【摘要】 为研究火鸡组织滴虫(Histomonas meleagridis)胞质烟酰胺腺嘌呤二核苷酸磷酸(NADP)依赖性苹果酸酶(ME 1)的抗原性及其在火鸡组织滴虫中的定位，本研究根据本实验室前期对火鸡组织滴虫ME 1基因的测序结果设计引物，以虫体cDNA为模板，经RT-PCR扩增火鸡组织滴虫ME 1 (HmME 1)基因，结果显示Hm ME 1基因全长1 182 bp，将其克隆至p ET28a(+)构建重组表达载体后转化BL21 (DE3)，经IPTG诱导表达后，利用Ni-NTA亲和层析柱纯化重组蛋白r Hm ME 1，经SDS-PAGE和western blot鉴定，结果显示，r Hm ME 1以可溶形式存在，相对分子量约为46 ku，且纯化的r Hm ME 1条带单一，浓度为0.4 mg/mL。将该纯化的r Hm ME 1免疫小鼠，制备该重组蛋白的鼠抗血清(多克隆抗体)，采用间接ELISA方法检测多克隆抗体的效价，结果显示制备的多克隆抗体效价为1∶409 600。采用western blot分别检测r Hm ME 1与制备的鼠源r Hm ME 1阳性血清和鸡源火鸡组织滴虫阳性血清的反应原性，以及鉴定虫体天然表达的Hm ME 1蛋白与制备的鼠源r Hm ME 1阳性血清的反应原性，结果显示r Hm ME 1蛋白能同时被鸡源火鸡组织滴虫阳性血清及鼠源r Hm ME 1阳性血清特异性识别，制备的鼠源r Hm ME 1阳性血清也可以与虫体天然表达的Hm ME 1蛋白反应。利用间接免疫荧光试验(IFA)对虫体内的Hm ME 1蛋白进行定位分析，结果显示Hm ME 1广泛存在于火鸡组织滴虫的胞质内。上述结果首次表明，Hm ME 1的反应原性及抗原性均较强，可以作为火鸡组织滴虫重组亚单位疫苗研制的潜在靶标，该结果为进一步研究火鸡组织滴虫的致病机制及疫苗研制奠定了基础。更多还原

【Abstract】 In order to study the antigenicity and localization of cytosolic NADP-dependent malic enzyme(ME 1) in Histomonas meleagridis(HmME 1), primers were designed according to the ME 1 gene sequence in transcriptome sequencing results of H.meleagridis. The Hm ME 1 gene amplified by RT-PCR had a total length of 1182bp. The Hm ME 1 gene was cloned into prokaryotic expression vector pET28a(+), and then induced by IPTG. SDS-PAGE analysis showed that r Hm ME 1 protein mainly expressed in supernatant and the molecular weight was about 46ku. The recombinant protein(rHmME 1) was purified by HIS tag chromatography and identified by SDS-PAGE and western blot. The concentration of the purified r Hm ME 1 was0.4mg/mL. The positive serum(polyclonal antibody) was prepared by immunizing the mouse with the purified r Hm ME 1 and the titer was detected by indirect ELISA. The titer of the serum antibody was 1 ∶409 600. Western blot results showed that r Hm ME 1could be specifically recognized by mouse positive serum anti-rHmME 1 and chicken positive serum anti-H.meleagridis, and the mouse positive serum anti-rHmME 1 could also react with the natural Hm ME 1 protein. The distributions of natural Hm ME 1protein was detected by indirect immunofluorescence localization(IFA), and the results showed that the protein was widely distributed in the cytoplasm. Our results indicated that Hm ME 1 had high antigenicity, and it might be a new candidate for the recombinant subunit vaccines, which lay a foundation for exploring the pathogenic mechanism of H.meleagridis.更多还原