A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome a...A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags （ESTs）, we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription.展开更多
利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹...利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western Blotting鉴定该单克隆抗体的特性和交叉性。利用双单抗夹心ELISA法检测大豆过敏原。结果表明:获得6株可稳定分泌鼠抗大豆主要过敏原Gly m Bd 30K蛋白的单克隆抗体,分别命名为1C10,1D12,2D1,4B4,5F9,6B12,其Ig亚型除1D12和4B4为IgG2a外,其余均为IgG1,且6株单抗效价均在10-5以上。ELISA和Western Blotting分析表明该6株单抗均能特异性识别大豆主要过敏原Gly m Bd 30K蛋白,并且建立双单抗夹心ELISA的方法可以准确检测出大豆过敏原的存在。鼠抗大豆主要过敏原Gly m Bd 30K蛋白抗原表位区蛋白的单克隆抗体的成功制备,以及双单抗夹心ELISA检测系统的建立,为大豆主要过敏原蛋白的检测奠定了基础,也可以为食品中大豆过敏原的检出提供依据。展开更多
基金This work was supported by the National Basic Research Program of China, the National Scientific and Technological Project and the National Natural Science Foundation under grant No. 2005 CB 121000, 2005 B A711A07 and 30471313, respectively.
文摘A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags （ESTs）, we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription.
文摘利用大豆主要过敏原Gly m Bd 30K蛋白抗原表位蛋白为免疫原免疫BALB/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤NS-1细胞融合。采用半固体培养基法和有限稀释法相结合的方法快速筛选获得稳定分泌的特异性杂交瘤细胞,用杂交瘤细胞株诱生小鼠腹水,应用蛋白A亲和层析法进行抗体纯化。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA、Western Blotting鉴定该单克隆抗体的特性和交叉性。利用双单抗夹心ELISA法检测大豆过敏原。结果表明:获得6株可稳定分泌鼠抗大豆主要过敏原Gly m Bd 30K蛋白的单克隆抗体,分别命名为1C10,1D12,2D1,4B4,5F9,6B12,其Ig亚型除1D12和4B4为IgG2a外,其余均为IgG1,且6株单抗效价均在10-5以上。ELISA和Western Blotting分析表明该6株单抗均能特异性识别大豆主要过敏原Gly m Bd 30K蛋白,并且建立双单抗夹心ELISA的方法可以准确检测出大豆过敏原的存在。鼠抗大豆主要过敏原Gly m Bd 30K蛋白抗原表位区蛋白的单克隆抗体的成功制备,以及双单抗夹心ELISA检测系统的建立,为大豆主要过敏原蛋白的检测奠定了基础,也可以为食品中大豆过敏原的检出提供依据。
