大豆主要过敏原β-伴大豆球蛋白β亚基的抗原表位分析及分段克隆

Analysis of antigenic epitopes and cloning of soybean major allergen β-conglycinin beta-subunit

利用生物信息学软件SWISS-MODEL对大豆主要过敏原β-伴大豆球蛋白β亚基建立出三级结构模型,并根据Disco Tope 2.0服务器预测出该模型的构象表位相关区段,采用分段克隆方式扩增目的基因的3个片段区域,利用PCR技术扩增目的基因全长及3个互相重叠的片段(A、B、C)将其连接至p MD18-T载体,并转化到大肠杆菌感受态JM109。经蓝白斑筛选,以及对重组质粒进行PCR和双酶切验证,测序结果与设计的基因序列一致,成功得到了目的基因片段的阳性克隆子,为β-伴大豆球蛋白β亚基分段表达及抗原区域位置的筛选与鉴定提供参考。

The model of tertiary of β-conglycinin beta-subunit was constructed by the bioinformation tool SWISS-MODEL,and the conformational epitopes were predicted by Disco Tope 2.0 server.The overlapping fragments of β-conglycinin beta-subunit genes were amplified by PCR and inserted into p MD-18 T vector,then preserved into competent cell of Escherichia coli JM109.The recombinant plasmids were evaluated by blue-white selection,PCR and double enzyme digestion,and DNA sequence analysis showed that the cloned sequence was the very fragment designed. The successful cloning of the genes can provide a reference for the study of the expresstion of β-conglycinin beta-subunit and screening of antigenic epitopes.