| 摘要: |
| 【目的】研究绵羊肝片吸虫（Fasciola hepatica，Fh）CatL1D蛋白的分子特征和免疫原性。【方法】利用RT-PCR技术扩增出CatL1D基因，用相关软件分析其编码蛋白的生物学信息。将CatL1D基因克隆入表达载体pET-32a(+)中，构建原核表达载体pET-CatL1D，进行Bam HⅠ/XhoⅠ双酶切和测序鉴定。将pET-CatL1D载体转化入大肠杆菌BL21(DE3)感受态细胞，IPTG诱导表达后进行SDS-PAGE和Western blot分析；以纯化后的重组蛋白CatL1D为抗原免疫小鼠，利用间接ELISA方法检测抗体效价，分析其免疫原性。【结果】成功克隆了CatL1D基因，该基因全长981 bp，编码326个氨基酸，其中第21-79位氨基酸为组织蛋白酶前肽抑制剂结构域，第108-321位氨基酸为木瓜蛋白酶家族半胱氨酸蛋白酶保守结构域；CatL1D蛋白含有6个酪蛋白激酶Ⅱ磷酸化位点、11个豆蔻酰化位点、1个蛋白激酶C磷酸化位点，含有10个优势抗原表位。成功构建了pET-CatL1D原核表达载体，诱导表达出CatL1D重组蛋白；SDS-PAGE和Western blot分析表明，重组蛋白CatL1D分子质量为51 ku，以包涵体的形式表达，具有较强的反应原性。间接ELISA法检测结果表明，重组蛋白CatL1D免疫小鼠血清抗体效价可达1∶102 400，证实其具有良好的免疫原性。【结论】Fh重组蛋白CatL1D具有较强的抗原性，有开发为早期诊断抗原和亚单位疫苗的潜力。 |
| 关键词: 肝片吸虫 组织蛋白酶L1D 分子特征 免疫原性 |
| DOI： |
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| 基金项目:国家重点研发计划项目（2017YFD0501200）；兵团国际科技合作项目（2016AH006） |
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| Immunogenicity of CatL1D protein in Fasciola hepatica |
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WANG Xifeng,MENG Qingling,QIAO Jun,et al
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| Abstract: |
| 【Objective】The molecular characteristics and immunogenicity of CatL1D protein in Fasciola hepatica (Fh) were studied.【Method】The CatL1D gene was amplified by RT-PCR and the biological information of its encoded protein was analyzed by online software.The CatL1D gene was cloned into the expression vector pET-32a(+) to construct the prokaryotic expression vector pET-CatL1D,and it was identified by BamHⅠ/XhoⅠ double enzyme digestion and sequencing.The pET-CatL1D vector was transformed into E.coli BL21 (DE3),the expression was induced by IPTG,and the expressed products were analyzed by SDS-PAGE and Western blot.The purified recombinant protein CatL1D was used as antigen to immunize mice.The antibody titer was detected by indirect ELISA and the immunogenicity was analyzed.【Result】The CatL1D gene was cloned successfully with 981 bp and encoding 326 amino acids.It had a cathepsin propeptide inhibitor domain at positions 21-79 and had a conserved papain family of cysteine protease domain at amino acids 108-321;The Cat1D protein contains six casein kinase Ⅱ phosphorylation sites,eleven myristoylation sites and a protein kinase C phosphorylation site;also containing ten dominant antigen epitopes.The pET-CatL1D prokaryotic expression vector was successfully constructed and the expression of CatL1D recombinant protein was induced.SDS PAGE and Western blot analysis showed that the molecular weight of recombinant protein CatL1D was 51 ku.It expressed as inclusion body and had good reactivity.The indirect ELISA result showed that the titer of serum antibody of mice immunized with recombinant protein CatL1D was 1∶102 400,which proved its good immuoogeneticity.【Conclusion】The Fh CatL1D recombinant protein is highly antigenic and has potential for the development of early diagnostic antigens and subunit vaccines. |
| Key words: Fasciola hepatica CathepsinL1D molecular characteristics immunogenicity |
