Methyltransferases (MTFs) are broad range of enzymes, which are ubiquitously expressed in diverse organisms ranging from bacteria to animals. MTFs proteins have been associated with various biological/cellular processes including transcriptional regulation, subcellular protein and RNA localization, signal transduction and DNA-damage repair. However, the role of MTFs in immune mechanism during host–parasite interaction has not been addressed yet. An open reading frame (764 bp) of methyltransferase-type 12 gene of H. contortus denoted as HcMTF-12, was successfully cloned using reverse transcriptase-polymerase chain reaction (RT-PCR) followed by prokaryotic expression in Escherichia coli BL21 (DE3 strain). The recombinant HcMTF-12 protein (rHcMTF-12) was about 47 kDa along with a fusion vector protein of 18 kDa. Immunoblot results identified the native protein MTF-12 with antibodies produced in rats against rHcMT-12, whereas rHcMTF-12 protein was recognized with sera of goat experimentally infected with H. contortus. Immunohistochemical analysis revealed that the native MTF-12 protein was mainly located in the periphery (cuticle) of parasite sections as well as within the pharynx and intestinal region. An immunofluorescence assay validated that rHcMTF-12 attached to the surface of goat PBMCs. Furthermore, the cytokines transcription of IL-2, IFN-γ and IL-4 transcripts of PBMCs incubated with rHcMTF-12 were enhanced in a dose-dependent manner. The secretion of TGF-β1 and IL-10 was significantly decreased. However, IL-6 production was not significantly different as compared to the control groups. Moreover, the migration activity and nitric oxide (NO) production by PBMCs were induced considerably, whereas the proliferation of PBMCs cells was negatively affected when incubated with the rHcMTF-12 protein. Our findings suggest that HcMTF-12 significantly mediated the functions of PBMCs, and it might be a potential candidate for therapeutic interventions against haemonchosis.

中文翻译：

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捻转血矛线虫新型甲基转移酶12型蛋白的鉴定及其对山羊PBMCs功能的影响

甲基转移酶（MTF）是广泛的酶，广泛存在于从细菌到动物的多种生物中。MTFs蛋白已与各种生物/细胞过程相关，包括转录调控，亚细胞蛋白和RNA定位，信号转导和DNA损伤修复。但是，尚未解决MTF在宿主与寄生虫相互作用期间在免疫机制中的作用。使用逆转录酶-聚合酶链反应（RT-PCR）成功克隆了Con。contortus甲基转移酶12型基因的开放阅读框（764 bp），然后在大肠杆菌BL21（DE3菌株）中进行原核表达）。重组HcMTF-12蛋白（rHcMTF-12）约为18 kDa的融合载体蛋白。免疫印迹结果鉴定了天然蛋白MTF-12，其具有在大鼠中产生的针对rHcMT-12的抗体，而rHcMTF-12蛋白则被实验性感染了Contortus的山羊血清所识别。免疫组织化学分析表明，天然MTF-12蛋白主要位于寄生虫部分的周围（表皮）以及咽和肠区域。免疫荧光分析证实rHcMTF-12附着在山羊PBMC的表面。此外，与rHcMTF-12一起孵育的PBMC的IL-2，IFN-γ和IL-4转录本的细胞因子转录以剂量依赖的方式增强。TGF-β1和IL-10的分泌明显减少。然而，与对照组相比，IL-6的产生没有显着差异。此外，与rHcMTF-12蛋白一起孵育时，PBMC的迁移活性和一氧化氮（NO）的产生被大量诱导，而PBMCs细胞的增殖受到负面影响。我们的发现表明，HcMTF-12显着介导了PBMC的功能，它可能是针对红血球病的治疗性干预的潜在候选者。