摘要
本实验首先从花生中提取总RNA,利用反转录聚合酶链式反应技术克隆了花生过敏原蛋白Ara h 6全c DNA,并以此为模板扩增出Ara h 6目的基因。将目的基因与p MD19-T Simple质粒进行重组后转入BL21(DE3)宿主表达菌中,异丙基-β-D-硫代吡喃半乳糖苷诱导产物表达,并利用镍离子亲和层析纯化表达产物。DNA测序结果显示Ara h 6基因片段全长为438 bp,编码145个氨基酸,与已知该蛋白DNA序列97%相同;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳结果显示表达产物分子质量为24 k D,与融合组氨酸标签的重组Ara h 6蛋白理论分子质量相符;质谱鉴定结果表明重组蛋白的一级结构与天然Ara h 6匹配度为100%;Western blotting结果显示融合蛋白能够为抗Ara h 6多克隆抗体所识别,具有免疫原性。
Ara h 6 is one of the major peanut allergens. Ara h 6 c DNA was synthesized from total RNA using Oligo primers by reverse transcription-polymerase chain reaction（RT-PCR） in order to provide a template for the PCR amplifi cation of Ara h 6 gene. The target gene was cloned into p MD19-T vector to construct a recombinant vector. Then the recombinant vector was transferred into the bacterial expression host BL21（DE3）. IPTG was used t o induce protein expression, and the expressed product was purifi ed by Ni affi nity chromatography. DNA sequence analysis showed that the full-length gene fragment of Ara h 6 was 438 bp and encoded 145 amino acids, which was 97% identical to the known DNA sequence. Sodium dodecyl sulfate-polyacryl amide gel electrophoresis（SDS-PAGE） results showed that the molecular weight of the expressed fragment was 24 k D, which matched with the theoretical value of the His-tagged fusion recombinant protein Ara h 6. Mass spectrometric results showed that the matching degree of structure was 100% between the recombinant protein and natural Ara h 6. Western blotting indicated that the protein could be recognized by anti-Ara h 6 polyclonal antibody, and has strong immunogenicity.
出处
《食品科学》
EI
CAS
CSCD
北大核心
2016年第3期125-130,共6页
Food Science
基金
“十二五”国家科技支撑计划项目(2011BAK10B03)
国家高技术研究发展计划(863计划)项目(2013AA102205)
江西省科技支撑计划项目(20133BBF60004)
