Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L anti...Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.展开更多
Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent o...Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the展开更多
The denaturational behaviour of bovine β-lactoglobulin B has been studied in solutions containing guanidine hydrochloride by differential scanning calorimetry. The experiments have shown that complete peaks of cold d...The denaturational behaviour of bovine β-lactoglobulin B has been studied in solutions containing guanidine hydrochloride by differential scanning calorimetry. The experiments have shown that complete peaks of cold denaturation can be recorded also in high concentration of protein solutions. The cold denaturation and the renaturation of the protein are reproducible, but the thermal denaturation is irreversible. The activation energy of thermal denaturation calculated is about 285 kJ/mol.展开更多
Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteris...Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column （250 mm×4.6 mm×5μm） and Shiseido CAPCELL PAK SG 300 C18 column （250 mm× 4.6 mm×5 μm） were used in the experiment. Phase A was composed of 0.1% （V/V） trifluoroacetic acid （TFA） in ultrapure water and Phase B （organic phase） was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.展开更多
The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often t...The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.展开更多
运用代谢组学技术研究表没食子素儿茶素没食子酸酯(epigallocatechin gallate,EGCG)⁃β⁃乳球蛋白(β⁃lacto⁃globulin,βlg)复合物对βlg致过敏反应小鼠血液中代谢物的变化,筛选出过敏反应与脱敏相关的潜在生物标志物,进一步分析其代谢通...运用代谢组学技术研究表没食子素儿茶素没食子酸酯(epigallocatechin gallate,EGCG)⁃β⁃乳球蛋白(β⁃lacto⁃globulin,βlg)复合物对βlg致过敏反应小鼠血液中代谢物的变化,筛选出过敏反应与脱敏相关的潜在生物标志物,进一步分析其代谢通路,探讨EGCG对βlg脱敏的代谢机制。采用多酚成分EGCG与βlg形成复合物进行脱敏,以βlg诱导小鼠发生过敏反应为模型组,观察空白组、βlg组、βlg⁃EGCG组小鼠过敏行为学体征,通过超高效液相色谱⁃四极杆静电场轨道阱质谱(ultra performance liquid chromatography⁃quadrupole⁃electrostatic field orbitrap mass spectrometry,UPLC⁃QE⁃MS)技术对小鼠血清进行代谢组学分析,并进一步采用主成分分析和偏最小二乘辨别分析探究各组代谢轮廓差异,筛选出差异代谢物。结果显示,βlg组和βlg⁃EGCG组的代谢谱图存在明显差异,并筛选出与过敏反应相关的40个差异代谢物,7条主要代谢通路。推测βlg致敏与EGCG脱敏作用机制可能涉及氨基酸代谢和脂类代谢等过程。展开更多
基金Sponsored by the Young Scholar Scientific Research Foundation of China CDC[2015A202]:The establishment of testing platform of quantitatively detecting main protein of cow milk by using protein chip technique
文摘Objective To research a protein chip method which can simultaneously quantitative detect β-Lactoglobulin （β-L） and Lactoferrin （Lf） at one time. Methods Protein chip printer was used to print both anti-β-L antibodies and anti-Lf antibodies on each block of protein chip. And then an improved sandwich detection method was applied while the other two detecting antibodies for the two antigens were added in the block after they were mixed. The detection conditions of the quantitative detection for simultaneous measurement of β-L and Lf with protein chip were optimized and evaluated. Based on these detected conditions, two standard curves of the two proteins were simultaneously established on one protein chip. Finally, the new detection method was evaluated by using the analysis of precision and accuracy. Results By comparison experiment, mouse monoclonal antibodies of the two antigens were chosen as the printing probe. The concentrations of β-L and Lf probes were 0.5 mg/mL and 0.5 mg/mL, respectively, while the titers of detection antibodies both of β-L and Lf were 1：2,000. Intra- and inter-assay variability was between 4.88% and 38.33% for all tests. The regression coefficients of protein chip comparing with ELISA for β-L and Lf were better than 0.734, and both of the two regression coefficients were statistically significant （r = 0.734, t = 2.644, P = 0.038; and r = 0.774, t = 2.998, P = 0.024）. Conclusion A protein chip method of simultaneously quantitative detection for β-L and Lf has been established and this method is worthy in further application.
基金supported by the Natural Science Foundation of China (30871752)the High-tech Industrialization Funding of Guangdong Province (2009B011300010)
文摘Objective To characterize the relationship between the refolding process of recombinant bovine β-1actoglobulin and its immunoreactivity for clinical purposes. To establish a spectral method which examine the extent of recombinant allergen renaturation. Methods The refolding process of recombinant bovine β-1actoglobulin was investigated by using circular dichroism, fluorescence and synchronous fluorescence spectra. IgE-binding capacity of recombinant protein was analyzed by ELISA. In addition, bioinformatic methods were used to explain the spectral characteristics and analyze the relationship between the conformational changes and the immunoreactivity of the protein during renaturation in vitro. Results Renaturation of recombinant bovine β-1actoglobulin resulted in a more compact structure resembling the natural counterpart with stronger IgE-binding capacity. Conclusion The degree of protein renaturation Results from this study may be of help for food future. correlated with the IgE-binding capacity of the protein. allergy therapy and development of vaccination in the
基金This project was supported by the National Natural Science Foundation of China
文摘The denaturational behaviour of bovine β-lactoglobulin B has been studied in solutions containing guanidine hydrochloride by differential scanning calorimetry. The experiments have shown that complete peaks of cold denaturation can be recorded also in high concentration of protein solutions. The cold denaturation and the renaturation of the protein are reproducible, but the thermal denaturation is irreversible. The activation energy of thermal denaturation calculated is about 285 kJ/mol.
基金Supported by the Project of Science & Technology Plans in Heilongjiang Province in the 11th Five-year Period (GB07B407)
文摘Reversed-phase high-performance liquid chromatography （RP-HPLC） method with C4 column and C18 column for analyzing β-lactoglobulin and α-lactalbumin in bovine milk was developed and the performance and characteristic of two columns were compared. Shiseido Proteonavi C4 column （250 mm×4.6 mm×5μm） and Shiseido CAPCELL PAK SG 300 C18 column （250 mm× 4.6 mm×5 μm） were used in the experiment. Phase A was composed of 0.1% （V/V） trifluoroacetic acid （TFA） in ultrapure water and Phase B （organic phase） was composed of 0.1% TFA in acetonitrile. Gradient elution was taken. Flow rate was 1 mL min-1. The detection wavelength was 215 nm. The injection volume was 20 μL and the column temperature was 30℃. The results showed that linear relationship was good and recovery of α-lactalbumin and β-lactoglobulin was 86.12%-104.38%, C18 column had stronger ability to resist acid and more stable, and the method with C4 column had excellent sensitivities and good separation.
文摘The rationale of this work is based on recent evidences suggesting that: 1) both qualitative and quantitative β-lactoglobulin (β-LG) polymorphism may be found in bovine milk;2) quantitative polymorphisms are often the result of expression gradients in multiple copies of a gene;3) the β-LG gene is duplicated in the dog and bovine genome;4) mammary genes are highly conserved across Mammalia. Thus, an investigation was conducted on ovine β-LG polymorphism checking phenotypic evidence for copy-number variants of β-LG in sheep. To the purpose, 206 milk samples were collected, during a small-scale survey within sheep farms breeding Southern Italian breeds. PAGIF screening of the samples revealed that approximately 50% individuals exhibited β-LG polymorphism and 4 different quantitative patterns, which were characterized in detail by a proteomic approach relying on combined chromatographic and mass spectrometric techniques. The expected figures based on the expression gradient models were compared with well-established α-globin gene arrangements in sheep. The different phenotypes suggest the presence of both duplicate and triplicate BLG haplotypes. The occurrence of a triplicate haplotype was supported by population data. The current study supports the helpfulness of up-to-date proteomics for inferring copy number polymorphisms through the characterization of the phenotypic expression.
文摘运用代谢组学技术研究表没食子素儿茶素没食子酸酯(epigallocatechin gallate,EGCG)⁃β⁃乳球蛋白(β⁃lacto⁃globulin,βlg)复合物对βlg致过敏反应小鼠血液中代谢物的变化,筛选出过敏反应与脱敏相关的潜在生物标志物,进一步分析其代谢通路,探讨EGCG对βlg脱敏的代谢机制。采用多酚成分EGCG与βlg形成复合物进行脱敏,以βlg诱导小鼠发生过敏反应为模型组,观察空白组、βlg组、βlg⁃EGCG组小鼠过敏行为学体征,通过超高效液相色谱⁃四极杆静电场轨道阱质谱(ultra performance liquid chromatography⁃quadrupole⁃electrostatic field orbitrap mass spectrometry,UPLC⁃QE⁃MS)技术对小鼠血清进行代谢组学分析,并进一步采用主成分分析和偏最小二乘辨别分析探究各组代谢轮廓差异,筛选出差异代谢物。结果显示,βlg组和βlg⁃EGCG组的代谢谱图存在明显差异,并筛选出与过敏反应相关的40个差异代谢物,7条主要代谢通路。推测βlg致敏与EGCG脱敏作用机制可能涉及氨基酸代谢和脂类代谢等过程。
