边缘革蜱Dm86基因表达及免疫原性分析

Expression of Dm86 Gene of Dermacenter Marginatus and Analysis of Immunogenicity

【目的】研究Bm86同系物Dm86基因及其编码蛋白,为抗边缘革蜱疫苗候选抗原提供基础。【方法】以边缘革蜱饱血雌蜱c DNA为模板扩增克隆Dm86基因,分析生物学特性及生活史各阶段表达水平,截短基因构建重组质粒,经IPTG诱导后SDS-PAGE鉴定,纯化蛋白,制备多克隆抗体进行Western Blotting鉴定。【结果】PCR扩增获得了1 773 bp目的片段,Dm86蛋白由591个氨基酸组成,分子量为64 857.81,理论等电点为6.78,酸性,具有不稳定性、亲水性和多个B细胞抗原表位;Dm86基因在边缘革蜱不同阶段均有不同程度的表达,饱血蜱阶段的表达量较高;重组蛋白大小为39kDa,以包涵体形式表达;抗体效价可达1∶25 600,能够识别重组蛋白。【结论】Dm86重组蛋白具有一定的反应原性和免疫原性。

【Objective】To understand Dm86 gene and its coding protein of Bm86 homologue,provide the basis for anti-Dermacenter marginatus vaccine candidate antigen.【Method】this experiment amplified the Dm86 gene using the cDNA of fulminated female ticks,truncated genes to construct recombinant plasmids,SDS-PAGE identified after IPTG induction,purified protein and prepared polyclonal antibodies for Western Blotting identification.【Result】The target fragment of 1,773 bp was obtained by PCR amplification. According to the analysis of biological characteristics,Dm86 protein was presumed to be composed of 591 amino acids,with molecular weight of 64857.81 and theoretical isoelectric point of 6.78. It was acidic,had unstable hydrophilicity and multiple B cell epitopes. Fluorescence quantitative PCR results showed that Dm86 gene was expressed to different degrees in different stages of ticks,and the expression level was higher in ticks at satiety stage. High antigenicity truncated gene building proteins. SDS-PAGE results showed that specific bands appeared around 39 kDa and expressed as an inclusion body. The titer of polyclonal antibody was 1∶25600,Western blot results show that,the recombinant protein can react with polyclonal antibody to produce specific bands.【Conclusion】Recombinant protein Dm86 has certain reactivity and immunogenicity,which can lay the foundation for the study of candidate antigens of vaccine against D. marginatus.