高，并且产生sIgA抗体滴度更早、更高。此外，LTMT与鞭毛蛋白协同作用能刺激小鼠机体产生更高的IgG和sIgA。在试验中，当鞭毛蛋白与ISA 206佐剂联合使用时，血清中IgG滴度大幅增高，且抗体持续期延长。口蹄疫抗原与ISA 206佐剂和F0NC-LTMT混合使用效果最佳，显示对FMDV的最好保护。【结论】数据表明，黏膜佐剂F0-LTMT发挥双重佐剂活性，皮下注射可显著提高FMDV全身特异性IgG和局部sIgA水平，显示其具有良好的应用前景。

关键词: 口蹄疫病毒, 非致病性大肠杆菌, 鞭毛蛋白, 佐剂

Abstract: 【Objective】Foot and mouth disease (FMD) is highly infections and spread wide. It has brought a great harm to the world economic and social development. The current vaccination is still the main and effective means of prevention and control of this disease. To enhance FMD Virus (FMDV) antigenicity, the Flagella (F0) was modified from the non-pathogenic Escherichia coli to construct recombinant F0 protein and evaluate the single F0 protein and fusion protein F0-LTMT adjuvant effectiveness on FMDV based on the research about flagella protein adjuvants from home and abroad.【Method】Four recombinant proteins of F0, F0-LTMT, F0NC, and F0NC-LTMT were expressed, and their biological activities were detected by GM1-ELISA. The FMDV immunogen was prepared by mixing inactivated FMDV with a single recombinant F0 at 5 μg per mouse, and additional ISA 206 adjuvant was set according to the proportion to evaluate the immune effect in mice. The Balb/c mice was subcutaneously inoculated with emulsified immunogen twice at intervals of 2 weeks. Sera and feces were collected, respectively, on the day 0, 14, 21, 28, 35 and 45, and distal intestine was also collected after immunization on day 45. Titers of IgG in serum were detected by blocking ELISA kit, specific sIgA in feces and intestinal liquid by indirect ELISA to evaluate the titers.【Result】SDS-PAGE and Western blot analyses showed that four different recombinant flagellin were successfully obtained. Their biological activities were verified by GM l-ELISA. In mice no significant levels of antibodies were detected in the single FMDV immunized group compared with higher IgG titer sIgA titers in flagellin adjuvant group. Moreover, the synergistic effect of LTMT and flagellin facilitated to produce more IgG and sIgA in mice. When flagellin combined with the ISA 206 adjuvant, the titer of serum IgG increased significantly and gave extended duration. The mixed immunogen of FMDV, ISA 206 and H0NC-LTMT displayed the best antibody titers.【Conclusion】F0-LTMT provided dual adjuvant effectiveness to FMDV, induced systemic specific IgG and sIgA in mice. It is a potential mucosal adjuvant for FMDV.

Key words: FMDV, Escherichia coli, flagellin, mucosal adjuvant

孙小涵，张碧成，张强，何孔旺，张雪寒. 非致病性大肠杆菌鞭毛蛋白对O型口蹄疫病毒的佐剂效果[J]. 中国农业科学, 2017, 50(9): 1714-1722.

SUN XiaoHan, ZHANG BiCheng, ZHANG Qiang, HE KongWang, ZHANG XueHan. Adjuvant Effects of Flagellin from Non-Pathogenic E.coli on FMDV[J]. Scientia Agricultura Sinica, 2017, 50(9): 1714-1722.

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