鸡Toll样受体15的生物信息学分析及组织表达

Bioinformatics Analysis and Tissue Expression of Chicken Toll-like Receptor 15

Toll样受体是参与机体天然免疫的一类模式分子,可以识别多种病原体相关分子模式,快速启动有效的免疫应答,在机体的抗感染免疫中发挥重要的作用。为了深入探讨鸡Toll样受体的免疫功能,为禽类相关疾病的预防和控制提供依据,试验通过RT-PCR技术克隆海兰褐鸡TLR15基因的全长阅读框,同时将其胞外区片段插入载体p ET32a并导入大肠杆菌BL21(DE3)中进行优化表达,经SDS-PAGE和Western Blot法检测融合蛋白,并将融合蛋白作为免疫原免疫昆明鼠后制备多克隆抗体,同时采用免疫组织化学的方法,对ch TLR15的组织表达进行检测。结果表明,海兰褐鸡TLR15编码基因全长2 607 bp,编码868个氨基酸残基,其中胞外区1 962 bp,重组蛋白分子量约为72 ku;免疫小鼠可以产生特异性的抗体,抗体效价为1∶10 000;免疫组化检测结果显示,ch TLR15主要在脾脏中表达,在肝脏和肺部表达较少。研究结果可为禽类TLR15相关的免疫调节机制分析提供物质基础,也可为后续抗感染生物制剂的开发奠定基础。

Toll-like receptor that involve innate immunity is one kind of model molecule,and it can recognize a wide range of pathogen-associated molecular patterns and has important effects on anti-infectious immunity. The TLR15 gene was cloned from the Hy-Line brown chicken by RT-PCR,the extracellular region of TLR15 gene was expressed in prokaryotic cells. The fusion protein was detected by SDS-PAGE and Western Blot assay,and were used to immunize Kunming-mice to prepare polyclonal antibodies. Then the expression of chTLR15 in the main organs was detected by immunohistochemical method. The results showed that the open reading frame of Hy-Line brown chicken TLR15 gene was composed of 2 607 bp in length encoding 868 amino acids,the extracellular region was 1 962 bp,the molecular weight of recombinant protein was regarding 72 ku,and the fusion protein was used for producing the antiserum in mice with the antibody titer 1 ∶ 10 000. TLR15 protein was mainly expressed in the spleen,but less in liver and lung. The study would provide basic materials for the molecular study on poultry TLR15,and construct a basis for the development of anti-infectious biological agents.