Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody
Determination of royal jelly freshness by ELISA with a highly specific anti-apalbumin 1, major royal jelly protein 1 antibody
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DOI：
10.1631/jzus.b1400223
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发表时间：
2015-02-01
影响因子：
5.1
通讯作者：
Xiao, Fa
中科院分区：
文献类型：
--
作者：
Shen, Li-rong;Wang, Yi-ran;Xiao, Fa
蜂王浆主要蛋白1（Major Royal Jelly Protein 1，MRJP 1）是皇家浆新鲜度的标志蛋白。通过生物信息学分析主要皇家蛋白家族的同源成员，合成了MRJP 1特异性肽（IKEALPHVPIFD），并用于制备多克隆抗MRJP 1抗体（抗SP-MRJP 1抗体）。Western blot分析表明，抗SP-MRJP 1抗体仅与RJ中的MRJP 1反应。相反，先前报道的针对重组MRJP 1的抗体（抗R-MRJP 1抗体）与RJ中MRJP家族的其他成员反应。利用抗SP-MRJP 1抗体的酶联免疫吸附试验（ELISA）表明，在40 A ℃下储存的RJ中MRJP 1含量在7、14、21、28、35、42和49 d分别显著降解37.3%、55.9%、58.0%、60.6%、65.7%、72.7%和73.1%，与新鲜RJ（0 d）中MRJP 1含量相比。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）图谱中MRJP条带的光密度分析表明，RJ中MRJP 1、MRJP 2、MRJP 3和MRJP 5的降解与储存时间呈强正相关（P < 0.0001）。结果表明，抗SP-MRJP 1抗体对MRJP 1具有高度的特异性，用该抗体建立的ELISA方法是一种灵敏、简便的检测RJ新鲜度和真伪的方法。
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 A degrees 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.