双向电泳分析链格孢霉过敏原提取液的致敏蛋白组分

I为7.5～8.5;34×103,PI为6.5～7.0;45×103,PI为7.0～8.0;57×103,PI为6.5～7.5.结论 双向电泳可用于链格孢霉过敏原提取液致敏蛋白组分的分析.     

过敏原蛋白组分sIgE检测及分析

目的 分析过敏性疾病患者血清中多种过敏原蛋白组分的sIgE,明确过敏原中的致敏蛋白组分,为临床过敏性疾病预防和治疗提供科学依据.方法 采用荧光酶联免疫法,用ImmunoCAP过敏原检测试剂检测受试者血液标本中过敏原蛋白组分及相关过敏原的特异性IgE(sIgE)水平,统计每种过敏原蛋白组分sIgE检出量和阳性率,并对主要致敏蛋白组分sIgE阳性率和相关过敏原的阳性率进行对比和分析.结果 40种过敏原蛋白组分的阳性率均小于过敏原的阳性率,致敏组分和交叉过敏组分共存于过敏原中.户尘螨、黄胡蜂毒、桃、猫毛、榛子、牛奶和鸡蛋的主要致敏蛋白组分阳性率高于过敏原阳性率的50%;百慕达草、意大利蜜蜂毒、桦树和花生的主要致敏蛋白组分阳性率介于过敏原阳性率的20%~50%之间;虾、狗毛、梯牧草和北艾草的主要致敏蛋白组分阳性率低于过敏原阳性率的20%.结论 过敏原蛋白组分检测能鉴别过敏原中真正致敏的蛋白组分,解决由于交叉反应所导致的过敏原特异性诊断难题,确保过敏性疾病特异性免疫治疗的精确性和有效性.     

桃过敏原组分检测蛋白质芯片的制备及验证

目的制备用于检测血清中针对几种桃过敏原组分抗体的蛋白质芯片,为针对桃过敏原组分过敏患者的临床诊断提供快速可靠的方法。方法合成6种常见的桃过敏原组分的编码基因,分别将其插入p GAPZαA酵母表达载体,AvrⅡ单酶切线性化后,电转毕赤酵母SMD1168,表达和纯化相应的桃过敏原组分蛋白,并制备蛋白质芯片;用蛋白芯片检测临床血清样本中针对桃过敏原组分蛋白的抗体,与ImmunoCAP法相比,验证蛋白质芯片检测的灵敏度和特异性。结果用酵母表达系统成功表达并纯化了4种桃过敏原组分Pru p 1、Pru p 3、Pru p 4和Pru p 7,制备了能进行质量控制的检测血清桃过敏原组分抗体的蛋白质芯片。检测了疑似桃过敏患者血清41例,结果以ImmunoCAP法为金标准,蛋白芯片检测血清Pru p 1、Pru p 3和Pru p 4抗体的灵敏度分别为40%、100%和100%,特异性为78%、50%和100%;对上述3种组分抗体的综合检测灵敏度为86%,特异性为96%。针对缺乏ImmunoCAP检测结果的Pru p 7抗体,蛋白芯片检测出6份阳性的血清样本,均为桃过敏患者。结论桃过敏原组分检测芯片在灵敏度和特异性方面与ImmunoCAP相当,血清用量少,优化后有望成为临床桃过敏检测的一种新方法。     

霍乱弧菌菌体蛋白双向电泳技术的建立

目的建立霍乱弧菌全菌体蛋白的双向电泳技术，获得分辨度高、重复性好的双向电泳图谱。方法利用适当的裂解液处理霍乱弧菌，提取全菌蛋白；采用pH梯度等电聚焦对全菌蛋白进行双向电泳；考马斯亮蓝染色后获得的双向电泳图谱，并利用ImageMaster 2D Elite5．0图象分析软件进行分析，所得的数据用SPSS15．0进行统计分析。结果得到了（1081±16）个蛋白斑点，蛋白主要集中在pI4．24～7．20之间，重复胶的匹配点数为（1057±28），匹配率为97．85％。结论建立了霍乱弧菌全菌双向电泳分析方法，2-DE图谱中蛋白位点的分辨率和重复性非常高，获得了较为理想、清晰的双向电泳图谱，为进一步研究其蛋白质组学奠定了基础。     

双向电泳技术在蛋白质组研究中的应用体会

双向电泳(two-dimensional electrophoresis,2-DE)作为蛋白质组研究的三大核心技术之一,是目前分离复杂蛋白质组分最常用的工具.双向电泳分离蛋白质的优势在于具有高分辨率、高重复性和高信息量,因此日益受到人们的广泛关注.其原理是第一向根据蛋白质的等电点(pI)分离蛋白质,第二向是等电点相同的蛋白质根据蛋白质的分子量(MW)不同,再次得到分离.     

针刺大鼠太溪 对穴位组织双向电泳图谱影响的实验研究

研究太溪穴位组织蛋白质特异性,探寻针刺对大鼠太溪穴位组织双向电泳图谱的影响.8周龄雄性Wistar大鼠12只,随机分为空白对照组(n=6)和针刺组(n=6).针刺组每日定时电针双侧太溪穴,空白对照组大鼠不予针刺.1周后切取太溪穴组织和非经非穴组织,进行双向电泳,比较分析太溪穴和非穴组织、太溪穴组织针刺前后的双向电泳图谱.大鼠太溪穴组织与非经非穴组织存在8个完全差异蛋白点,针刺前后太溪穴组织胶图中蛋白点分布发生显著变化,同时可见蛋白点数量由285升至326.说明:太溪穴位组织较非穴位组织存在蛋白质差异性,针刺可引起太溪穴区组织蛋白质组发生显著变化.     

应用于双向电泳技术的不同软骨蛋白提取方案对比

目的探索可兼顾双向电泳凝胶图像质量和结果保真性的软骨蛋白提取方案。方法取股骨髁软骨(n=17)。分别用软骨组织直接提取总蛋白(软骨组织组)、软骨组织提取总蛋白后CPC处理(软骨+CPC组)、直接分离软骨细胞提取总蛋白(直接软骨细胞组)或培养软骨细胞提取总蛋白(培养软骨细胞组)同步进行2-DE,对比不同方案产生凝胶图像质量和结果保真性的差异。结果软骨组织组不能形成等电聚焦。软骨+CPC组可形成等电聚焦,但蛋白点数量偏少。直接软骨细胞组可获得与培养软骨细胞组媲美的高质量等电聚焦和凝胶图像,且在高分子量和偏碱区域分离出培养软骨细胞组缺如的部分蛋白点,质谱结果显示这些蛋白分别为VI型胶原、TGF-β2和annexin等骨关节炎病因学相关蛋白。结论从软骨组织直接提取软骨细胞用于2-DE的方案在解决等电聚焦难题的同时,还避免了细胞培养对实验结果保真性的影响,是软骨相关疾病样本的2-DE研究优化处理方案。     

恶性胶质瘤细胞SHG-44诱导分化后的差异蛋白质组双向电泳分析

目的用双向电泳分析诺帝诱导胶质瘤细胞SHG-44分化后的差异蛋白质组,为进一步了解这些差异蛋白质的作用打下基础。方法将诺帝诱导胶质瘤细胞SHG-44分化后的总蛋白及其相应的空白对照组细胞总蛋白进行双向电泳分离,重复3次后用PDQuest7.1软件比较分析蛋白质表达差异并获得差异蛋白质的相对分子质量、等电点等信息。结果诺帝诱导胶质瘤细胞SHG-44分化后有23个差异蛋白点,其中21个蛋白点表达下调,2个蛋白点表达上调。结论诺帝诱导胶质瘤细胞SHG-44分化后大部分蛋白质表达下调,推测诺帝诱导胶质瘤细胞SHG-44分化时蛋白表达以抑制作用为主。     

利用双向电泳比较3种提取户尘螨蛋白的方法

为改进户尘螨过敏原提取方法,在采用Coca^ s液、裂解液及Trizol法提取纯种户尘螨螨体蛋白后,用二喹啉甲酸(BCA)检测法测定蛋白质含量,双向电泳比较上述蛋白提取方法的效果。结果显示,在蛋白质回收率方面,裂解液优于Trizol法优于Coca^ s液。经过双向电泳,Coca^ s液提取的蛋白仅仅有少量低分子量的蛋白点;用裂解液提取的蛋白可见明显增多低分子量的蛋白点,但无中分子量的蛋白点;Trizol法提取的蛋白可见中分子量的蛋白点如174～178ku和133．0ku的蛋白质。另外,纯种户尘螨螨体经Trizol液提取后进行双向电泳,在考染和银染时均出现5个高丰度的特殊蛋白点,一个为酸性中分子量蛋白,位置孤立的;另有4个位置彼此非常接近的中性蛋白点。比较后结论为使用Coca^ s液提取的尘螨蛋白质浓度低,蛋白点少。使用裂解液提取的尘螨蛋白质浓度虽然较高,但是没有中分子量的蛋白点,不能全面反映尘螨的过敏原。使用Trizol法提取的蛋白质浓度中等,有中分子量蛋白点,反映的蛋白点更加全面。纯种户尘螨螨体经Trizol液提取后进行双向电泳,在考染和银染时出现的特征性蛋白点,作为一种双向电泳的指纹图谱,在鉴定此种纯种户尘螨螨体时具有一定的价值。     

Application of two-dimensional electrophoresis in the research of retinal proteins of diabetic rat

Diabetes mellitus （DM） is a chronic disease which is associated with numerous serious health complications such as diabetic retinopathy, and is the leading cause of new cases of blindness in adults at the age of 20-74 years old. The aim of the study was to establish and optimize a two-dimensional polyacrylamide gel electrophoresis （2-DE） technique for retina proteomics to improve the resolution and reproducibility, and to observe the proteomic changes of retinal tissues in diabetic and normal rats. Proteins were extracted from retinal tissues of normal and 8 weeks diabetic SD rats and used in two-dimensional electrophoresis. Various conditions of retina proteomic 2-DE were adjusted, optimized and protein spots of differential expression were obtained through analysis of 2-DE images with PDQuest software. By choosing appropriate sample amount, using pre-cast IPG dry strips （pH 5-8） and casting 12% equal gel, satisfactory 2-DE images of retina were obtained and a steady 2-DE technique was established. In this way, we found 36 spots in 2-DE gel of diabetic retinas that exhibited statistically significant variations, including up-regulation of 5 proteins in diabetic rat retinas, down-regulation of 23, and disappearance of 8, in comparison with normal tissues. The differences of protein expression were observed in retinas between diabetic and normal rats. Our established 2-DE technique of retina proteins could be effectively applied in proteomics of retina diseases. Cellular ＆ Molecular Immunology. 2007;4（1）：65-70.     

Comprehensive and accurate mutation scanning of the CFTR gene by two-dimensional DNA electrophoresis

The large number of possible disease-causing mutations in the 27 exons of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has severely limited direct diagnosis of cystic fibrosis (CF) patients and carriers by mutation detection. Here we show that in principle testing for mutations in the CFTR gene can be both substantially facilitated and made virtually complete, by two-dimensional DNA electrophoretic separation of polymerase chain reaction (PCR) amplified exons on the basis of size and basepair sequence in denaturing gradient gels. Under a single optimized set of conditions we were able to obtain a pattern of spots representing all 27 exons of the CFTR gene and to readily detect 17 out of 17 identified sequence variations in 9 different exons in DNA from 11 CF patients and carriers. Our results demonstrate the potential of 2-dimensional DNA electrophoresis for comprehensive mutation analysis of the CFTR gene. The approach serves as a model for comprehensive diagnosis of the many other large disease genes for which a variety of mutations have also which been reported. © 1996 Wiley-Liss, Inc.     

Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry

Despite improvements in treatment of patients with head and neck squamous cell carcinoma (HNSCC) over the last two decades, the survival rate of these patients has not increased significantly. One of the major factors in the poor outcome of the disease is regional metastasis. To better understand the mechanisms of this process at the protein level, we performed two-dimensional electrophoresis (2-DE) and mass spectrometry using SELDI ProteinChip technology to identify proteins differentially expressed in two HNSCC cell lines, UMSCC10A and UMSCC10B, from the same patient. UMSCC10A was derived from the primary tumor and UMSCC10B from a metastatic lymph node. The differentially expressed proteins were excised from the gels. Following in-gel digestion by trypsin, mass profiles of the peptides were generated. Proteins were identified by submitting the peptide mass profiles to a public available NCBInr databases (www.proteometrics.com). Two membrane-associated proteins, annexin I and annexin II, and glycolytic protein enolase-α were found to be upregulated, and calumenin precursor down-regulated, in metastatic cell line UMSCC10B. The identity of these proteins was confirmed by analyzing additional peptide mass fingerprints obtained by endoproteinase lysine-C digestion. The results were also validated by Western blotting analysis. Our results showed that enolase-α, annexin-I and annexin-II might be important molecules in head and neck cancer invasion and metastasis. The results also suggest an important complementary role for proteomics in identification of molecular abnormalities important in cancer development and progression. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification and characterisation of the IgE-binding proteins 2S albumin and conglutin gamma in almond (Prunus dulcis) seeds

BACKGROUND: Almond proteins can cause severe anaphylactic reactions in susceptible individuals. The aim of this study was the identification of IgE-binding proteins in almonds and the characterisation of these proteins by N-terminal sequencing. METHODS: Five sera were selected from individuals with a positive reaction to food challenge. Sodium dodecylsulphate-polyacrylamide gel electrophoresis and immunoblotting were performed on almond seed proteins. Purified IgE-binding proteins were tested for immunoblot inhibition with sera pre-incubated with extracts of hazelnut and walnut. RESULTS: N-terminal sequences of the 12-, 30- and 45-kD proteins were obtained. The 45- and 30-kD proteins shared the same N terminus, with 60% homology to the conglutin gamma heavy chain from lupine seed (Lupinus albus) and to basic 7S globulin from soybean (Glycine max). The sequences of the N-terminal 12-kD protein and of an internal peptide obtained by endoproteinase digestion showed good homology to 2S albumin from English walnut (Jug r 1). Immunoblot inhibition experiments were performed and IgE binding to almond 2S albumin and conglutin gamma was detected in the presence of cross-reacting walnut or hazelnut antigens. CONCLUSIONS: Two IgE-binding almond proteins were N-terminally sequenced and identified as almond 2S albumin and conglutin gamma. Localisation and conservation of IgE binding in a 6-kD peptide obtained by endoproteinase digestion of 2S albumin was shown.     

The apoE polymorphism studied by two-dimensional, high-resolution gel electrophoresis of serum

The apoE polymorphism of human very low density lipoprotein (VLDL) was studied by the two-dimensional high-resolution gel electrophoresis technique of O'Farrell, which combines isoelectric focusing and SDS-polyacrylamide gel electrophoresis. With whole serum, six major patterns and two "sub-variants" of apoE were observed. A series of twin pairs (21 monozygotic (MZ) and 13 dizygotic (DZ) pairs) as well as unrelated people were analyzed. Both members of MZ pairs always exhibited the same pattern, whereas DZ twins were often discordant. The patterns observed would be consistent with the hypothesis that three apoE isopeptides are coded for by three different alleles at one single locus. In this small series, all three postulated homozygous patterns, namely apoE-II, apoE-III and apoE-IV as well as the three heterozygous patterns apoE-11, 111; apoE-III, IV and apoE-ll, IV were seen.     

Mouse serum amyloid A (SAA) proteins isolated by two-dimensional electrophoresis: characterization of isotypes and the effect of separate and combined administrations of cytokines,dexamethasone and lipopolysaccharide (LPS) on serum levels and isotype distribution

Hydrophobic interaction chromatography and two-dimensional electrophoresis were used to isolate and characterize mouse SAA, and to study the in vivo effect of separate or combined administrations of cytokines, dexamethasone (DEX) and LPS on mouse SAA. Four SAA spots containing partial amino acid sequence in accordance with mouse apoSAA1 and apoSAA2/SAA_SJL/J pI 5.9 were demonstrated in serum. One of these proteins represents a previously undescribed, acidic acute-phase mouse SAA protein. Both DEX and interferon-gamma (IFN-γ) proved to be capable of increasing SAA serum levels. In contrast to what has been shown in previous in vivo studies, administration of IL-6 did increase the SAA levels to nearly the same magnitude as IL-1, and the effect of IL-6 and LPS on SAA production was not significantly altered by the addition of DEX. Irrespective of the inflammatory stimuli that was administered, a non-selective production of SAA1 and SAA2 was observed in most groups, including the group that received IL-6. The results illustrate that data obtained about mouse SAA are highly dependent on which models, isolation and identification methods are used.     

二维条形码技术在实验室信息管理系统（LIS）中的应用

目的利用二维条形条码技术,减轻实验室信息管理系统(LIS)瓶颈,提高网络速度,为LIS增加更多功能(例如智能审核,图像传输等)创造良好的条件。方法设计与制作二维条码标签,以Windows2000AdvancedServer或Windows2000Server为服务器操作系统;以有极佳性价比的MicrosoftSQLServer2000数据库;以PowerBuilder8.0为前台开发工具;以Windows2000Professional或WindowsXPRrofesional为客户端操作系统;把客户端联接成网并入LIS服务器,再与医院信息系统(HIS)联接。结果通过DENSO二维条码数据采集器BHT103Q扫描标本容器上的二维条码,直接获得全部病人资料(包括基本资料和检验信息)。最后将测定结果发送回HIS。结论通过使用二维条形码技术,减少LIS数据库的访问量和网络数据流量,从而有效减轻LIS瓶颈,提高了系统速度。为以后完善和丰富LIS的功能创造了良好的条件。