【作者】 沈立荣； 王一然； 翟量； 周文秀； 李玫璐； 柳丹丹； 肖发；

【Author】 Lirong Shen;Yiran Wang;Liang Zhai;Wenxiu Zhou;Meilu Li;Dandan Liu;Fa Xiao;Department of Food Science and Nutrition,Fuli Institute of Food Science,Zhejiang Key Laboratory for Agro-Food Processing,Zhejiang University;

【机构】 浙江大学食品科学与营养系馥莉食品科学研究院浙江省农产品与食品加工重点实验室；

【摘要】 近年来国际上已认可蜂王浆主蛋白1(MRJP1)是代表蜂王浆新鲜度生物标志物。为此,我们通过蜂王浆主蛋白家族氨基酸序列的同源性分析。筛选出了1段MRJP1的特异性多肽(IKEALPHVPIFD),并以此多肽为抗原免疫大白兔,制备了一种MRJPl的特异性多克隆抗体(antiSP-MRJP1 antibody)。Wester blot分析结果显示,该抗体只能与蜂王浆中的主蛋白1产生免疫印迹,而另一种采用MRJPP1蛋白编码基因表达的重组MRJP1蛋白作为抗原制备的多抗则会与王浆主蛋白家族的所有蛋白发生交叉反应。以MRJP1特异性多抗作为一抗的ELISA方法分别检测在40℃下保温不同时间的鲜王浆,结果显示,与冷冻保存的鲜王浆相比,保温7天、14天、21天、28天、35天、42天和49天的鲜王浆中的MRJP1蛋白含量分别下降了37.26%、55.91%、57.96%、60。60%、65.71%、72.73%和73.12%。于此同时,我们将经上述保温处理的鲜王浆进行了SDSPAGE蛋白电泳分析,然后用成像仪对所得蛋白电泳图像进行不同蜂王浆主蛋白条带灰度分析。结果显示,与。与冷冻保存的鲜王浆相比,经保温处理的鲜王浆中的蜂王浆主蛋白家族成员MRJP1、MRJP2、MRJP3、MRJP5蛋白含量的下降程度与保温时间成正相关(p<0.0001)。该结果证实,我们所制备的MRJP1特异性多抗对MRJP1蛋白具有高度的专一性,采用此多抗为一抗的ELISA方法是一种非常实用和应用与质量标准的蜂王浆新鲜度检测新技术。更多还原

【Abstract】 Major royal jelly protein 1(MRJP1),designated apalbumin 1 has been regarded as freshness marker of royal jelly(RJ).A MRJP1 specific peptide(IKEALPHVPIFD) identified with analysis of homologous members of the major royal protein family was synthesized,and used to raise polyclonal anti-MRJP1antibody(anti-SP-MRJP1 antibody).Western blot analysis showed that anti-SP-MRJP antibody only reacted with MRJP1 in RJ.In contrast,the antibody against recombinant MRJP1(anti-R-MRJP1 antibody) reacted cross with other MRJP family members in RJ.Enzyme-Linked Immunosorbent Assay using anti-SP-MRJP1 antibody demonstrated that MRJP1 content in RJ stored under 40℃ degraded 37.26%,55.91%,57.96%,60.60%,65.71%,72.73%and 73.12%at 7 d,14 d,21 d,28 d,35 d,42 d and 49 d,respectively,when compared to MRJP1 content in fresh RJ(0 day).Optical density analysis of MRJP bands from SDS-PAGE profile demonstrated that the degradation of MRJP1,MRJP2,MRJP3,MRJP5 in RJ strongly and positively correlated with the period of storage(p <0.0001).Our results indicated anti-SPMRJP antibody was highly specific for MRJP1,and ELISA using the antibody is a practical tool to determine freshness and authenticity of RJ.更多还原