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1.
We report on the development of a novel alternative method for the assessment of floral origin in honey samples based on the study of honey proteins using immunoblot assays. The main goal of our work was to evaluate the use of honey proteins as chemical markers of the floral origin of honey. Considering that honeybee proteins should be common to all types of honey, we decided to verify the usefulness of pollen proteins as floral origin markers in honey. We used polyclonal anti-pollen antibodies raised in rabbits by repeated immunization of Sunflower (Elianthus annuus) and Eucalyptus (Eucalyptus sp.) pollen extracts. The IgG fraction was purified by immunoaffinity. These antibodies were verified with nitrocellulose blotted pollen and unifloral honey protein extracts. The antibodies anti-Sunflower pollen, bound to the 36 and 33 kDa proteins of Sunflower unifloral honey and to honey containing Sunflower pollen; and the antibodies anti-Eucalyptus sp. pollen bound to the 38 kDa proteins of Eucalyptus sp. unifloral honey in immunoblot assays. Satisfactory results were obtained in differentiating between the types of pollen analyzed and between Sunflower honey and Eucalyptus honey with less cross reactivity with other types of honey from different origin and also with good sensitivity in the detection. This immunoblot method opens an interesting field for the development of new antibodies from different plants, which could serve as an alternative or complementary method to the usual melissopalynological analysis to assess honey floral origin.  相似文献   

2.
Royal jelly (RJ) is a thick, milky material produced by both the hypopharyngeal and the mandibular glands of nurse honeybees. The main proteins of RJ, named apalbumins or major royal jelly proteins (MRJPs), have multiple biological functions. Apalbumin1 is the most abundant glycoprotein of RJ. In this study, Bacmid- apalbumin1 was constructed for Apis cerana cerana using the newly established Bac-to-Bac/BmNPV baculovirus expression system (BES). This procedure allowed us to obtain the recombinant A. cerana cerana ( Acc) apalbumin1 (r Accapalbumin1) from the hemolymph of silkworm larvae through the BmNPV bacmid system, 96 h postinfection. The r Accapalbumin1 was then purified by Ni-NTA spin columns and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. A 55 kDa protein with good solubility was then obtained. The peptide Ile-Phe was identified from trypsin production of r Accapalbumin1. Such a peptide has been reported to have an antihypertensive ability. Our results have therefore potential applications in biomedical research and open new perspectives for the study of apalbumins.  相似文献   

3.
In this study, the proteins contained in royal jelly (RJ) produced by Africanized honeybees and European honeybees (Apis mellifera) haven been analyzed in detail and compared using two-dimensional gel electrophoresis, and the N-terminal amino acid sequence of each spot has been determined. Most spots were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular MRJP3, in terms of molecular weights and isoelectric points between the two species of RJ. Furthermore, during the determination of the N-terminal amino acid sequence of each spot, for the first time, MRJP4 protein has been identified, the existence of which had been only implied by cloning of its cDNA sequence. The presence of heterogeneous bands of glucose oxidase was also identified. Thus, the results suggest that two-dimensional gel electrophoresis provides a suitable method for the qualitative analysis of the proteins contained in RJ derived from different honeybee species.  相似文献   

4.
Starch phosphorylase (SP) in immature mungbean (Vigna radiata L. cv KPS1) seed soluble extract was detected by in situ activity staining and identified by MALDI-TOF mass analysis. After in situ SP assay on native-PAGE, a major starch-enzyme complex was located on the gel zymogram in a dose-dependent manner. This complex depicted two major SP-activity related proteins, 105 kDa and 55 kDa, by SDS-PAGE. The mass and predicted sequence of the tryptic fragments of the isolated 105 kDa protein, analyzed by MALDI-TOF spectroscopy and bioinformatic analysis, confirmed it to be mungbean SP as a result of high similarity to the L-SP of known plant. Polyclonal antibodies raised from the 55 kDa recognized both the 105 kDa and the 55 kDa proteins on the Western blot and neutralized partial SP activity, indicating that the two proteins were immunologically related. The 55 kDa protein possess high similarity to the N-terminal half of the 105 kDa SP was further confirmed. The SP activity and the activity stained protein density in mungbean soluble extract decreased as the seed size increased during early seed growth. These data indicate that mungbean 105 kDa SP and SP activity-related 55 kDa were identified in the developing mungbean.  相似文献   

5.
We report the development of a rapid, specific, and sensitive enzyme-linked immunoassay (ELISA) for the evaluation of sunflower pollen in honey as a method alternative to melissopalynology, which is considered the standard technique for the evaluation of floral origin of honey. Two 33-36 kDa proteins, identified as characteristic of sunflower pollen, were isolated and used as coating antigens in the competitive ELISA. We verified its analytical performance by evaluating reproducibility, specificity, and exactitude in relation to melissopalynology. The competitive ELISA developed during this work is able to quantify sunflower pollen in honey, with a detection limit of 10%, showing linear response between 10 and 90%. The method afforded low cross reactivity with honey from other floral origin, thus evidencing an adequate selectivity. We also observed a significant correlation (r = 0.975; p < 0.001) when the proposed ELISA was referenced to melissopalynology. Hence, we conclude that the competitive ELISA constitutes a valuable and feasible alternative for authentication of sunflower honey. This work opens the possibility to develop similar assays for other pollen types.  相似文献   

6.
To compare the protein complement of royal jelly (RJ) from high RJ producing honeybees ( Apis mellifera L.), a strain of A. mellifera artificially selected for increased RJ production from Italian honeybees in China for more than two decades was compared to those of native Italian honeybees ( A. mellifera L.) and Carnica honeybees ( A. mellifera C.); the protein in RJ from these three strains of honeybees was partially identified by using a combination of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS), and a protein engine identification tool applied to the honeybee genome. The results showed that 152, 157, and 137 proteins were detected in the three species of RJ; among which 57, 57, 51 high abundant proteins ere identified, respectively. Most identifited spots, 45, 45, 41, were assigned to major royal jelly proteins (MRJPs). Remarkable differences were found in the heterogeneity of the MRJPs, in particular, MRJP3. Also, 3-glucose oxidase, 1-peroxiredoxin (PRDX), and 1-glutathione S-transferase (GST) S1 were identified in three RJ samples. Furthermore, during the determination of the peptides mass fingerprinting (PMF) of each spot, for the first time, PRDX and GST S1 proteins have been identified in RJ. Thus, the results suggest that the protein complement of high RJ producing honeybees is not different compared to native Italian honeybees, while a difference remains between Carnica honeybees.  相似文献   

7.
Novel royal jelly proteins identified by gel-based and gel-free proteomics   总被引：1，自引：0，他引：1  
Royal jelly (RJ) plays an important role in caste determination of the honeybee; the genetically same female egg develops into either a queen or worker bee depending on the time and amount of RJ fed to the larvae. RJ also has numerous health-promoting properties for humans. Gel-based and gel-free proteomics approaches and high-performance liquid chromatography-chip quadruple time-of-flight tandem mass spectrometry were applied to comprehensively investigate the protein components of RJ. Overall, 37 and 22 nonredundant proteins were identified by one-dimensional gel electrophoresis and gel-free analysis, respectively, and 19 new proteins were found by these two proteomics approaches. Major royal jelly proteins (MRJPs) were identified as the principal protein components of RJ, and proteins related to carbohydrate metabolism such as glucose oxidase, α-glucosidase precursor, and glucose dehydrogenase were also successfully identified. Importantly, the 19 newly identified proteins were mainly classified into three functional categories: oxidation-reduction (ergic53 CG6822-PA isoform A isoform 1, Sec61 CG9539-PA, and ADP/ATP translocase), protein binding (regucalcin and translationally controlled tumor protein CG4800-PA isoform 1), and lipid transport (apolipophorin-III-like protein). These new findings not only significantly increase the RJ proteome coverage but also help to provide new knowledge of RJ for honeybee biology and potential use for human health promotion.  相似文献   

8.
Cross-reactivity of antibodies against almond major protein (AMP, a legumin), the major almond allergen, with cereal proteins may cause problems in detecting almond contaminants in cereal products when antibody-based assays are used. Rabbit polyclonal IgG antiserum produced against AMP was used to test cross-reactivity with protein extracts from maize, a cereal commonly found in breakfast and snack foods. Gradient SDS-PAGE followed by Western blotting was performed, and two cross-reactive proteins were detected by chemiluminescence. A fraction of maize proteins purified by elution from an IgG anti-AMP affinity column followed by electrophoreseis and immunoblotting showed a high degree of cross-reactivity with a minor 50 kDa protein of maize, as well as low cross-reactivity with the 27 kDa gamma-zein. The 50 kDa cross-reactive protein was identified as the 50 kDa gamma-zein by immunoreaction with anti-50 kDa gamma-zein antiserum. Notably, the 50 kDa maize gamma-zein also reacted with IgE from pooled human sera from patients with self-reported severe almond allergies. The high immunoreactivity of the 50 kDa gamma-zein should be considered in maize quality improvement programs, and such notable cross-reactivity is of relevance in the design of antibody-based assays for almond allergen detection.  相似文献   

9.
The main goal of the present study is to develop an immunoanalytical method for the quality control of orange juice products. Peptides from various parts (juice, albedo, and flavedo) of citrus fruits (orange, mandarin, grapefruit, and lemon) were analyzed and isolated by SDS-PAGE. Antisera were developed in mice against the protein pool of orange juice and peel and tested by Western blot analysis. Using these antisera, some juice- and peel-specific peptides were detected. One of the antibodies in the antiserum developed against peel proteins recognized a single peel-specific peptide with a molecular mass of 28 kDa in 10000-fold dilution. It did not give any positive reactions against the sample prepared from the juice. The 24 and 27 kDa juice-specific peptides were isolated in electrophoretically pure form, and polyclonal antibodies were developed against them in mice. The anti-27 kDa antibody reacted with a 29 kDa protein in the peel sample, and it gave a positive reaction against the 27 kDa peptide of the juice. The antibodies developed in the course of the present work seem to be useful for determining the juice content in commercial citrus beverages and for evaluating the peel contamination in them.  相似文献   

10.
Whole, unprocessed Nonpareil almonds were subjected to a variety of heat processing methods that included roasting (280, 300, and 320 degrees F for 20 and 30 min each; and 335 and 350 degrees F for 8, 10, and 12 min each), autoclaving (121 degrees C, 15 psi, for 5, 10, 15, 20, 25, and 30 min), blanching (100 degrees C for 1, 2, 3, 4, 5, and 10 min), and microwave heating (1, 2, and 3 min). Proteins were extracted from defatted almond flour in borate saline buffer, and immunoreactivity of the soluble proteins (normalized to 1 mg protein/mL for all samples) was determined using enzyme linked immunosorbent assay (ELISA). Antigenic stability of the almond major protein (amandin) in the heat-processed samples was determined by competitive inhibition ELISA using rabbit polyclonal antibodies raised against amandin. Processed samples were also assessed for heat stability of total antigenic proteins by sandwich ELISA using goat and rabbit polyclonal antibodies raised against unprocessed Nonpareil almond total protein extract. ELISA assays and Western blotting experiments that used both rabbit polyclonal antibodies and human IgE from pooled sera indicated antigenic stability of almond proteins when compared with that of the unprocessed counterpart.  相似文献   

11.
The low prices of some nonmilk proteins make them attractive as potential adulterants in dairy products. An optical biosensor (BIACORE 3000) was used to develop a direct and combined biosensor immunoassay (BIA) for the simultaneous detection of soy, pea, and soluble wheat proteins in milk powders. Affinity-purified polyclonal antibodies raised against the three protein sources were immobilized in different flow channels (Fcs) on the biosensor chip (CM5). Dissolved milk powders were injected (20 microL injections at 20 microL min(-1)) through the serially connected Fcs, and the antibody-bound plant proteins were detected directly. The total run time between samples, including a regeneration step with 5 microL of 10 mM HCl, was 5 min. The limits of detection in milk powder were below 0.1% of plant protein in the total milk protein content. The antibodies also recognized some proteins from other plant sources, which made this BIA even more suitable as a broad screening assay for nonmilk proteins.  相似文献   

12.
The gene of the most abundant protease inhibitor in potato cv. Elkana was isolated and sequenced. The deduced amino acid sequence of this gene showed 98% identity with potato serine protease inhibitor (PSPI), a member of the Kunitz family. Therefore, the most abundant protease inhibitor was considered to be one of the isoforms of PSPI. The PSPI group represents approximately 22% of the total amount of proteins in potato cv. Elkana and is composed of seven different isoforms that slightly differ in isoelectric point. Antibodies were raised against the two most abundant isoforms of PSPI. The binding of these antibodies to PSPI isoforms and protease inhibitors from different groups of protease inhibitor in potato showed that approximately 70% of the protease inhibitors present in potato juice belong to the Kunitz family.  相似文献   

13.
The objective of this study was to analyze the origin of proteins of a Chardonnay wine. Three various polyclonal antibodies raised against must, yeast, and bacteria proteins were produced. For microorganisms, only the secreted macromolecules were used. To this end, yeast and bacteria were cultured in a model medium under conditions close to those of winemaking. Results obtained using these specific antibodies indicate that most of the wine proteins came from grapes and many of them were glycoproteins. Some proteins of this Chardonnay wine came from the yeast; they were released during the alcoholic fermentation and consisted of high molecular weight mannoproteins. In contrast, no bacteria proteins were detected in this Chardonnay wine.  相似文献   

14.
Proteins associated with starch synthase (SS) activities were identified in immature mungbeans (Vigna radiata L. cv KPS1). Seed soluble extract was separated by native-PAGE and subjected to in situ activity staining. The gel zymogram located starch-enzyme complex bands. The soluble extract was also partitioned by preparative-IEF and screened for SS activity using radioactive assay. IEF fractions eluted within pH 4-6 revealed enriched SS activity of 145-fold. Parallel comparison of the protein profiles among the activity stained enzyme complex and the active isoelectric focused fractions on SDS-PAGE depicted three SS-activity-related proteins with molecular size of 32, 53, and 85 kDa. The 85 kDa protein, however, was identified to be methionine synthase by MALDI-TOF analysis and should be a protein physically associated with the active SS. Polyclonal antibodies raised from eluted native enzyme complex neutralized up to 90% activity and antigenically recognize the other 53 and 32 kDa proteins on Western blot. Antibodies raised from the two individual denatured proteins were able to neutralize SS activities near 60% separately, indicating that the 53 kDa and 32 proteins associated with SS activity are potentially involved in starch biosynthesis during mungbean seed development.  相似文献   

15.
Globulin-P, the polymerized 11S amaranth globulin, is composed of 280 kDa unitary molecules (UM, 23%) and aggregates larger than 500 kDa (A, 70%). Antibodies against these proteins were prepared to study their surface characteristics and to assess their homology with other storage proteins. Results showed that globulin-P unitary molecules and aggregates had similar reactive surfaces. A polypeptide of 56 kDa was found to be the most reactive to the antibodies assayed, followed by the acidic polypeptides. Such results support previous information, according to which these polypeptides appeared to be the most exposed on the molecule surface. Globulin-P fraction presented cross-reactivity with the remaining amaranth protein fractions: 11S-globulin, glutelins, and albumins. Globulin-P and 11S-globulin showed similar reactive surfaces whereas glutelin and albumins presented a lower cross-reactivity. The reactivity of the glutelin fraction depended on its sequence. Globulin-P fraction presented cross-reactivity with quinoa globulins, and to a lesser extent with globulins of sunflower and rice. Moreover, the anti-Gp serum was unable to detect either conformational or sequence epitopes in globulins of soybean, wheat, buckwheat, rice, and rye.  相似文献   

16.
To elucidate the molecular mechanism of the allergenicity of soybean P34 protein recognized as the most allergenic protein in soybean, the protein was expressed in Escherichia coli transformed with a plasmid carrying P34 cDNA. SDS-PAGE pattern showed that the molecular weight of the recombinant P34 was approximately 2 kDa less than that of the native soybean P34. The difference in the molecular mass between these two proteins could be due to the native P34 in soybean being glycosylated at position Asn(170), whereas the recombinant protein generated in E. coli lacks this post-translational modification. Immunoblot analysis showed that both soybean and recombinant P34 proteins cross-reacted not only with polyclonal and monoclonal antibodies produced against P34 and crude soybean protein but also with patients' sera. The results suggest that the recombinant P34 is immunologically reactive, indicating that both proteins have similar epitope structures. Thus, the recombinant P34 produced by the E. coli expression system can be used as a standard allergen for molecular design to reduce the allergenic structure.  相似文献   

17.
Polyclonal antibodies raised against the plasmin-released 1-28 phosphopeptide from bovine beta-casein [i.e., beta-CN(f1-28)4P] specifically recognized the tryptic beta-casein 1-25 and 2-25 peptides, whatever the degree of phosphorylation, but were unresponsive to the shortened beta-casein 16-22 phosphopeptide. These antibodies were able to recognize the parent bovine beta-casein as well as the homologous water buffalo protein, but they could not detect the homologous counterparts from ovine and caprine milks. Such antibodies were used in competitive enzyme-linked immunosorbent assays to monitor the plasmin-mediated release of the 1-28 phosphopeptide from beta-casein and to evaluate the residual native beta-casein in bovine cheese sampled during ripening. Applications of these polyclonal antibodies are suggested mainly for estimating the age of hard cheeses and, possibly, for tracing the presence of bovine casein in fresh ovine and caprine cheeses.  相似文献   

18.
Contamination of food products with pepsin resistant allergens is generally believed to be a serious threat to patients with severe food allergy. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to measure pepsin resistant hazelnut protein in food products. Capturing and detecting rabbit antibodies were raised against pepsin-digested hazelnut and untreated hazelnut protein, respectively. The assay showed a detection limit of 0.7 ng/mL hazelnut protein or <1 microg hazelnut in 1 g food matrix and a maximum of 0.034% cross-reactivity (peanut). Chocolate samples spiked with 0.5-100 microg hazelnut/g chocolate showed a mean recovery of 97.3%. In 9/12 food products labeled "may contain nuts", hazelnut was detected between 1.2 and 417 microg hazelnut/g food. It can be concluded that the application of antibodies directed to pepsin-digested food extracts in ELISA can facilitate specific detection of stable proteins that have the highest potential of inducing severe food anaphylaxis.  相似文献   

19.
Jelly curd used for a popular summer drink in Taiwan is prepared by extracting the pericarpial portion of jelly fig (Ficus awkeotsang Makino) achenes. The two most abundant proteins found in jelly curd have been identified as a pectin methylesterase and a chitinase. A method was developed to purify the next abundant protein by 40% ammonium sulfate precipitation and flowing through Mono Q chromatography. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses, the purified protein migrated as a polypeptide of 20 kDa in the absence of beta-mercaptoethanol but split into a minor polypeptide of 20 kDa and a major polypeptide of 27 kDa in the presence of this reducing agent. Two cDNA fragments encoding precursor polypeptides of two putative thaumatin-like protein isoforms were obtained by polymerase chain reaction cloning and subsequently overexpressed in Escherichia coli to generate recombinant proteins for antibody preparations. Immunological detection and mass spectrometric analyses indicated that the two split polypeptides were thaumatin-like protein isoforms encoded by the two cloned cDNA fragments.  相似文献   

20.
A study on the possible degradation of amitraz, bromopropylate, coumaphos, chlordimeform, cymiazole, flumethrin, and tau-fluvalinate during the storage of honey was carried out by HPLC. Except amitraz, the other acaricides are stable in this medium for at least 9 months. Degradation studies of amitraz in honey and beeswax were carried out; the degradation products detected in both matrices were 2,4-dimethylphenylformamide (DMF) and N-(2,4-dimethylphenyl)-N'-methylformamidine (DPMF). The reaction rate constants and the half-lives of the amitraz degradation in honey and wax were calculated. Amitraz was nearly completely degraded within 1 day in beeswax and within 10 days in honey. When amitraz-spiked combs are recycled into new beeswax, DMF was found to be the principal degradation product left in pure wax.  相似文献   

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