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首页> 外文期刊>Journal of Parasitology >Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice.
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Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice.

机译：鉴定了Fasciola hepatica重组15-kDa脂肪酸结合蛋白T细胞表位，可保护兔和小鼠的实验性筋膜病。

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摘要

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.
机译：已经证明，用肝片吸虫（Fasciola hepatica）的脂肪酸结合蛋白（FABP）进行疫苗接种可在小鼠，兔子和绵羊中提供显着水平的抗激发感染的保护。重组15 kDa FABP（rFh15）已被纯化，并且也显示是一种免疫保护分子。从rFh15分子序列2中，鉴定出12个和10个推定的T细胞表位，第一个是氨基酸序列IKMVSSLKTKIT的Fh15Ta，第二个是氨基酸序列VKAVTTLLKA的Fh15Tb。将合成的寡核苷酸分别克隆到pGEX-2TK表达载体中。通过与过量还原型谷胱甘肽竞争性洗脱，使用谷胱甘肽S-转移酶（GST）亲和纯化过表达的融合蛋白。这些GST融合蛋白在弗氏佐剂中乳化以进行兔免疫，或在凝血酶消化后进一步纯化为肽。纯化的12-mer和10-mer肽在弗氏佐剂中乳化以在兔中免疫，或在佐剂适应（ADAD）系统中使用，然后在小鼠和兔中用肝炎隐丝菌进行激发感染。与接受GST免疫的对照组相比，在接受疫苗攻击的兔子中，经GST表位治疗的兔子的保护水平最高（分别降低了Fh15Ta 48.2％和Fh15Tb 59.1％）。此外，与单独使用Fh15Tb（41％）或GST（64％）相比，用Fh15Ta免疫的那些未成熟吸虫的数量更高（84％）。佐剂中用推定的T细胞抗原决定簇免疫的兔子与Fh15Ta相比，吸虫率降低了13％，不成熟的吸虫率最高（46％）。在用致死数量的尾cer虫攻击的接种疫苗的小鼠中，用完全ADAD-GST-Ta处理的CD-1和BALB / c小鼠在感染后47天时，所有组的存活率最高（40％）。因此，Fh15Ta和Fh15Tb多肽表位作为抗肝炎镰刀菌的潜在疫苗值得进一步研究。在小鼠中进行的抗体同种型研究显示，疫苗接种产生了混合的Th1 / Th2反应。

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