| 用高度特异性王浆主蛋白1多克隆抗体ELISA法检测蜂王浆新鲜度(英文) |
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| 作者姓名： | Li-rong SHEN Yi-ran WANG Liang ZHAI Wen-xiu ZHOU Liang-liang TAN Mei-lu LI Dan-dan LIU Fa XIAO |
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| 作者单位： | Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, Department of Food Science and Nutrition,Zhejiang University |
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| 基金项目： | supported by the Public Beneficial Scientific&Technical Plan of Zhejiang(No.2011C22039);the Important Scientific & Technical Plan of Zhejiang(No.2011C12023);the Important Scientific & Technical Innovation Project of Hangzhou(No.20131812A25);the Foundation of Fuli Institute of Food Science of Zhejiang University(No.KY201404);the National Natural Science Foundation of China(No.31271848) |
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| 摘 要： | 目的:为蜂王浆主蛋白1(MRJP1)的快速检测和鉴别提供科学依据,为蜂王浆的质量控制提供技术支持。创新点:首次比较了MRJP1特异性多克隆抗体与MRJP1重组表达蛋白多克隆抗体对王浆主蛋白家族的免疫反应差异,验证了蜂王浆中MRJP1蛋白降解与保温时间的相关性,建立了以MRJP1作为蜂王浆新鲜度生物标志物的快速检测方法。方法:通过蜂王浆主蛋白家族蛋白的氨基酸序列同源性分析,筛选出MRJP1的特异性多肽区域,进行人工合成,免疫兔子后取血清制备成特异性多克隆抗体。用蛋白质印迹法(Western blot)检测了MRJP1特异性多克隆抗体与MRJP1重组表达蛋白多克隆抗体对王浆主蛋白家族的免疫反应。以新鲜蜂王浆为对照品,用MRJP1特异性抗体酶联接免疫吸附剂测定(ELISA)法和变性电泳胶灰度扫描法分别测定保温(40°C)7~49天的蜂王浆中MRJP1含量的变化,并进行了相关性分析。结论:MRJP1的特异性抗体对MRJP1蛋白具有专一的免疫识别特性,可特异性地检测代表蜂王浆新鲜度的MRJP1含量变化,并鉴别蜂王浆的真伪。
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| 关 键 词： | 新鲜度 蜂王浆 王浆主蛋白1 ELISA 高度特异性 |
| 收稿时间： | 2014 Aug 17 |
Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody
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| Li-rong SHEN,Yi-ran WANG,Liang ZHAI,Wen-xiu ZHOU,Liang-liang TAN,Mei-lu LI,Dan-dan LIU,Fa XIAO.Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody
[J].Journal of Zhejiang University Science,2015,16(2):155-166. |
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| Authors: | Li-rong SHEN;Yi-ran WANG;Liang ZHAI;Wen-xiu ZHOU;Liang-liang TAN;Mei-lu LI;Dan-dan LIU;Fa XIAO;Zhejiang |
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| Institution: | Li-rong SHEN;Yi-ran WANG;Liang ZHAI;Wen-xiu ZHOU;Liang-liang TAN;Mei-lu LI;Dan-dan LIU;Fa XIAO;Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, Department of Food Science and Nutrition,Zhejiang University; |
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| Abstract: | Major royal jelly protein 1 (MRJP1), designated apalbumin 1, has been regarded as a freshness marker of royal jelly (RJ). A MRJP1-specific peptide (IKEALPHVPIFD) identified by bioinformatics analysis of homologous members of the major royal protein family was synthesized and used to raise polyclonal anti-MRJP1 antibody (anti-SP-MRJP1 antibody). Western blot analysis showed that anti-SP-MRJP1 antibody only reacted with MRJP1 in RJ. In contrast, the previously reported antibody against recombinant MRJP1 (anti-R-MRJP1 antibody) reacted with other members of MRJP family in RJ. Enzyme-linked immunosorbent assay (ELISA) using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored at 40 °C significantly degraded by 37.3%, 55.9%, 58.0%, 60.6%, 65.7%, 72.7%, and 73.1% at 7, 14, 21, 28, 35, 42, and 49 d, respectively, when compared with MRJP1 content in fresh RJ (0 d). Optical density analysis of MRJP bands from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles demonstrated that the degradation of MRJP1, MRJP2, MRJP3, and MRJP5 in RJ was strongly and positively correlated with the period of storage (P<0.0001). Our results indicated anti-SP-MRJP1 antibody was highly specific for MRJP1, and ELISA using the antibody is a sensitive and easy-to-use method to determine the freshness and authenticity of RJ. |
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| Keywords: | Freshness Royal jelly Major royal jelly protein 1 (MRJP1) Enzyme-linked immunosorbent assay (ELISA) High specific antibody |
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