摘要
为获得重组花生过敏原Ara h2。通过RT-PCR合成cDNA,并以此为模板进行PCR扩增目的基因Ara h2,扩增产物经纯化后克隆至pMD19-TSimple载体中,构建重组质粒pMD19-T-Ara h2。上述重组质粒经酶切纯化后定向克隆到pGEX-4T-1表达载体中,构建原核表达载体pGEX-4T-1-Ara h2,并转化表达宿主菌BL21-codonPlus(DE3)-RIPL中,经IPTG诱导表达。SDS-PAGE电泳结果表明,该表达蛋白大小约为46kD,与理论值相符。通过Glutathione Sepharose 4B凝胶亲和层析方法纯化融合蛋白GST-Ara h2,获得融合蛋白纯度约为90%。Western blotting分析表明,经纯化的融合蛋白能与抗Ara h2兔血清发生特异性反应,说明该蛋白具有良好的免疫原性。
Ara h 2 is one of paramount allergens in peanut.The goal of the present study was to biosynthesize recombinant peanut allergen Ara h 2.Peanut cDNA was synthesized from total RNA using Oligo primers by RT-PCR in order to provide a template for the PCR amplification of Ara h 2 gene.The purified amplification products were cloned into the pMD19-T simple vector to construct a recombinant vector carrying Ara h 2 gene,named pMD19-T-Ara h 2.The recombinant plasmid was digested by Nco I and Hind III.The purified digestion products were then ligated to the pGEX-4T-1 expression vector and transformed into the BL21-codonPlus（DE3）-RIPL for 24 h expression under IPTG induction.The target product,GST-Ara h 2 fusion protein,was purified with Glutathione Sepharose 4B.The results of SDS-PAGE and western-blotting showed that GST-Ara h 2 fusion protein was 46 kD in size,with 90% purity and could specifically react with anti-Ara h 2 sera from rabbits,indicating that the recombinant protein has high specificity of immune reaction.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2010年第15期203-207,共5页
Food Science
基金
南昌大学食品科学与技术国家重点实验室目标导向项目(SKLF-MB-200807)
江西省主要学科学术和技术带头人培养项目([2004]234号)
教育部新世纪优秀人才支持计划项目(NCET-08-07-04)
