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

发布时间：2018-11-15 11:21

【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5μg/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。
[Abstract]:[objective] Foot-and-mouth disease (foot and mouth disease,FMD) is highly infectious and widely spread, which brings great harm to the world economy and social development. At present, vaccination is still the main and effective means of prevention and control. In order to enhance the immune effectiveness of foot-and-mouth disease vaccine, based on the deep understanding and research of flagellin adjuvant at home and abroad, the flagellum of non-pathogenic Escherichia coli was modified, and the flagellin (flagellin,) was studied. F) and different recombinant flagellins with or without LTMT adjuvant for FMDV (foot and mouth disease virse,FMDV. [methods] pCold-F0,pCold-F0-LTMT,pCold-F0NC, was obtained by expression in vitro. The biological activities of pCold-F0-LTMT and pCold-F0NC-LTMT proteins were detected by indirect ELISA method. The vaccine was prepared according to 5 渭 g / mouse protein and FMDV inactivated antigen. At the same time, the Balb/c mice were inoculated subcutaneously with or without ISA 206 adjuvant. The mice were immunized twice every time for 2 weeks. The blood samples were collected before and after immunization, and the feces of mice were collected on day 14, 21, 2835 and 45, respectively. After 45 days, the posterior segment of small intestine was taken, and the titer of serum IgG antibody was detected by blocking ELISA method. Indirect ELISA assay was used to detect the specific secretory IgA (secretory IgA,SIgA antibody titers in mouse feces and intestinal lotions in order to evaluate the immune potency. [results] SDS-PAGE and Western blot analysis showed that four different recombinant flagellins were successfully expressed. The biological activities of pCold-F0-LTMT and pCold-F0NC-LTMT were verified by GM l-ELISA method. The fusion proteins F0-LTMT and F0NC-LTMT retained the ability to bind to GM l. The results of animal experiment showed that the IgG titer of FMD antigen group was higher than that of FMD antigen group, and the titer of sIgA antibody was earlier in FMD antigen group than in FMD antigen group. Higher. In addition, the synergistic effect of LTMT and flagellin could stimulate the production of higher IgG and sIgA. in mice. In the experiment, when the flagellin was combined with ISA 206 adjuvant, the serum IgG titer was significantly increased and the antibody duration was prolonged. Foot-and-mouth disease antigen combined with ISA 206 adjuvant and F0NC-LTMT showed the best protection to FMDV. [conclusion] the data showed that the mucosal adjuvant F0-LTMT had double adjuvant activity. Subcutaneous injection can significantly improve the level of systemic specific IgG and local sIgA of FMDV, which shows that it has a good application prospect.
【作者单位】：江苏省农业科学院兽医研究所/农业部兽用生物制品工程技术重点实验室;吉林农业大学动物科学技术学院;
【基金】：江苏省自主创新资金(cx(15)1060) 国家自然科学基金(31572503)
【分类号】：S855.3

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