Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive a...Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.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
【目的】鹰嘴桃是岭南地区的特色水果,因其良好的风味和品质被评为“岭南十大佳果”。通过分析鹰嘴桃过敏原蛋白的抗原表位,为过敏原重组抗原的制备提供研究基础,也为阐述食品加工过程中鹰嘴桃过敏原的特性变化和致敏特性提供研究依据...【目的】鹰嘴桃是岭南地区的特色水果,因其良好的风味和品质被评为“岭南十大佳果”。通过分析鹰嘴桃过敏原蛋白的抗原表位,为过敏原重组抗原的制备提供研究基础,也为阐述食品加工过程中鹰嘴桃过敏原的特性变化和致敏特性提供研究依据。【方法】利用磷酸缓冲液提取鹰嘴桃冻干粉中的粗蛋白,采用SDS-PAGE蛋白电泳方法鉴定并联合蛋白质谱方法分析粗蛋白中存在的过敏原,通过蛋白数据库UniProt进行筛选比对,并采用生物信息学方法分析致敏蛋白的理化性质、空间结构和抗原表位等生物学特性。【结果】从鹰嘴桃中鉴定出7种致敏蛋白(A0A251RBV3、P86888、M5X697、M5WV03、M5WTQ8、Q2I6V8、Q9LED1),主要归属于病程相关蛋白(Pru p 1)、类甜蛋白(Pru p 2)、非特异性脂质转移蛋白家族(Pru p 3)和赤霉素调节蛋白(Pru p 7)4类过敏原蛋白。7种鹰嘴桃过敏原蛋白具有较高稳定性,分子量为6.91~26.04 kD,脂肪族氨基酸指数为29.37~81.54。M5WTQ8和Q2I6V8过敏原蛋白为酸性蛋白,其余过敏原蛋白为碱性蛋白。除Q9LED1蛋白外,其余过敏原蛋白均为亲水蛋白。筛选抗原表位、亲水性和柔韧性大于0且表面可及性大于1的区域,并结合二、三级蛋白结构分析蛋白键能较低的区域,获得鹰嘴桃过敏原的抗原表位分别为Pru p 1(EIP、GSQ、KEN、NL、KG、EIK、HPD)、Pru p 2(TGDQKPQ、SP、NQ、PPNDKPETCPPT、DDKSS、RP)、Pru p 3(RT、VN)和Pru p 7(AGY、GTYGN、LKNSKGN)。【结论】通过对鹰嘴桃过敏原蛋白的结构、亲水性、表面可及性、柔韧性和抗原指数进行分析,获得过敏原蛋白的多个抗原表位,可为鹰嘴桃过敏原在食品加工中的致敏特性研究提供研究基础。展开更多
基于巴斯德毕赤酵母表达系统探讨了屋尘螨过敏原Der p 1的重组表达与纯化。首先,采用毕赤酵母GS115表达密码子优化的全长编码基因PreProDer p 1,其产量可达100 mg/L,进一步共表达分子伴侣实现产量提高到140 mg/L,并实现3 L反应器发酵产...基于巴斯德毕赤酵母表达系统探讨了屋尘螨过敏原Der p 1的重组表达与纯化。首先,采用毕赤酵母GS115表达密码子优化的全长编码基因PreProDer p 1,其产量可达100 mg/L,进一步共表达分子伴侣实现产量提高到140 mg/L,并实现3 L反应器发酵产量提高到1 g/L。其次,对上述重组蛋白发酵液分别使用阳离子交换层析及亲和层析进行了纯化工艺优化,目的蛋白得率达到60.7%。本研究为后续PreProDre p 1诊断试剂盆的开发提供了参考。展开更多
基金supported by the National Natural Science Foundation of China (No: 81230079, 81102414, 81227802)the Natural Science Basic Research Plan in Shaanxi Province of China (Program No. 2017JQ8024)
文摘Cow's milk allergy is mainly observed in infants and young children. Most allergic reactions affect the skin, followed by the gastrointestinal and respiratory systems. Conventional diagnosis is based on positive allergy studies and evaluation of parameters including IgE and IgG1 levels, acute allergic skin response and anaphylactic shock reactions. We developed a cell membrane chromatographic(CMC)method based on human mast cells(HMC-1) for screening potential allergens in infant formula milk powders(IFMP). HMC-1 cell membranes were extracted and mixed with silica to prepare cell membrane chromatography columns(10 mm ? 2 mm i.d., 5 mm). Under the conditions of 0.2 mL/min flow rate and214 nm detection wavelength, human breast milk showed no retention. However, IFMP showed clear retention. The retained fractions were collected and analyzed through matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS). Four major milk proteins, i.e., α-casein, β-casein, α-lactalbumin, and β-lactoglobulin A, were identified. Furthermore, these proteins and β-lactoglobulin B showed clear retention on HMC-1/CMC columns. To test the degranulation effects of the five proteins, histamine and β-hexosaminidase release assays were carried out. All five proteins induced HMC-1 cells to release histamine and β-hexosaminidase. Also, we established a reversed phase liquid chromatographic(RPLC) method for the determination of the five proteins in IFMP and the results showed that 90% proteins in IFMP were α-casein and β-casein. We concluded that cow's milk proteins may be potential allergens and caseins cause more β-casein allergic risk than other proteins. This conclusion was consistent with other studies.
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
文摘【目的】鹰嘴桃是岭南地区的特色水果,因其良好的风味和品质被评为“岭南十大佳果”。通过分析鹰嘴桃过敏原蛋白的抗原表位,为过敏原重组抗原的制备提供研究基础,也为阐述食品加工过程中鹰嘴桃过敏原的特性变化和致敏特性提供研究依据。【方法】利用磷酸缓冲液提取鹰嘴桃冻干粉中的粗蛋白,采用SDS-PAGE蛋白电泳方法鉴定并联合蛋白质谱方法分析粗蛋白中存在的过敏原,通过蛋白数据库UniProt进行筛选比对,并采用生物信息学方法分析致敏蛋白的理化性质、空间结构和抗原表位等生物学特性。【结果】从鹰嘴桃中鉴定出7种致敏蛋白(A0A251RBV3、P86888、M5X697、M5WV03、M5WTQ8、Q2I6V8、Q9LED1),主要归属于病程相关蛋白(Pru p 1)、类甜蛋白(Pru p 2)、非特异性脂质转移蛋白家族(Pru p 3)和赤霉素调节蛋白(Pru p 7)4类过敏原蛋白。7种鹰嘴桃过敏原蛋白具有较高稳定性,分子量为6.91~26.04 kD,脂肪族氨基酸指数为29.37~81.54。M5WTQ8和Q2I6V8过敏原蛋白为酸性蛋白,其余过敏原蛋白为碱性蛋白。除Q9LED1蛋白外,其余过敏原蛋白均为亲水蛋白。筛选抗原表位、亲水性和柔韧性大于0且表面可及性大于1的区域,并结合二、三级蛋白结构分析蛋白键能较低的区域,获得鹰嘴桃过敏原的抗原表位分别为Pru p 1(EIP、GSQ、KEN、NL、KG、EIK、HPD)、Pru p 2(TGDQKPQ、SP、NQ、PPNDKPETCPPT、DDKSS、RP)、Pru p 3(RT、VN)和Pru p 7(AGY、GTYGN、LKNSKGN)。【结论】通过对鹰嘴桃过敏原蛋白的结构、亲水性、表面可及性、柔韧性和抗原指数进行分析,获得过敏原蛋白的多个抗原表位,可为鹰嘴桃过敏原在食品加工中的致敏特性研究提供研究基础。
文摘基于巴斯德毕赤酵母表达系统探讨了屋尘螨过敏原Der p 1的重组表达与纯化。首先,采用毕赤酵母GS115表达密码子优化的全长编码基因PreProDer p 1,其产量可达100 mg/L,进一步共表达分子伴侣实现产量提高到140 mg/L,并实现3 L反应器发酵产量提高到1 g/L。其次,对上述重组蛋白发酵液分别使用阳离子交换层析及亲和层析进行了纯化工艺优化,目的蛋白得率达到60.7%。本研究为后续PreProDre p 1诊断试剂盆的开发提供了参考。
