金华火腿蛋白质的双向电泳研究
摘要
金华火腿作为地方传统特色食品,如何有效控制和评判其品质特性,一直以来都是相关的科研人员、企业乃至地方政府所关注的焦点。目前火腿的品质鉴定一般集中在外表样式、气味及风味等方面,而对优质火腿品质评价和控制尚缺乏规范有效的方法。蛋白质作为火腿中最主要的成分,它的变化对火腿品质的影响很大,因此从蛋白质的角度研究火腿的品质特性具有重要的意义。双向凝胶电泳技术是目前研究蛋白质的较核心的技术,利用双向电泳技术筛选出与火腿品质相关的特征蛋白的变化,为研究火腿的品质评价打下基础。
本研究通过对火腿样品制备和双向电泳条件等相关技术参数进行比较和优化,建立了火腿双向电泳的技术方法；并以“雪纺蒋”、“冠美”、“宗泽”、“金字”四种品牌金华火腿为研究对象,对不同品牌的金华火腿进行了差异蛋白质组学的初步研究。并对“雪纺蒋”、“冠美”两种品牌金华火腿货架期内进行了差异蛋白质组学研究,主要实验结果如下：
1、在本实验已经建立的生鲜猪肉双向电泳条件下,通过对火腿肌肉组织样品制备图谱的比较,发现采用凝胶层析脱盐柱-冷冻干燥-裂解法制备火腿肌肉样品能有效去除样品中的盐分,并达到了提高蛋白的抽提率的效果,具有较高的分辨率和良好的重复性。
2、通过对胶条pH、蛋白上样量、等电聚焦参数以及染色方法等条件的优化,建立了火腿肌肉蛋白质双向电泳的方法。结果表明,采用pH5-8、蛋白质上样300 ug、渐进式升压的方式能获得较好的等电聚焦效果,减少横条纹；改进后的银染能检测到更多的低丰度蛋白,适于凝胶分析。
3、应用建立的火腿肌肉组织双向电泳方法对四种不同品牌的金华火腿蛋白样品进行差异蛋白质组学分析。SDS-PAGE凝胶电泳结果表明,差异蛋白主要分布在18.4KD～35KD和45KD～116KD之间。通过双向电泳分离,“雪纺蒋”、“冠美”、“宗泽”、“金字”四种品牌金华火腿肌肉组织的双向电泳凝胶的平均蛋白点数分别为1238±7、1126±6、1067±9、1035±10,经PDQuest8.0软件对比筛选分析得到“雪纺蒋”和“冠美”、“雪纺蒋”和“宗泽”、“雪纺蒋”和“金字”、“冠美”和“宗泽”、“冠美”和“金字”、“宗泽”和“金字”显著差异的蛋白点分别有11、12、11、8、6、6个。
4、应用建立的火腿肌肉组织双向电泳方法对“雪纺蒋”、“冠美”牌金华火腿不同货架期内蛋白样品进行差异蛋白质组学分析。不同货架期的差异蛋白主要分布在18.4 KD～35 KD和45 KD～116 KD之间。对“雪纺蒋”牌金华火腿相邻货架期的图谱进行统计分析,从中分别筛选出10、8、5、12、10个具有显著差异的蛋白点。对“冠美”牌金华火腿相邻货架期的图谱进行统计分析,从中分别筛选出11、8、7、8、4个具有显著差异的蛋白点。本实验初步找到的“雪纺蒋”“冠美”牌金华火腿货架期内每相邻两个时间点之间的差异蛋白质点,可以分为三类：(1)在货架期内表达量基本不变的蛋白质。此类蛋白质对火腿货架期内品质变化的影响很小,几乎可以忽略不计。(2)随着货架期时间的延长表达量下降或者不再表达的蛋白质。这类蛋白质可能参与了蛋白质的分解,形成了更小分子量的蛋白质及其他的小分子物质。(3)随着货架期时间的延长表达量提高的蛋白质。这类蛋白质可能是大分子蛋白质分解产生的,分解产生的蛋白质与之前原有的蛋白质分子相加和,使得这部分蛋白质量有所增加。
Jinhua ham as a local traditional food, how to effectively control and evaluate its quality characteristics has always been the focus for relevant scientific, corporate and even local governments. The current identification methods for quality of ham generally concentrated on the exterior style, such as odor and flavor, while the high-quality ham quality evaluation still lacks standardized and effective methods. Protein is the main compotent for Jinhua ham, and its changes effect the quality of Jinhua ham, so it is meanful for studying protein of Jinhua ham. Two-dimensional gel electrophoresis technology is the core of proteomics, and they provide a new way for ham quality evaluation. With Two-dimensional gel electrophoresis technology, we will search the characteristics protein associated with the ham-quality, and it can offer the basis for further study of differences in the different ham quality.
By optimizing the ham sample preparation methods, two-dimensional electrophoresis conditions and other related technical parameters, we established two-dimensional electrophoresis methods for Jinhua ham. Then we compared the maps of "Xuefangjiang," "Guanmei", "Zongze," and "Jinzi" ham. We chose "Xuefangjiang," "Guanmei" ham in different shelves, and studied the changes of their maps, and the main results are as follows:
1, At the basic of the general two-dimensional electrophoresis parameters of pork, we found gel filtration desalting columns is the better method through comparing maps with different methods. This method can remove salt and improve the rate of protein extraction with high resolution and good reproducibility.
2, By optimizing the pH, the sample volume, isoelectric focusing parameters and staining methods and so on, we established two-dimensional electrophoresis methods for Jinhua ham. The results show that the pH 5～8, the content of proteins 300ug, progressive boost mode can get better isoelectric focusing effects and reduce the horizontal stripes, and the improved silver staining method can detect more low abundance proteins, and it is suitable for gel analysis.
3, Using the established 2-DE for ham muscle tissue, we analyzed four different brands of Jinhua ham. SDS-PAGE gel electrophoresis showed that differences in protein mainly distributed between 18.4KD and 66.2KD and between 45KD and 116KD. Separated by two-dimensional electrophoresis, the average protein spots of "Xuefangjiang", "guanmei", "Zongze" and "Jinzi" separately are 1238±7,1126±6,1067±9,1035±10. By software analysis we obtained significant different spots that between "Xuefangjiang" and "guanmei", between "Xuefangjiang" and "Zongze", between "Xuefangjiang" and "Jinzi", between "guanmei" and "Zongze", between "guanmei" and "Jinzi", between "Zongze" and "Jinzi" separately are 11,12,11,8,6,6.
4, Using the established 2-DE for ham muscle tissue, we analyzed Jinhua ham with different shelf life. SDS-PAGE gel electrophoresis showed that differences in protein mainly distributed between 18.4KD and 66.2KD and between 45KD and 116KD. "Xuefangjiang" ham with different shelves were analyzed, the significant different protein points between adjacent two shellf time were separately 10,8,5,12,10. The "Guanmei" ham with different shelves were analyzed, the significant different protein points that between adjacent two shellf time were separately 11,8,7,8,4. During shelf time the different protein points can be divided into three categories:(1) The amount of the expressed proteins are almost the same during shelf life. The effect of this kind of proteins on the quality of ham is very small, almost negligible. (2) During shelf life some expressed proteins decreased or disappeared. Such proteins may be involved in degradation, and form smaller proteins or other small molecules. (3) During shelf life some expressed proteins increased. Such proteins may be come from the degradation of protein with large molecules, and this part of proteins increased with the add of original proteins and made this part of proteins increased.
本研究通过对火腿样品制备和双向电泳条件等相关技术参数进行比较和优化,建立了火腿双向电泳的技术方法；并以“雪纺蒋”、“冠美”、“宗泽”、“金字”四种品牌金华火腿为研究对象,对不同品牌的金华火腿进行了差异蛋白质组学的初步研究。并对“雪纺蒋”、“冠美”两种品牌金华火腿货架期内进行了差异蛋白质组学研究,主要实验结果如下：
1、在本实验已经建立的生鲜猪肉双向电泳条件下,通过对火腿肌肉组织样品制备图谱的比较,发现采用凝胶层析脱盐柱-冷冻干燥-裂解法制备火腿肌肉样品能有效去除样品中的盐分,并达到了提高蛋白的抽提率的效果,具有较高的分辨率和良好的重复性。
2、通过对胶条pH、蛋白上样量、等电聚焦参数以及染色方法等条件的优化,建立了火腿肌肉蛋白质双向电泳的方法。结果表明,采用pH5-8、蛋白质上样300 ug、渐进式升压的方式能获得较好的等电聚焦效果,减少横条纹；改进后的银染能检测到更多的低丰度蛋白,适于凝胶分析。
3、应用建立的火腿肌肉组织双向电泳方法对四种不同品牌的金华火腿蛋白样品进行差异蛋白质组学分析。SDS-PAGE凝胶电泳结果表明,差异蛋白主要分布在18.4KD～35KD和45KD～116KD之间。通过双向电泳分离,“雪纺蒋”、“冠美”、“宗泽”、“金字”四种品牌金华火腿肌肉组织的双向电泳凝胶的平均蛋白点数分别为1238±7、1126±6、1067±9、1035±10,经PDQuest8.0软件对比筛选分析得到“雪纺蒋”和“冠美”、“雪纺蒋”和“宗泽”、“雪纺蒋”和“金字”、“冠美”和“宗泽”、“冠美”和“金字”、“宗泽”和“金字”显著差异的蛋白点分别有11、12、11、8、6、6个。
4、应用建立的火腿肌肉组织双向电泳方法对“雪纺蒋”、“冠美”牌金华火腿不同货架期内蛋白样品进行差异蛋白质组学分析。不同货架期的差异蛋白主要分布在18.4 KD～35 KD和45 KD～116 KD之间。对“雪纺蒋”牌金华火腿相邻货架期的图谱进行统计分析,从中分别筛选出10、8、5、12、10个具有显著差异的蛋白点。对“冠美”牌金华火腿相邻货架期的图谱进行统计分析,从中分别筛选出11、8、7、8、4个具有显著差异的蛋白点。本实验初步找到的“雪纺蒋”“冠美”牌金华火腿货架期内每相邻两个时间点之间的差异蛋白质点,可以分为三类：(1)在货架期内表达量基本不变的蛋白质。此类蛋白质对火腿货架期内品质变化的影响很小,几乎可以忽略不计。(2)随着货架期时间的延长表达量下降或者不再表达的蛋白质。这类蛋白质可能参与了蛋白质的分解,形成了更小分子量的蛋白质及其他的小分子物质。(3)随着货架期时间的延长表达量提高的蛋白质。这类蛋白质可能是大分子蛋白质分解产生的,分解产生的蛋白质与之前原有的蛋白质分子相加和,使得这部分蛋白质量有所增加。
Jinhua ham as a local traditional food, how to effectively control and evaluate its quality characteristics has always been the focus for relevant scientific, corporate and even local governments. The current identification methods for quality of ham generally concentrated on the exterior style, such as odor and flavor, while the high-quality ham quality evaluation still lacks standardized and effective methods. Protein is the main compotent for Jinhua ham, and its changes effect the quality of Jinhua ham, so it is meanful for studying protein of Jinhua ham. Two-dimensional gel electrophoresis technology is the core of proteomics, and they provide a new way for ham quality evaluation. With Two-dimensional gel electrophoresis technology, we will search the characteristics protein associated with the ham-quality, and it can offer the basis for further study of differences in the different ham quality.
By optimizing the ham sample preparation methods, two-dimensional electrophoresis conditions and other related technical parameters, we established two-dimensional electrophoresis methods for Jinhua ham. Then we compared the maps of "Xuefangjiang," "Guanmei", "Zongze," and "Jinzi" ham. We chose "Xuefangjiang," "Guanmei" ham in different shelves, and studied the changes of their maps, and the main results are as follows:
1, At the basic of the general two-dimensional electrophoresis parameters of pork, we found gel filtration desalting columns is the better method through comparing maps with different methods. This method can remove salt and improve the rate of protein extraction with high resolution and good reproducibility.
2, By optimizing the pH, the sample volume, isoelectric focusing parameters and staining methods and so on, we established two-dimensional electrophoresis methods for Jinhua ham. The results show that the pH 5～8, the content of proteins 300ug, progressive boost mode can get better isoelectric focusing effects and reduce the horizontal stripes, and the improved silver staining method can detect more low abundance proteins, and it is suitable for gel analysis.
3, Using the established 2-DE for ham muscle tissue, we analyzed four different brands of Jinhua ham. SDS-PAGE gel electrophoresis showed that differences in protein mainly distributed between 18.4KD and 66.2KD and between 45KD and 116KD. Separated by two-dimensional electrophoresis, the average protein spots of "Xuefangjiang", "guanmei", "Zongze" and "Jinzi" separately are 1238±7,1126±6,1067±9,1035±10. By software analysis we obtained significant different spots that between "Xuefangjiang" and "guanmei", between "Xuefangjiang" and "Zongze", between "Xuefangjiang" and "Jinzi", between "guanmei" and "Zongze", between "guanmei" and "Jinzi", between "Zongze" and "Jinzi" separately are 11,12,11,8,6,6.
4, Using the established 2-DE for ham muscle tissue, we analyzed Jinhua ham with different shelf life. SDS-PAGE gel electrophoresis showed that differences in protein mainly distributed between 18.4KD and 66.2KD and between 45KD and 116KD. "Xuefangjiang" ham with different shelves were analyzed, the significant different protein points between adjacent two shellf time were separately 10,8,5,12,10. The "Guanmei" ham with different shelves were analyzed, the significant different protein points that between adjacent two shellf time were separately 11,8,7,8,4. During shelf time the different protein points can be divided into three categories:(1) The amount of the expressed proteins are almost the same during shelf life. The effect of this kind of proteins on the quality of ham is very small, almost negligible. (2) During shelf life some expressed proteins decreased or disappeared. Such proteins may be involved in degradation, and form smaller proteins or other small molecules. (3) During shelf life some expressed proteins increased. Such proteins may be come from the degradation of protein with large molecules, and this part of proteins increased with the add of original proteins and made this part of proteins increased.
引文
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