多房棘球蚴LDH基因的克隆表达及免疫原性研究

发布时间：2018-10-24 14:10

【摘要】：目的克隆表达青海省多房棘球蚴(Echinococcus multilocularis,Em)乳酸脱氢酶(lactate dehydrogenase,LDH)基因,鉴定EmLDH重组蛋白的免疫原性,初步评价其免疫诊断价值。方法采用RT-PCR方法克隆EmLDH基因,将其连接入pET15b表达载体中,构建重组表达质粒pET15b-EmLDH,转化至E.coli Rosetta(DE3)感受态细胞进行诱导表达。通过SDS-PAGE检测重组蛋白的表达形式,Ni-IDA树脂亲和层析纯化重组蛋白。通过Western blotting法鉴定EmLDH重组蛋白的免疫原性,ELISA法检测泡型包虫病患者(57例)、囊型包虫病患者(33例)和正常人(50例)血清,初步评价EmLDH重组蛋白的免疫诊断效果。结果成功克隆了EmLDH基因并表达纯化出相应的重组蛋白。Western blotting结果显示,EmLDH重组蛋白可被泡型和囊型包虫病患者血清识别,而不被正常人血清识别。ELISA结果显示,EmLDH重组蛋白对泡型和囊型包虫病患者血清的诊断敏感性分别为84.21%和84.85%。结论 EmLDH重组蛋白具有较高的免疫原性,对包虫病具有较好的免疫诊断价值。
[Abstract]:Objective to clone and express the lactate dehydrogenase (lactate dehydrogenase,LDH) gene of Echinococcus multilocularis,Em in Qinghai province, identify the immunogenicity of EmLDH recombinant protein and evaluate its immunological diagnostic value. Methods the EmLDH gene was cloned by RT-PCR and ligated into the pET15b expression vector. The recombinant expression plasmid pET15b-EmLDH, was transformed into E.coli Rosetta (DE3)-receptive cells for induction expression. The expression of recombinant protein was detected by SDS-PAGE, and the recombinant protein was purified by Ni-IDA resin affinity chromatography. The immunogenicity of EmLDH recombinant protein was identified by Western blotting method. The serum samples of 57 patients with alveolar hydatid disease, 33 patients with cystic hydatid disease and 50 normal controls were detected by ELISA method. The immunological diagnostic effect of EmLDH recombinant protein was evaluated preliminarily. Results the EmLDH gene was cloned successfully and the corresponding recombinant protein. Western blotting was expressed and purified. The results showed that the recombinant EmLDH protein could be recognized by the sera of patients with vesicular and cystic hydatidosis. ELISA results showed that the diagnostic sensitivity of EmLDH recombinant protein to sera of patients with vesicular and cystic hydatidosis was 84.21% and 84.85%, respectively. Conclusion the recombinant EmLDH protein has high immunogenicity and has a good value for immunological diagnosis of hydatid disease.
【作者单位】：青海大学生态环境工程学院;青海大学附属医院;
【基金】：青海省科技项目(No.2016-ZJ-746)资助~~
【分类号】：R383.33

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