论文部分内容阅读
目的克隆、表达和纯化鸡蛋主要过敏原Gald1区基因,并对其免疫学活性进行鉴定。方法用生物信息学方法对鸡蛋主要过敏原Gald1基因进行抗原表位预测,设计特异性引物,用PCR的方法分别扩增Gald1N端和C端基因,将其分别连接到原核表达载体PET32a上。重组质粒转入大肠杆菌,用IPTG诱导表达,通过镍亲和柱层析法纯化重组蛋白,最后用Western-blotting检测重组蛋白的免疫原性。结果表达的蛋白均以可溶性为主,N端融合蛋白相对分子质量为28600,C端融合蛋白相对分子质量为26800,纯化的N端和C端蛋白均有较好的免疫原性,C端的免疫原性强于N端。结论成功地克隆和表达了鸡蛋主要变应原基因Gald1的N端和C端,为鸡蛋过敏的诊断和免疫治疗奠定了基础。
Objective To clone, express and purify the Gald1 gene of egg major allergen and identify its immunological activity. Methods The bioinformatics method was used to predict the epitopes of the Gald1 gene. The specific primers were designed. The Gald1 N-terminal and C-terminal genes were amplified by PCR and ligated to the prokaryotic expression vector PET32a respectively. Recombinant plasmids were transformed into E. coli and induced with IPTG. The recombinant protein was purified by nickel affinity column chromatography. Finally, the immunogenicity of the recombinant protein was detected by Western-blotting. The results showed that all of the expressed proteins were soluble. The relative molecular mass of the N-terminal fusion protein was 28600 and the relative molecular mass of the C-terminal fusion protein was 26800. The purified N-terminal and C-terminal proteins had good immunogenicity and C-terminal immunity The original is stronger than the N terminal. Conclusion The N-terminal and C-terminal of Gald1, the major allergen gene of egg, were successfully cloned and expressed, which laid the foundation for the diagnosis and immunotherapy of egg allergy.