Immunological Characterization of Honey Proteins and Identification of MRJP 1 as an IgE-Binding Protein

We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.     

植物源过敏蛋白

植物过敏原是最普遍的一类过敏原,也是与人类关系最密切的一类过敏原。本文简单介绍了植物中蛋白质类过敏原的种类、特性及低敏植物性食品开发等研究新进展。     

Identification of major royal jelly proteins in the brain of the honeybee Apis mellifera

The consumption of royal jelly (RJ) determines the differences between castes and behavioral development in the honeybee Apis mellifera. However, it is not known whether the proteins of RJ are related to these differences, or which proteins are responsible for the changes. To understand the functions of RJ proteins that are present in other tissues of the bee, in addition to hypopharyngeal gland, we used a polyclonal antibody anti-MRJP1 to investigate the presence of this protein in nervous system of honeybee. This study showed the presence of three polypeptides (p57, p70 and p128) in specific tissues of bee brain. Mushroom body, optic lobe and antennal lobe neuropils all contained proteins recognized by anti-MRJP1. Proteomic analysis showed that the three polypeptides are correlated with proteins of the MRJP family. p57 is correlated with MRJP1, p70 with MRJP3, while p128 may be an oligomeric form or a new polypeptide. Immunostaining of the brain and hypopharyngeal gland revealed differential expression of MRJPs in various brain regions and in different honeybee castes and subcastes. The identification and localization of these MRJPs contribute to the elucidation of the biological roles of this protein family.     

蜜蜂王浆主蛋白（MRJPs）基因家族结构与功能概述

王浆主蛋白（Major royal jelly proteins,MRJPs）是王浆中的水溶性蛋白,为王浆蛋白的主要组分。因结构与功能的相似性,各王浆蛋白编码基因构成一个基因家族。目前该基因家族已鉴定出9个成员,依次命名为mrjp 1~9。该家族为单系群,拥有共同的祖先—yellow-e3,各成员之间具有较高的同源性。随着进化的进行,该家族逐步进化出营养及其它多种生物学功能。本文从该基因家族成员的鉴定、基因和蛋白质的结构特征、进化、功能以及其表达调控等多个方面进行综述,以期为相关的研究和应用提供帮助。     

蜜蜂蜂王浆主蛋白(MRJPs)的研究进展

蜜蜂的蜂王浆主蛋白具有为蜂王和幼虫提供营养、影响蜂群社会行为及调节个体生理机能等作用,作为蜂王浆的主要成分对其他机体也可产生多方面的生物学功能。因此,近年来蜂王浆主蛋白的相关研究备受关注。本文针对蜂王浆主蛋白的发现、种类、功能、系统进化及其基因表达情况进行了系统综述。     

Antiapoptotic role of major royal jelly protein 8 of honeybee (Apis mellifera) venom

The dominant protein components of honeybee royal jelly (RJ) are major royal jelly proteins (MRJPs), which exhibit various biological properties. However, the biological basis of why bee venom contains MRJPs and what role MRJPs play in bee venom remains to be elucidated. This study reports the antiapoptotic role of MRJP 8 of Apis mellifera venom (AmMRJP 8) in melittin-treated mammalian cells. Recombinant AmMRJP 8 reduced caspase-3 activity and melittin-induced cell apoptosis. Additionally, recombinant AmMRJP 8 decreased the production levels of H₂O₂ and proinflammatory molecules. These results indicate that MRJP in bee venom plays a role in cell protection in bee venom-induced inflammatory responses.     

Origin and function of the major royal jelly proteins of the honeybee (Apis mellifera) as members of the yellow gene family

In the honeybee, Apis mellifera, the queen larvae are fed with a diet exclusively composed of royal jelly (RJ), a secretion of the hypopharyngeal gland of young worker bees that nurse the brood. Up to 15% of RJ is composed of proteins, the nine most abundant of which have been termed major royal jelly proteins (MRJPs). Although it is widely accepted that RJ somehow determines the fate of a female larva and in spite of considerable research efforts, there are surprisingly few studies that address the biochemical characterisation and functions of these MRJPs. Here we review the research on MRJPs not only in honeybees but in hymenopteran insects in general and provide metadata analyses on genome organisation of mrjp genes, corroborating previous reports that MRJPs have important functions for insect development and not just a nutritional value for developing honeybee larvae.     

Antioxidant capacity of major royal jelly proteins of honeybee (Apis mellifera) royal jelly

Major royal jelly proteins (MRJPs) of honeybee royal jelly (RJ) exhibit antimicrobial and antioxidant activities. Although MRJPs of Apis mellifera RJ (AmMRJPs) responsible for antibacterial activity have been identified, AmMRJPs with antioxidant effects remain to be elucidated. Here we identified and compared the antioxidant activities of purified recombinant AmMRJPs 1–7, which are expressed in baculovirus-infected insect cells. Antioxidant assays of recombinant AmMRJPs 1–7 against H₂O₂ revealed that AmMRJPs reduce caspase-3 activity and oxidative stress-induced cell apoptosis and lead to increased cell viability. Consistent with these results, AmMRJPs 1–7 exhibit 1,1-diphenyl-2-picrylhydrazyl radical-scavenging activity and protect against oxidative DNA damage. These results indicate that AmMRJPs play a role as antioxidants in A. mellifera RJ.     

Antimicrobial activity of major royal jelly protein 8 and 9 of honeybee (Apis mellifera) venom

Honeybee venom is a complex mixture of toxic components, including major royal jelly protein (MRJP) 8 and 9. MRJP 8 and MRJP 9 are allergens, and MRJP 8 reduces melittin-induced cell apoptosis. However, their functional roles are poorly understood, and their antimicrobial activities have not been determined. In this study, the antimicrobial role of MRJP 8 and MRJP 9 of honeybee (Apis mellifera) venom (AmMRJP 8 and AmMRJP 9) was demonstrated. The presence of AmMRJP 8 and AmMRJP 9 in the secreted venom was observed using antibodies against recombinant AmMRJP 8 and AmMRJP 9 produced in baculovirus-infected insect cells. Recombinant AmMRJP 8 and AmMRJP 9 exhibited an inhibitory activity against microbial serine proteases. Consistent with their inhibitory activity, they induced structural damage by binding to microbial surfaces, resulting in a broad-spectrum antimicrobial activity against bacteria and fungi. They had little effect on hemolysis. Therefore, AmMRJP 8 and AmMRJP 9 could function as antimicrobial agents in honeybee venom.     

Ethanol‐soluble proteins from the royal jelly of Xinjiang black bees

Royal jelly is a nutritious food that has beneficial effects to human health. However, the functional substances remain unclear. Herein, we fractioned the royal jelly proteins of Xinjing black bees according to the Osboren method. Two main proteins from the ethanol‐soluble fraction were purified and identified. RJG‐1 was determined as glucosylceramidase, and RJG‐2 was major royal jelly protein 1 (MRJP1). RJG‐1 showed potent cytotoxicity toward various mammalian cells, and caused quick disruption of cell membranes. With glucosylceramidase activity, RJG‐1 may degrade the glucosylceramide of the cell membranes and disrupt the membrane structure, thereby resulting in cell necrosis. This study extends our knowledge about the composition and function of royal jelly, and is significant for the application of royal jelly.     

不同人工代花粉对蜂群群势和生产性能的影响

早春将群势、蜂王年龄和质量基本一致的25群意大利蜜蜂Apis melliferaL.,随机分为5个处理,分别饲喂纯花粉和4种不同的人工代用花粉,观测它们对蜂群群势和生产性能的影响。结果表明,含有添加剂的各代用花粉组与纯花粉组的蜂群的蜂子数量差异均不显著,但均显著高于纯豆粕组(P<0.01);各组产蜜量随着纯花粉所占比例的减少而呈现递减的趋势,但饲喂含有60%花粉+40%豆粕+添加剂1组与纯花粉组没有明显的差异;含有添加剂的各代用花粉组的王台接受率均显著高于纯花粉组(P<0.01);饲喂含有添加剂的各代用花粉组与饲喂纯花粉组相比,王浆产量及蜂王浆中的10-HDA、水分、蛋白质的含量及酸度均没有差异。     

The effects of royal jelly protein on bone mineral density and strength in ovariectomized female rats

[Purpose]Sex hormones deficiency leads to dramatically bone loss in particular postmenopausal women. Royal jelly has anti-osteoporosis effect due to maintain bone volume in that condition. We hypothesized that royal jelly protein (RJP, a latent residue after extracting royal jelly) also prevents bone deficient in ovariectomized (OVX) female rats, the animal model of postmenopausal women. [Methods]Female Sprague-Dawley rats (n = 30, 6 weeks age old) were sham operated (Sham; sham operated group, n = 7), OVX control group (OC, n = 7), OVX with low RJP intake group (ORL, n = 8), and OVX with high RJP intake group (ORH, n = 8) during 8 weeks experimental periods. In the end point of this experiment, the bone samples (lumbar spine, tibia, and femur) were surgically removed under anesthesia. These bone samples were evaluated bone mineral density (BMD) and bone strength.[Results]BMD of lumbar spine in RJP intake groups (ORL, ORH) were higher than that in OC group (p < 0.05 and p < 0.01) in RJP intake volume dependent manner. BMD of tibial proximal metaphysis and diaphysis in RJP intake groups were also higher than these in OC group (p < 0.01 and p < 0.01 / p < 0.05 and p < 0.001). In addition, breaking force of femur in RJP intake groups were significantly increase compared with that in OC group (p < 0.001 respectively). [Conclusion]These findings indicate that RJP contribute to prevent sex hormone related bone abnormality     

Production of Ochratoxin A by Aspergillus ochraceus Isolated in Japan from Moldy Rice

A simple thin-layer chromatography-fluorodensitometric method for quantitative analysis of ochratoxin A was developed. This method proved to be of use in investigating the production of the toxin and the nutritional factors affecting the toxin production by two strains of Aspergillus ochraceus isolated from moldy rice in Japan. These fungi produced large amounts of ochratoxin A in a nutrient solution containing 1% l-phenylalanine and 2% yeast extract.     

重组苦荞麦过敏蛋白TBa的原核表达及其免疫活性鉴定

TBa ［tartary buckwheat allergen］是苦荞麦中的一种主要过敏蛋白.根据长度为585 bp 的TBacDNA序列,以pET-28a为表达载体并选择合适的酶切位点合成上、下游引物,采用基因克隆技术构建重组表达载体pET-28a-TBa.进一步将重组质粒转入大肠杆菌BL21(DE3) 中进行表达.从而获得以包涵体形式存在的TBa目的蛋白.该目的蛋白经Ni ²⁺ -NTA琼脂糖柱亲和纯化及SDS-PAGE分析显示, 纯度达到95% 以上.用透析复性的方法将目的蛋白重折叠,其复性产率可达到约68%.Western印迹证实,目的蛋白N端带有6个组氨酸标签.ELISA检测表明,通过基因重组及表达获得的重组苦荞麦过敏蛋白,与天然苦荞种子中的该蛋白具有相似的免疫学活性,与荞麦过敏病人血清中的IgE有特异性的结合.     

蜜蜂属六蜂种间MRJP2基因C-端重复序列的比较分析

构建了中华蜜蜂（Apis cerana cerana,中蜂）8日龄工蜂头部cDNA文库,获得了中蜂王浆主蛋白2（major royal jelly protein 2,MRJP2）的全长cDNA序列,该序列长1 605bp,包含一个编码468个氨基酸的开放阅读框（open reading frame,ORF）。在中蜂MRJP2的cDNA序列的C-端,首次发现存在串联重复片段长度多态性（variable numbers of tandem repeat,VNTR）。克隆并测定了蜜蜂属Apis内其他5个种的MRJP2基因的C-端重复序列,结果表明: 在蜜蜂属的其他5个种中,C-端重复片段的核心序列是以碱基高度突变方式而表现出个体之间的多态性,而重复片段长度基本一致。中蜂与西方蜜蜂A. mellifera,大蜜蜂A. dorsata与黑大蜜蜂A. laboriosa,以及小蜜蜂A. florea与黑小蜜蜂A. andreniformis分别形成3个进化枝。中蜂和西方蜜蜂与大蜜蜂和黑大蜜蜂之间的亲缘关系较近,而与小蜜蜂和黑小蜜蜂的亲缘关系较远。     

饲喂胡萝卜和蜂王浆对黄粉虫繁殖力的影响

采用多因素对比饲养试验,设计不同黄粉虫TemibriomolitorL.发育时期分别补饲胡萝卜、不同浓度的蜂王浆,研究胡萝卜和蜂王浆对黄粉虫繁殖力的影响。结果表明:各处理组比对照组羽化齐一,羽化率增高,产卵速度加快,总产卵量增多,良卵率、孵化率增高。幼虫期和成虫期都补饲2%的蜂王浆,可使产卵量极显著高于对照97.79%、孵化率极显著地高于对照8.90%;而只在成虫期补充2%的蜂王浆,也可使产卵量极显著高于对照90.65%、有效地提高孵化率6.66%。     

Ameliorative Effects of Propolis and Royal Jelly against CCl4-Induced Hepatotoxicity and Nephrotoxicity in Wistar Rats

Carbon tetrachloride (CCl₄) is known to have hepatotoxic and nephrotoxic effects. During the two-month CCl₄ exposure of Wistar rats, propolis extract (PE) and royal jelly (RJ) were added in order to test the potential protective effect against hepato-renal injury. Ketonuria, proteinuria, high creatinine and urea levels are the result of CCl₄-induced nephrotoxicity. Severe disorders of hematological indicators indicate anemia; high values of leukocytes indicate inflammatory condition. Cytogenetic impairments in hepatocytes, aggregation of platelets, and hypoproteinemia indicate severe liver impairment. Results suggest a more significant protective role of RJ compared to PE. Both extracts regulated proteinuria, ketonuria, hypoproteinemia and reduced platelet aggregation in the hepatic circulation. The increase in the number of erythrocytes (RBC) suggest protective effects against anemia; the decrease in the number of leukocytes can be linked to anti-inflammatory effects. PE and RJ have a beneficial effect against hepato-renal injury, anemia and anti-inflammatory conditions caused by CCl₄.     

利用VNTR分子标记鉴定蜜蜂群内蜂王交配次数和雄蜂母系来源

如何准确测定蜂王交配次数和雄蜂母系来源,是研究蜜蜂亚家系行为生物学的关键。本研究利用王浆主蛋白（MRJPs）的串联重复序列多态性（VNTR）分子标记分别鉴定了蜂王单雄人工授精、双雄人工授精和自然交尾的中华蜜蜂Apis cerana cerana蜂群中的蜂王交配次数和雄蜂母系来源。结果表明： 在蜂王单雄人工授精和双雄人工授精蜂群中,蜂王的交配次数分别为1和2;在蜂王自然交尾的2个蜂群中,蜂王的交配次数分别为8和5。另外,经鉴定发现：在以上实验蜂群中,所有雄蜂都是由蜂王产的未受精卵发育而来。因此,作为一种分子标记,蜜蜂MRJPs VNTR能简单、有效地鉴定蜂群内蜂王的交配次数和雄蜂母系来源。     

意蜂蜂王浆超氧化物歧化酶的分离纯化及部分性质

以意蜂Apis mellifera蜂王浆为材料,经过硫酸铵分段盐析,DEAE-Sepharose 柱层析和Sephacryl S-200凝胶过滤,得到纯化的超氧化物歧化酶(SOD),纯化倍数104.00,比活力53.05 U/mg。该SOD经SDS-PAGE显示单一蛋白带。温度对该酶活力的影响较小。Cu、Zn、Fe和Mn等元素含量测定发现该酶只含有Cu和Zn。酶经圆二色谱测定后,其α螺旋、β折叠和无规则卷曲蛋白构型的含量分别为26.1%、53.8%和22.0%。等电聚焦电泳测得酶的等电点为4.69、4.85和5.01。NR/R单向和双向SDS-PAGE表明该酶含有链内二硫键。氨基酸组成分析发现该酶由约402个氨基酸残基组成,其中Asp、Gly、Leu、Ala、Glu和Val的含量较高。脲可抑制SOD活性,并使其紫外光谱发生变化,荧光发射峰强度变小。溴乙酸（BrAc）抑制酶的活力,使其紫外光谱发生变化,荧光发射峰强度变小。二巯基苏糖醇（DTT）使酶的活力发生变化,紫外吸收峰增大,荧光发射峰变小。