抗Ⅳ型登革病毒NS1单克隆抗体的制备及特性鉴定

目的:研制Ⅳ型登革病毒(DENV-4)非结构蛋白1(NS1)特异性的单克隆抗体(mAbs),并鉴定其特异性。方法:在表达具有良好抗原性的重组DENV4-NS1蛋白的基础上,用重组DENV4-NS1蛋白和灭活的DENV-4免疫BALB/c小鼠,取免疫小鼠的脾细胞与小鼠骨髓瘤细胞进行融合,用间接ELISA法筛选阳性的杂交瘤细胞,并结合免疫荧光测定法(IFA)和Western blot分析对mAb的特异性进行鉴定;通过竞争抑制试验对mAb识别的抗原位点进行分析。结果:获得15株特异性针对DENV4-NS1的mAbs,与其他3型DENV的NS1不产生交叉反应。mAb的Ig亚类测定均为IgG1。竞争抑制试验显示,15株mAbs存在2个不同识别抗原位点。结论:成功地获得特异性针对DENV4-NS1蛋白的mAbs,为进一步研究DENV4-NS1蛋白的结构与功能及研制DENV-4的临床诊断试剂奠定了基础。     

利用小鼠感染模型筛选与鉴定幽门螺杆菌外膜蛋白抗原

目的:利用小鼠感染模型筛选与鉴定幽门螺杆菌SS1株的外膜蛋白抗原。方法:提取SS1株的外膜蛋白进行双向电泳,用幽门螺杆菌感染的小鼠血清作免疫印迹实验,将阳性反应蛋白点进行质谱鉴定分析,将肽质量指纹谱数据输入互联网上的蛋白质数据库进行检索。结果:获得32种抗原相关蛋白。通过与已有报道的幽门螺杆菌感染人抗原比较分析,发现大部分典型的保护性抗原在本实验中都可以检测到。结论:幽门螺杆菌感染的小鼠模型适用于人用保护性幽门螺杆菌抗原的筛选;而且此研究中得到的相关抗原蛋白对于寻找与鉴定幽门螺杆菌未知保护性抗原也有参考价值。     

丙烯酰胺暴露对幼鼠胼胝体PLP和MBP表达的影响

目的:检测断乳期幼鼠胼胝体髓鞘蛋白PLP和MBP的表达,探讨丙烯酰胺(acrylamide,ACR)染毒对幼鼠胼胝体部髓鞘发育的影响。方法:断乳期幼鼠随机分为对照组(0 mg/kg)、低(18 mg/kg)和高(36 mg/kg)剂量组,每组12只,从出生后第22~42 d进行灌胃染毒。观测幼鼠步态的变化,用免疫组化方法和免疫荧光双标记法检测幼鼠胼胝体髓鞘蛋白脂蛋白(myelin PLP,PLP)和髓鞘碱性蛋白(myelin basic protein,MBP)的表达。结果:ACR染毒后幼鼠的步态评分均增加,差异有统计学意义(P0.01),免疫组织化学检测结果显示,与对照组相比较,ACR高剂量组幼鼠大脑PLP和MBP表达减少,差异有统计学意义(P0.05)。免疫荧光双标技术检测结果与免疫组织化学检测结果一致,即ACR染毒后胼胝体PLP和MBP均表达减少。结论:ACR染毒可能会通过减少PLP和MBP的表达,抑制胼胝体髓鞘的形成而影响神经系统发育。     

牛磷酸烯醇式丙酮酸羧激酶单克隆抗体的制备

目的制备牛磷酸烯醇式丙酮酸羧激酶(PEPCK)单克隆抗体(m Ab),并对其特异性进行初步鉴定。方法构建含PEPCK基因的重组质粒,将其转化大肠杆菌,并进行诱导表达,对表达产物PEPCK重组蛋白进行纯化,以PEPCK重组蛋白为抗原,免疫BALB/c小鼠,取脾细胞与小鼠骨髓瘤细胞融合获得杂交瘤细胞,用间接ELISA筛选阳性克隆,获得稳定分泌抗牛PEPCK m Ab的细胞株,并用Western blot法,免疫组织化学染色和免疫沉淀鉴定其特异性。结果成功获得稳定分泌抗牛PEPCK m Ab的杂交瘤细胞株。Western blot法、免疫组织化学和免疫沉淀结果表明,该抗体能与牛PEPCK蛋白特异性结合。结论成功获得抗牛PEPCK m Ab。     

抗人精胺氧化酶单克隆抗体的制备与鉴定

目的:制备小鼠抗人精胺氧化酶(SMO)单克隆抗体(mAb),并验证其在Western blot,免疫组织化学等方法中的应用价值.方法:用pET-15b/SMO质粒转化BL2 (DE3)后,IPTG诱导表达带6×His标签的SMO重组蛋白并用Ni-NTA树脂亲和层析纯化.用SMO重组蛋白免疫BALB/c小鼠,取免疫小鼠的脾细胞与Sp2/0骨髓瘤细胞杂交融合,获得能高效合成和分泌抗SMO mAb的杂交瘤细胞株.所得抗体的特异性和效价用ELISA和Western blot法以及免疫组织化学技术检测.结果:成功建立了稳定分泌鼠抗人SMO的mAb杂交瘤细胞株,获得的mAb效价高、特异性强,可用于ELISA,Western blot,免疫组织化学等分析技术.结论:成功制备出高特异性并能用于多种生物分析技术的SMO mAb.     

抗人ASGPR大亚基异构体蛋白H1b多克隆抗体的制备和鉴定

目的:制备小鼠抗人ASGPR大亚基异构体蛋白H1b的多克隆抗体,并鉴定其特异性.方法:合成H1b特异性多肽,以匙孔血蓝蛋白(KLH)作为载体构建多肽免疫原,通过致敏小鼠,制备小鼠抗人H1b蛋白的多克隆抗体;ELISA法检测抗体效价,通过Western blot和免疫组织化学检测,鉴定抗体的特异性与适用范围.结果:得到小鼠抗人H1b蛋白的多克隆抗体,效价达1:105.纯化后的抗体用于Western blot可高特异性识别真核表达的H1b蛋白,并可用于免疫组织化学实验.结论:成功制备小鼠抗人H1b蛋白的多克隆抗体,该抗体具有较高的效价以及特异性,能区分ASG-PR大亚基的两种剪接异构体,从而为进一步研究H1b蛋白的生理功能及其在人类疾病中的意义提供了有利工具.     

呼吸道合胞病毒 L 蛋白单克隆抗体的制备

目的：制备人呼吸道合胞病毒L蛋白单克隆抗体，并对其进行性质鉴定。方法：选取RSV-A2 L蛋白上具有亲水性可能的loop区段，将这些目的片段展示在HBc149上进行原核表达并纯化。重组蛋白免疫6~8周BALB/c雌性小鼠，根据免疫后血清的ELISA和WB分析结果选择L3935和L4099组小鼠进行脾脏免疫。融合后，用间接ELISA法筛选和多次克隆化，获得稳定分泌抗L蛋白单克隆抗体细胞株，并结合ELISA、免疫荧光和免疫印迹对所获得的单抗进行鉴定。结果：获得11株L蛋白特异性单克隆抗体，亚类鉴定结果IgG1、IgG2a和IgG3单抗各3株，IgG2b单抗2株。结论：成功制备了L蛋白特异性单克隆抗体，也为RSV感染的免疫和发病机制研究奠定基础。     

抗ARL-1单克隆抗体的制备及初步应用

目的: 制备抗醛糖还原酶相似蛋白(aldosereductaselikeprotein, ARL- 1)的单克隆抗体 (mAb)并进行初步应用。方法: 用纯化的ARL- GST蛋白免疫BALB/c小鼠, 采用杂交瘤技术制备mAb。间接ELISA法、Westernblot及免疫组织化学染色法, 对mAb进行鉴定及初步应用。结果: 获得 1株可稳定分泌抗ARL -1蛋白mAb的杂交瘤细胞系。mAb的Ig亚类为IgG2b, 轻链属于κ型。腹水mAb的效价为: 1∶4. 096×105。Westernblot的结果表明, ARL 1蛋白在被检测的肝癌组织中呈高表达, 而在相应的癌旁组织中不表达。免疫组织化学染色的结果表明, ARL -1蛋白在肝癌组织中呈高表达且主要定位在胞质内。结论: 成功地制备 1株抗ARL -1蛋白的mAb。初步应用的结果表明, ARL- 1蛋白在肝癌组织中呈高表达, 为进一步研究ARL -1蛋白的功能, 以及其与肝癌的确切关系奠定了基础。     

小鼠脑缺血再灌注对铁及其调节蛋白在神经胶质细胞内分布的影 …

应用免疫组织化学，荧光双标记和组织化学方法，对双侧颈总动脉反复暂缺血造成再灌流损伤的小鼠部分脑区，颞叶大脑皮质，海马，胼胝体，丘脑和纹状体，中的铁，铁蛋白和转铁蛋白的分布进行了观察。     

鸡肺表面活性蛋白A的表达及其多克隆抗体的制备

目的表达鸡肺表面活性蛋白A(cSP-A)重组蛋白,并制备鼠抗cSP-A多克隆抗体。方法人工合成cSP-A的凝集素糖识别区(CRD)基因(cSP-A-CRD),亚克隆到pGEX-6P-1载体中进行原核表达,对表达产物进行鉴定。切胶纯化法纯化重组蛋白,并免疫小鼠制备抗cSP-A多克隆抗体,ELISA检测抗体效价,Western blot法、免疫荧光组织化学染色和ELISA鉴定该抗体的特异性和适用范围。结果获得鼠抗cSP-A血清,效价达到105;Western blot法结果表明多抗血清具有良好的特异性和反应性,并可用免疫荧光组织化学技术检测鸡肺上皮细胞表达的cSP-A和用间接ELISA检测鸡肺灌洗液中存在的水溶性cSP-A蛋白。结论成功表达了cSP-A重组蛋白并制备了小鼠抗cSP-A多克隆抗体。     

肺腺癌和癌旁组织蛋白质表达谱差异的研究

通过比较肺癌患者癌组织和癌旁组织的二维凝胶电泳图谱(two-dimensional electrophoresis,2DE),以寻找肺癌相关蛋白、肺癌患者癌组织和癌旁组织,采用固相pH梯度(immobilized pH gradient,IPG)2DE分离总蛋白质,凝胶经银染显后,ImageMaster 2D图像分析软件进行比较分析、识别差异表达的蛋白质并进行质谱鉴定获取肺癌相关蛋白信息。结果分析得到肺癌组织蛋白点709个,癌旁组织蛋白点722个,选择表达量差异达3倍以上的40个,进行胶内酶解,基质辅助电离解析飞行时间质谱(MALDI TOF MS)获得肽质量指纹图谱(PMF),搜寻蛋白质数据库,得到7个肺癌标志物的候选蛋白,但这些差异蛋白点在肺癌发生发展中的作用需要进一步研究。     

小鼠小脑前叶后叶和旁绒球叶的蛋白组学比较分析

目的 研究小脑不同区域所含蛋白质表达的差异.方法 提取小脑前叶、后叶和旁绒球叶的蛋白质,通过二维凝胶电泳分离蛋白,用PDQuest图像分析软件寻找电泳图谱中的差异蛋白,MALDI TOF/TOF串联质谱分析蛋白质,所得的质谱图通过GPS软件在Mascot数据库中检索并鉴定蛋白质.结果 在小脑3个不同区域鉴定了5种差异蛋白,丙酮酸激酶同工酶M2在小脑前叶和后叶的表达量比旁绒球叶的高;核不均一核糖核蛋白L和真核翻译延伸因子2在小脑后叶的表达量少,在小脑前叶和旁绒球叶未检测到;表达序列蛋白AI429145在小脑后叶的表达量高,但在小脑前叶和旁绒球叶未检测到;三磷酸腺苷酶氢离子转运体V0亚单位D异构体1在旁绒球叶的表达量比小脑前叶和后叶的高.结论 这些蛋白质在小脑不同区域表达量上的差异可能有助于理解小脑不同区域的特殊功能和疾病.     

胎脑与成年脑皮质组织的蛋白质组学分析

目的 应用蛋白质组技术研究成年和胚胎脑皮质蛋白质组表达差异,初步探索在胎脑与成年人中脑蛋白的变化情况.方法 收集成人无病变区正常脑皮质1份和水囊引产胎脑组织脑皮质2份(3月胎脑组织和5月胎脑组织),提取总蛋白,通过双相电泳分离蛋白,每组样品均重复电泳,使用PDQuest7.0图像分析软件分析电泳图谱,得到具有表达差异的蛋白质,分别选取在大脑发育过程中逐步降低和逐步升高的差异点及在胎脑成脑蛋白表达中差异两倍以上的蛋白点,应用胶内酶切和基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization-time of flight-time of flight,MALDI-TOF-TOF)进行鉴定,得到肽指纹图谱,并查阅蛋白质数据库得到蛋白质的相关信息.结果 (1)成人脑组织、3月胎脑组织和5月胎脑组织的脑组织蛋白图谱分别测得642、511和527个点,图象匹配率达87%.成人脑蛋白图谱中碱性蛋白点明显多于胎脑的;(2)成人脑组织、3月胎脑组织和5月胎脑组织的脑组织单独特有蛋白分别为:172、171和152个;(3)与成人脑组织比较,3月胎脑组织和5月胎脑组织的差异蛋白质分别为131和115个;差异在2倍以上的蛋白点为60和40个,其中低表达的蛋白点分别有24和17个,高表达蛋白点为36和23个;(4)白蛋白、磷酸丙糖异构酶等8种蛋白表达均有不同的变化.脂肪酸结合蛋白7和未命名蛋白只在3月胎脑高表达,而5月胎脑和成人组均无表达;核酮糖1,5-二磷酸羧化酶/加氧酶和转导素只在成人脑中高表达,胎脑无表达.血清白蛋白却随着大脑发育逐渐降低.ATP合成酶、线粒体F0复合体亚基、磷酸丙糖异构酶随着大脑发育而逐渐升高.结论 人类从胚胎期到成人发育过程中脑部蛋白质可能会发生明显改变,这些差异蛋白的发现为研究脑发育机理提供了极其有益的线索.

Abstract：

Objective To study the differences of protein expression levels in the brain cortex of human fetus and adult with proteomics technique, and provide preliminary data on the change of proteins during brain development. Methods Proteins extracted from human temporal lobes in fetal (3 month and 5 month respectively) and adult (30 years old) brain were separated by two-dimensional gel electrophoresis (2DE).The proteins were then stained with colloidal Coomassie blue to produce a high-resolution map of the proteiome.The differential protein spots were analyzed by PDQuest 7.0 software and 8 spots,which were gradually reduced or gradually increased in brain development process and the protein spots of difference over two-fold in the brain,were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/TOF).Results (1) On average, 642, 511 and 527 protein spots could be obtained in the temporal lobes of adult, 3 month and 5 month fetus. The matching rate of images was 87%. The basic proteins in adult brain were obviously much more than that in the fetus; (2) There were 172, 171 and 152 singular protein spots in temporal lobes of adult, 3 month and 5 month fetus respectively. (3) Compared with adult, there were 131 and 115 different protein spots in the 3 month and 5 month fetus respectively. There were 60 and 40 protein spots with more than 2 fold difference, among which 24 and 17 were down-regulated, and 36 and 23 were up-regulated respectively. (4) There was different expression in proteins such as serum albumin, triosephosphate isomerase, etc. in the 3 groups. Fatty acid binding protein 7 and unnamed proteins were only highly expressed in the 3 month brain;ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit and transducin beta-1 subunit were up-regulated in adult brain. Serum albumin decreases gradually with brain development. However, ATP synthase, mitochondrial F0 complex, and triosephosphate isomerase increase gradually with brain development. Conclusion The proteins of human brain cortex were obviously changed from embryonic stage to adult. The differentially displayed proteins may provide further insight into the understanding of development of human brain.     

蛋白质组学检测β-原肌球蛋白在结肠癌组织中的表达

目的：通过比较结肠腺癌与正常结肠黏膜的蛋白质组表达差异,寻找与结肠腺癌发生相关的蛋白质,筛选结肠癌诊断的分子标志物。方法: 运用蛋白质组学技术,对8例结肠腺癌组织和8例正常结肠黏膜组织进行胶内差异双向电泳（2-D）,选择差异表达超过2倍的蛋白质进行MALDI-TOF/TOF质谱分析和生物信息学分析,并对结肠癌组织中表达下调的蛋白β-原肌球蛋白(TM β)进行Western blotting验证。结果: 成功建立结肠腺癌和正常结肠黏膜的双向凝胶电泳图谱,结肠腺癌和正常黏膜组织凝胶电泳图谱中平均蛋白质斑点数分别为3 289和3 066,其中表达差异超过2倍的斑点共有31个,质谱分析和数据库检索共鉴定出18种蛋白质,包括cytokeratin 8、cytokeratin 10、S100A6、TM β、protein disulfide isomerase 等。从功能分析,这些差异蛋白与癌细胞的发生、增殖、分化、转移等相关;TM β在结肠癌组织中的表达水平Western blotting结果与电泳结果一致。结论: 蛋白质组学能有效地分离和鉴定结肠腺癌组织与正常结肠组织间的差异表达蛋白质,结肠癌组织中表达下降的TMβ,可能成为结肠癌诊断的分子标记物。     

Proteomic analysis of synovial fluid as an analytical tool to detect candidate biomarkers for knee osteoarthritis

We conducted research to detect the proteomic profiles in synovial fluid (SF) from knee osteoarthritis (OA) patients to better understand the pathogenesis and aetiology of OA. Our long-term goal is to identify reliable candidate biomarkers for OA in SF. The SF proteins obtained from 10 knee OA patients and 10 non-OA patients (9 of whom were patients with a meniscus injury in the knee; 1 had a discoid meniscus in the knee, and all exhibited intact articular cartilage) were separated by two-dimensional electrophoresis (2-DE). The repeatability of the obtained protein spots regarding their intensity was tested via triplicate 2-DE of selected samples. The observed protein expression patterns were subjected to statistical analysis, and differentially expressed protein spots were identified via matrix-assisted laser desorption/ionisation-time of flight/time of flight mass spectrometry (MALDI-TOF/TOF MS). Our analyses showed low intrasample variability and clear intersample variation. Among the protein spots observed on the gels, there were 29 significant differences, of which 22 corresponded to upregulation and 7 to downregulation in the OA group. One of the upregulated protein spots was confirmed to be haptoglobin by mass spectrometry, and the levels of haptoglobin in SF are positively correlated with the severity of OA (r = 0.89, P < 0.001). This study showed that 2-DE could be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA. Spots of interest identified by mass spectrometry, such as haptoglobin, may be associated with OA severity.     

蛋白质组学检测结直肠腺瘤及早期恶变患者血清的差异蛋白质变化及意义

目的： 通过比较分析结直肠腺瘤及早期恶变患者血清蛋白质的表达差异,寻找结直肠癌早期诊断的血清标记物。方法：建立结直肠腺瘤及腺瘤早期恶变患者血清蛋白双向电泳图谱,比较分析差异蛋白质斑点,切取酶解行MALDI-TOF／TOF质谱分析鉴定。结果：成功建立结直肠腺瘤及早期恶变患者血清蛋白双向电泳图谱,其平均蛋白质点分别为1672±73和1732±46,早期恶变组表达差异大于1.5倍的蛋白质斑点有28个,其中15个下调,13个上升;质谱分析鉴定出23个蛋白质,鉴定率达82.14%;合并重复鉴定的蛋白质,共获得的差异蛋白13种,其中8种为上调蛋白,5种为下调蛋白,分为6类,包括蛋白酶抑制剂、补体系统、免疫球蛋白、角质素、信号转导蛋白及未知蛋白。结论：蛋白质组学技术能灵敏分析结直肠腺瘤及早期恶变患者血清蛋白质的异常改变,本研究鉴定的蛋白质可能成为结直肠癌早期诊断的血清肿瘤标记物。     

Proteome analysis of Helicobacter pylori: major proteins of type strain NCTC 11637

Proteome analysis involves the simultaneous resolution and display of proteins produced by an organism, followed by the quantitation, characterisation and identification of these proteins. As part of an ongoing study mapping and comparing the proteins expressed by various strains of the pathogenic bacterium Helicobacter pylori, we have resolved and identified 93 of the most abundant proteins expressed by type reference strain NCTC 11637. Proteins were separated by two-dimensional gel electrophoresis and stained with Coomassie G250. Intensely-stained spots were excised and digested with trypsin, and the resulting peptides were characterised by mass spectrometry. Proteins were then identified by correlating actual peptide profiles with theoretical profiles generated from published nucleotide sequences. Ninety-three of the most intensely-stained protein spots were identified as the products of 35 genes, giving a ratio of 2.7 protein gene-products per gene. The products of the tsaA, pfr, ureA and ureB genes were amongst several proteins present in multiple isoforms. Peptide mass fingerprinting data were used to identify probable post-translational protein modifications. These results suggest that H. pylori proteins are subject to a high degree of post-translational modification. Comparative proteomics of H. pylori strains should greatly assist in investigating the pathogenic properties of this bacterium.     

肺鳞癌患者和健康人血清的差异蛋白质组学研究

目的：比较肺鳞癌患者和健康人血清蛋白质组的差异,为筛选肺鳞癌血清分子标志物提供依据。方法：以5例健康人和5例I期肺鳞癌患者的血清为样本,采用15%的聚丙稀酰胺凝胶电泳(SDS-PAGE)分离血清蛋白质,Bandleader凝胶图像分析软件识别两种血清样本差异的蛋白质条带,电喷雾-四极杆-串联质谱（ESI-Q-TOF MS/MS）鉴定差异蛋白质条带中的蛋白质。Western印迹检测差异蛋白质结合珠蛋白-2（haptoglobin-2,HP-2)在肺鳞癌患者和健康人血清中的表达。结果：建立了健康人和肺鳞癌患者血清样品的一维凝胶电泳图谱,图像分析识别了4条差异蛋白质条带,质谱鉴定出29种非冗余的血清蛋白质。Western印迹证实了HP-2在肺鳞癌患者和健康人血清中的差异表达。结论：29种血清差异蛋白质为筛选肺鳞癌的血清分子标志物提供了实验依据。     

Biomarkers of metastatic potential in cultured adenocarcinoma clones

Two-dimensional isoelectric focusing and gel electrophoresis followed by mass spectrometry were used to detect, measure, and identify changes in protein expression correlated with differences in the metastatic potential of cultured rat mammary adenocarcinoma cells. MTC is a non-metastatic cell clone derived from a primary tumor. MTLn2 and MTLn3 are low and high metastatic potential cell clones derived from lung metastases of the primary tumor. A total of 1,500 proteins was detected. The patterns of protein expression of MTLn2 and MTLn3 cells were similar. Only five spots had a threefold or greater statistically significant difference in staining intensity between MTLn2 and MTLn3 cells, whereas 70 spots differed between MTC and MTLn3 cells. Twenty spots were selected for further study, ten that had a positive correlation of staining intensity with metastatic potential and ten that had a negative (inverse) correlation. Of the 17 unique proteins that were identified, five have often been cited as tumor biomarkers. These included the positive biomarkers nucleophosmin (NPM) and 14-3-3 protein sigma and the negative biomarkers raf kinase inhibitor protein (RKIP), peroxiredoxin-2, and galectin-1. The only identified protein that was markedly higher in MTLn3 cells than in the less-metastatic MTLn2 cells was 14-3-3 protein sigma. The results indicate that increased metastatic potential is associated with positive and negative changes in expression of particular proteins. Proteins that are positively correlated with metastatic potential may prove more useful as clinical biomarkers, but those with negative correlations may still provide useful information about underlying mechanisms of metastatic spread.     

Analysis of differentially expressed proteins involved in hand,foot and mouth disease and normal sera

We implemented 2-D DIGE technology on proteins prepared from serum obtained from children with hand, foot and mouth disease (HFMD) and controls, to study the differentially expressed proteins in control and HFMD serum samples. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/ time-of-flight mass spectrometry (MALDI-TOF/TOF MS) analysis. We identified 30 proteins from mild HFMD samples and 39 proteins from severe HFMD samples, compared with the normal controls. 25 proteins among them (14 up-regulated and 11 down-regulated proteins) are found in both HFMD groups. Classification analysis and protein-protein interaction map showed that they associate with multiple functional groups, including transporter activity and atalytic activity. These findings build up a comprehensive profile of the HFMD proteome and provide a useful basis for further analysis of the pathogenic mechanism and the regulatory network of HFMD.