平榛主要过敏原Cor h 1的克隆表达、纯化及免疫学鉴定

Cloning,expression,and identification the gene of Cor h 1 from Corylus Heterophylla

目的克隆、表达榛(平榛)主要变应原基因Cor h1,检测其免疫学活性。方法提取平榛的总RNA,设计带有酶切位点的特异性引物,采用RT-PCR方法扩增目的基因,将其克隆入T载体中进行测序和分析。采用RT-PCR获得整个短的开放阅读框并将目的基因克隆到大肠杆菌表达载体pET28a中进行表达。通过Ni2+亲和层析,对重组蛋白进行纯化,采用免疫印迹(Western-blot)方法检测其IgE结合活性。结果克隆获得了平榛的主要过敏原Cor h1的基因,该基因被GenBank收录,登陆号为EU195058。基因开放阅读框为486个碱基(包括终止密码子),编码161个氨基酸。该序列编码的蛋白等电点为6.16,相对分子质量约为17600。结论成功地克隆和表达了平榛主要变应原Cor h1,蛋白具有良好的免疫原性。

Objective To clone,express,and identify the the gene of Cor h 1 from Corylus heterophylla.Methods The Cor h 1 gene was amplified by PCR and cloned into pMD18-T for sequencing.Then the interesting gene was subcloned into pET-28a for expression.Results The length of gene was 486 bp,coding 161 amino acids.The E-coli strain could express a recombinant protein with a molecular weight of 17 600.Conclusion The gene of Cor h 1 is successfully cloned and expressed in this study,and the recombinant protein possesses ...