摘要
目的:克隆花生主要过敏原Ara h 8基因,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Ara h 8基因;将反转录的基因连入pMD19-T Simple Vector,提质粒酶切鉴定并测序。将测序正确的片段连入原核表达载体pET-32a(+)上,并转入BL21(DE3)宿主表达菌中;IPTG诱导表达;通过Ni2+亲和层析(FPLC)纯化目的蛋白Ara h 8;Western blot检测该重组蛋白的免疫原性。结果:测序结果表明克隆的花生Ara h 8基因片段全长为474 bp,编码157个氨基酸,与GenBank中蛋白序列100%相同。重组蛋白纯化后经SDS-PAGE鉴定,目的蛋白大小与理论值相符。Western blot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:成功克隆并表达纯化了花生过敏原Ara h 8,该基因表达的重组蛋白具有良好的免疫原性。
Objective：To clone,express and purify the gene of the major allergen Ara h 8 from peanut（Arachis hypogaea L.） and preliminarily characterize the allergenicity of purified recombinant protein.Methods：The ORF of Ara h 8 was cloned and inserted into the expression vector pET-32a（＋）.The vector was transformed into Escherichia coli BL21（DE3） and the protein expression was induced by IPTG.Ni2＋ chelating affinity chromatography was used to purify the recombinant Ara h 8 protein.The allergenicity of Ara h 8 was examined by Western blot.Results：The cloned ORF which contained 474 bp and encoded 157 amino acids was authenticated to be Ara h 8.The recombinant Ara h 8 protein induced by IPTG was consistent with the actual value.The affinity between recombinant Ara h 8 and IgE antibodies from pooled peanut-allergic patient serum was identified by Western blot.Conclusion：Peanut recombinant Ara h 8 protein is obtained with allergenicity.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2011年第4期352-355,共4页
Chinese Journal of Immunology
基金
国家自然科学基金(No.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
深圳大学创新团队基金资助项目(200904)
关键词
花生
过敏原
ARA
H
8
克隆表达
纯化
免疫印迹
Arachis hypogaea L.； Allergen； Ara h 8； Cloning and expression； Purification； Western blot
