大肠杆菌毒素LT的B亚基与犬新孢子虫截断的rROP2蛋白融合可诱导小鼠Th2/Th17混合免疫反应

Alceu Gonçalves Santos Junior, Renan Eugênio Araújo Piraine, Lívia Budziarek Eslabão, Leandro Quintana Nizoli, Rodrigo Casquero Cunha, Fabrício Rochedo Conceição, Fábio Pereira Leivas Leite
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摘要

犬新孢子虫ROP2蛋白是一种很有前途的控制新孢子虫病的疫苗抗原候选物。在这项研究中,我们在小鼠模型中评估了对大肠杆菌热不稳定B部分毒素(LTB)形成的嵌合体的免疫反应及其与截断的犬链球菌ROP2的关联。重组蛋白在大肠杆菌中表达,保留了特异性抗体识别的抗原表位。用自然感染牛的血清和实验感染鼠的血清分别用免疫印迹法和ELISA法验证了抗原性。免疫原性通过小鼠血清总IgG、IgG1和IgG2a同型进行评价。采用qPCR检测各组脾细胞IL-10、IL-12、IL-17A和IFN-y的转录水平。嵌合构建的rLTB/tROP2调节了接种后第14天抗rROP2总抗体滴度的显著升高(p<0.05),而rROP2在疫苗强化后直至实验结束诱导的总IgG滴度均升高(p<0.05)。rROP2刺激两组脾细胞均诱导IL-10(18-27倍)和IL-12(7-12倍)转录显著升高(p<0.05), IL-17(40-3000倍)转录显著升高(p<0.0001)。rLTB/tROP2刺激后,IL-10(46-76倍)、IL-12(4-25倍)和IL-17(40-400倍)转录显著升高(p<0.05)。我们的研究结果表明,这些重组蛋白能够调节混合的Th2/Th17免疫反应,这表明它们可能是一种有希望用于控制犬瘟的疫苗。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
THE B SUBUNIT OF Escherichia coli TOXIN LT FUSED WITH TRUNCATED rROP2 PROTEIN OF Neospora caninum INDUCES MIXED Th2/Th17 IMMUNE RESPONSE IN MICE
Neospora caninum ROP2 protein is a promising vaccine antigen candidate for controlling neosporosis. In this study, we evaluated the immune response against a chimera formed by Escherichia coli heat labile B portion of the toxin (LTB) and its association with a truncated N. caninum ROP2 in a murine model. The recombinant proteins were expressed in E. coli, maintaining antigenic epitopes that were recognized by specific antibodies. Antigenicity was verified by western blot using serum from naturally infected cattle, and by ELISA using serum from mice infected experimentally with N. caninum. Immunogenicity was evaluated by total serum IgG, IgG1 and IgG2a isotypes in mice vaccinated with the experimental vaccines. Splenocyte transcription of the cytokines IL-10, IL-12, IL-17A and IFN-y were analyzed by qPCR. The chimeric construction rLTB/tROP2 modulated a significant increase (p<0.05) in total antibody titer against rROP2 by day 14 post inoculation, whereas the rROP2 induced higher (p<0.05) total IgG after the vaccine boost until the end of the experiment. Splenocytes from both groups stimulated with the rROP2 induced significant (p<0.05) higher IL-10 (18-27 folds), and IL-12 (7-12 folds) transcription, with a significant (p<0.0001) fold transcription increase for IL-17 (40-3000 folds). When stimulated with rLTB/tROP2, a significant (p<0.05) higher IL-10 (46-76 folds), IL-12 (4-25 folds) and IL-17 (40-400 folds) transcription were observed. Our results exhibit evidence that these recombinant proteins were able to modulate a mixed Th2/Th17 immune response, suggesting they may be a promising vaccine to be used for the control of N. caninum.
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