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Position: Home > Articles > Gene Cloning and Prokaryotic Expression of Peanut Allergen Ara h 6 FOOD SCIENCE 2016,37 (3) 125-130

花生过敏原蛋白Ara h 6基因克隆和原核表达

作  者：
詹少德;邱昌将;朱盼;吴志华;陈红兵
单  位：
浙江纺织服装职业技术学院;广东省疾病预防控制中心;南昌大学食品科学与技术国家重点实验室
关键词：
花生;过敏原;Ara h 6;基因克隆表达;质谱鉴定
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。
译  名：
Gene Cloning and Prokaryotic Expression of Peanut Allergen Ara h 6
作  者：
ZHAN Shaode;QIU Changjiang;ZHU Pan;WU Zhihua;CHEN Hongbing;State Key Laboratory of Food Science and Technology, Sino-German Joint Research Institute, Nanchang University;Zhejiang Fashion Institute of Technology;Center for Disease Control and Prevention of Guangdong Province;
关键词：
peanut;;allergen;;Ara h 6;;recombinant expression;;mass spectrometry
摘  要：
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.

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