摘要
目的:克隆花生主要过敏原Arah6基因,诱导表达并纯化该蛋白,检测其免疫活性。方法:提取花生总RNA,设计特异性引物,RT-PCR克隆花生Arah6的基因,将测序正确的片段连入原核表达载体pET-28a上,并转入BL21(DE3)宿主表达菌中,IPTG诱导表达,通过Ni2+亲和层析柱纯化目的蛋白,Western-blotting检测该重组蛋白的免疫原性。结果:测序结果表明花生Arah6目的片段全长为438bp,编码145个氨基酸,与GenBank中蛋白序列100%相同。该基因诱导表达的产物纯化后经SDS-PAGE鉴定为17kDa。Western-blotting结果表明该蛋白与花生过敏病人混合血清中IgE结合,具有免疫原性。结论:成功克隆花生过敏原Arah6的基因,该基因表达的重组蛋白具有良好的免疫原性。
Objective： To clone and express the gene of the major allergen Ara h6 from peanut（Arachis hypogaea L） and preliminarily characterize the resulting purified recombinant protein.Method：The ORF of Ara h6 was cloned and inserted into the expression vector pET-28a.The vector was transformed into Escherichia coil BL21 and the protein expression was induced by IPTG.Ni^2＋ chelating affinity chromatography was used to purify the recombinant Ara h6 protein. The allergenicity of Ara h6 was examined by Western-blotting. Result： The cloned ORF witch contained 438bp and encoded 145 amino acids was authenticated to be Ara hd.High yield recombinant Ara h6 was obtained upon IPTG induction and was purified by Ni^2＋ chelating affinity chromatography. The affinity between recombinant Ara h6 and IgE antibodies from pooled peanut-allergic patient sera was identified by Western- blotting.Conclusion：Peanut recombinant Ara h6 protein was obtained with allergenicity.
出处
《食品工业科技》
CAS
CSCD
北大核心
2010年第10期173-175,181,共4页
Science and Technology of Food Industry
基金
国家自然科学基金(30871752)
深圳出入境检验检疫局科技计划项目(SZ2008105)
深圳市深港创新圈项目
