梅花鹿致敏与休眠鹿茸干细胞差异蛋白表达的2D-DIGE分析

旨在对梅花鹿(Cervus nippon)致敏鹿茸干细胞与休眠鹿茸干细胞表达蛋白进行差异筛选、鉴定及生物信息分析,为深入探讨鹿茸独特的再生分子调节机制奠定基础。本研究采用双向荧光差异凝胶电泳(Two-dimensional fluorescence difference in gel electrophoresis,2D-DIGE)分离蛋白样品;利用DeCyder 7.2分析软件对2D-DIGE图像进行统计学分析寻找差异表达蛋白;利用MALDI-TOF-MS(Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry)鉴定差异蛋白,通过Mascot软件搜索NCBInr数据库寻找匹配的蛋白;采用PANTHER(Protein Analysis Through Evolutionary Relationships)软件对差异蛋白进行聚类分析,REACTOME数据库分析差异蛋白所参与的信号通路。结果得到了致敏鹿茸干细胞与休眠鹿茸干细胞2D-DIGE图谱,致敏鹿茸干细胞与休眠鹿茸干细胞蛋白丰度相比较,比值≥1.1倍以及比值≤-1.1倍(P0.05)的差异蛋白点有159个,其中110个上调表达,49个下调表达,EDA(Extended data analysis)分析得到了多个Marker蛋白,质谱鉴定了84个差异蛋白质点,48个为阳性结果,共来自27种蛋白质。并对已鉴定蛋白进行了GO分析以及信号通路富集分析。致敏鹿茸干细胞与休眠鹿茸干细胞蛋白差异明显,质谱鉴定获得了来自多种可能与鹿茸再生相关的差异蛋白。由此可知,鹿茸再生是鹿茸干细胞从休眠到致敏的转化过程,需要多种蛋白分子以及信号通路的综合调控。     

野牛草破眠机理的蛋白质组学分析

以野牛草种子为材料,以不同收获年份(2006年和2008年)去离子水浸泡和相同收获年份不同试剂(离子水浸泡和添加外源激素)浸泡为处理,应用蛋白质双向电泳技术和质谱技术鉴定了野牛草种子打破休眠过程中蛋白质表达的差异,从而探索破除野牛草种子休眠的分子机理.对2-DE图谱的分析表明,处理和对照中共检测到34个差异表达丰度2倍以上的蛋白质点；对其中的20个蛋白质点进行MALDI-TOF-MS分析,共有16个蛋白质点得到有效鉴定；功能分析表明,这些蛋白质主要参与了能量代谢、电子传递、光合作用、转录调控、信号传导、蛋白合成和生长发育等过程.     

水牛卵母细胞成熟前后的差异蛋白质组的分析

本试验以水牛卵母细胞为研究对象,通过基于双向电泳-质谱技术的蛋白质组学研究手段,鉴定卵母细胞成熟前后表达量存在变化的蛋白质并进行验证。通过优化方法建立水牛卵母细胞蛋白质双向电泳分离的技术体系,通过双向电泳(2-DE)获得成熟前后的卵母细胞蛋白质电泳图谱,软件分析得到差异表达蛋白质,对差异蛋白质进行飞行时间质谱(MALDI-TOF/TOF)分析,部分差异蛋白质合成抗体进行Western blot验证。结果表明,2组卵母细胞样品均获得约300个蛋白质斑点的双向电泳图谱。经ImageMaster软件比对分析,共发现在水牛卵母细胞成熟前后有27个差异蛋白质,其中表达上调15个,表达下调12个。将差异蛋白质斑点胶内酶解后用于MALDITOF/TOF飞行时间质谱鉴定,成功鉴定了6个蛋白质,包括主要穹窿蛋白(MVP)、热激蛋白60(HSP60)、Ras应答结合原件蛋白1(RREB1)等。Western blot结果表明,HSP60蛋白表达与双向电泳结果一致。本试验发现一批在卵母细胞成熟前后的差异表达蛋白质并进行表达量验证,推测HSP60蛋白可能在体外成熟时起到保护卵母细胞和物质转运的作用。     

盐碱胁迫下紫花苜蓿叶片蛋白组的初步分析

以紫花苜蓿（Medicago sativa）品种"WL343HQ"为材料,对其幼苗进行150 mmol·L^-1的Na₂CO₃和NaHCO₃混合盐碱胁迫,采用iTRAQ技术结合反相液相色谱与液相串联色谱,分析盐碱胁迫叶片中蛋白表达的变化,并对获得的差异蛋白进行生物信息学分析。结果表明,在盐碱胁迫处理下共鉴定到318个显著差异蛋白,包括172个上调蛋白和146个下调蛋白,这些差异蛋白的功能涉及多种代谢途径,其中与光合作用相关的蛋白质表达量总体下调,与苯丙素生物合成、苯丙氨酸代谢和类黄酮生物合成相关的蛋白质表达量总体上调。通过蛋白组学分析技术可有效筛选紫花苜蓿叶片中差异表达蛋白,可为深入研究紫花苜蓿应对盐碱胁迫的分子机制提供一定的理论基础。     

牦牛卵泡液差异蛋白质组双向电泳图谱的构建与质谱分析

从蛋白质水平了解牦牛季节性繁殖规律,利用双向电泳与质谱鉴定技术分析牦牛卵泡液与血浆蛋白质组分变化。以青海高原牦牛卵泡液与血浆为研究对象,采用双向电泳技术构建牦牛卵泡液与血浆蛋白质双向电泳图谱,银染后利用Image Master 2D Platinum软件分析并采用MALDI-TOF-MS进行质谱鉴定。用试剂盒ProteoExtract Albumin/IgG Removal Kit去除高丰度蛋白质后,利用2-DE技术获得了分辨率较高的卵泡液与血浆蛋白质电泳图谱,卵泡液与血浆蛋白质图谱对比分析共发现了24个差异表达蛋白质点,其中2个蛋白质点表达上调,22个蛋白质点表达下调。经质谱分析,最终成功鉴定出8个蛋白质点、5个未知蛋白质点。本研究成功构建了蛋白质图谱及分离鉴定的差异蛋白质,为从蛋白质水平揭示牦牛卵泡发育规律及了解卵母细胞发育的微环境提供了试验依据。     

雏鸡冷应激前后血清中差异蛋白质组学研究

本试验旨在比较分析雏鸡冷应激前后血清中蛋白质的表达差异,并对重点差异蛋白质进行鉴定。将30只雏鸡随机分为3组,分别为冷应激组、冷适应组和常温对照组,收集各组雏鸡的血液制备血清后进行双向凝胶电泳(2-DE),以获得血清蛋白质表达的2-DE图谱,对2-DE图谱进行差异分析(利用PDQuest 8.0软件),随后采用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)技术对差异表达蛋白质进行鉴定,并利用蛋白质印迹(Western blot)方法进行验证。结果显示:通过2-DE对常温对照组、冷应激组和冷适应组雏鸡血清进行分析,得到了比较完整的差异蛋白质数据,共找到差异蛋白质点23个。采用MALDI-TOF-MS技术分析其中几个重复性好且较为明显的蛋白质点,成功鉴定出4个差异蛋白质,其中2个为果糖二磷酸醛缩酶C(ALDOC),是参与葡萄糖、能量代谢通路供能的相关蛋白;随后,对差异蛋白质ALDOC进行Western blot验证,所得结果与2-DE的结果相一致。结果表明,雏鸡冷应激前后血清中蛋白质的表达具有明显的差异,这些蛋白质的表达差异可能与冷应激有关。     

家蚕近等基因系感染浓核病毒(BmDNV-Z)前后中肠组织蛋白的差异表达分析

家蚕浓核病毒(BmDNV)是危害家蚕的主要病毒之一。为鉴定与家蚕抗病毒相关的蛋白质,以对BmDNV完全不感染的家蚕品种兰10(L10)为供体亲本,将其抗性基因导入家蚕敏感品种菁松(JS)中,构建抗性近等基因系NIL(BC6F2)。对JS及其近等基因系NIL分别用家蚕浓核病毒镇江株(BmDNV-Z)添毒,二者分别表现为高度敏感和完全不感染。对JS和NIL在病毒感染前后中肠组织蛋白双向电泳(2D-PAGE)图谱中的差异蛋白点进行串联质谱(MALDI-TOF-TOF)分析,共鉴定了41个差异表达的蛋白点,其中有35个蛋白点可能与病毒的诱导相关,有6个蛋白点可能与家蚕的组成抗性相关。鉴定的差异表达蛋白包括糖酵解酶类、能量代谢酶类、参与基因表达的蛋白质以及参与其它细胞功能的蛋白质等。选取10个差异表达蛋白点进行基因表达定量PCR分析,进一步确证了这些蛋白质在JS及其近等基因系NIL之间以及在病毒诱导前后的差异表达。例如:精氨酸激酶在NIL和JS中均可被诱导表达;V-ATP合成酶及热激蛋白HSP70只在NIL中被特异性诱导表达;烯醇化酶在NIL中的表达水平显著高于JS,且不能被病毒感染所诱导,是一个典型的组成抗性相关蛋白。鉴定出的41个差异表达蛋白点有可能参与了家蚕对BmDNV-Z的抗性。     

家蚕性别相关血液蛋白质组的一维电泳-液相色谱-质谱分析

分别从家蚕5龄幼虫和蛹期不同发育阶段提取雌、雄蚕血液总蛋白质,采用一维电泳-液相色谱-质谱(1DE-LC-MS)技术分析家蚕血液蛋白质的差异性表达及差异蛋白组分。在家蚕血液蛋白质含量与种类方面检测到与性别及发育相关的差异性表达,数据库检索结果共获得96个相匹配的候选蛋白质,剔除冗余部分后鉴定其中的27个蛋白质组分,分别属于13种蛋白质或其亚基。雌、雄蚕之间的差异组分主要出现在分子质量70~100 kD之间,其中芳基贮存蛋白、卵黄蛋白原、抗胰凝乳蛋白酶因子为雌特异性表达组分。30K蛋白家族是家蚕血液中高丰度表达的蛋白组分。这些差异蛋白质的鉴定与功能分析为研究家蚕性别发育调控机制提供了新的信息。     

分娩前后奶牛血浆差异表达蛋白初步分析

本研究运用双向电泳方法分析奶牛产前及产后1周血浆蛋白的差异表达,为理解围产期免疫抑制以及分娩前后奶牛的生理状态提供参考。血浆样品经过热SDS法处理后,使用pH4～pH7的胶条,在硝酸银染色的7.5%～17.5%的梯度聚丙烯酰胺凝胶上,利用PDQuest6.0图像分析软件检测到239个蛋白质点。比较奶牛产前及产后1周血浆蛋白差异表达情况发现,表达量变化3倍以上的点有12个。MADIL-TOF/TOF质谱鉴定出2种蛋白:白蛋白和结合珠蛋白。结合珠蛋白在产后1周的奶牛血浆中的含量显著增加,反映了产后奶牛的生理变化。但是需要结合其他标记共同作为疾病诊断的标记。     

感染马传染性贫血病毒的巨噬细胞差异表达蛋白质组分析

为研究马传染性贫血病毒(EIAV)感染其主要靶细胞马巨噬细胞(eMDM)后与细胞蛋白的相互作用,本研究采用EIAV强毒株EIAVDLV34感染48 h后的马外周血单核细胞分化的eMDM,并以未感染eMDM为对照,提取细胞的蛋白质样品,进行双向凝胶电泳(2-DE)分离,并分析凝胶中差异蛋白点。结果共检测出19个表达差异的蛋白点(ratio1.4,p0.05),其中感染组相对于对照组有7个蛋白质上调表达,12个蛋白质呈现下调表达。将差异蛋白进行串联质谱分析进行鉴定,并通过生物信息学方法对这19个差异表达蛋白进行了蛋白互作用分析。此外,对其中5个比较重要蛋白的mRNA水平进行了荧光定量PCR分析,其表达变化与2-DE结果一致。本研究为进一步分析EIAV与其宿主细胞eMDM的相互作用提供了参考。     

Differences Between High‐ and Low‐Motility Buffalo Sperm Identified by Comparative Proteomics

This study was undertaken to investigate differences in protein expression between high‐ and low‐motility sperm of swamp buffalo. The research used two‐dimensional gel electrophoresis (2DE) coupled to matrix‐assisted laser desorption/ionization time‐of‐flight tandem mass spectrometry (MALDI‐TOF/TOF‐MS) to analyse the different proteins. The results showed 18 different expression protein spots between high‐ and low‐motility buffalo sperm; eight of these proteins were up‐regulated in low‐motility sperm, five were down‐regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl‐CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low‐motility sperm and provide clues for finding molecular markers associated with sperm motility.     

Alteration in the Expression of Proteins in Unexplained Recurrent Pregnancy Loss Compared with in the Normal Placenta

The placenta is a unique pregnancy-related tissue and plays a key role in occurrence of unexplained recurrent pregnancy loss (URPL). Abnormal placentation might play a key role in occurrence of URPL. Therefore, the purpose of this study was to compare the human placental proteome between URPL placentas and normal placental matched for gestational week. Total placental proteins were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) technique was used for separation of the placental proteomes. Protein spots differentially expressed between URPL and normal placentas were selected and identified by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF/TOF) technique after being digested in the gel. Moreover, quantitative real-time PCR and Western blot techniques were used to confirm the differential expression mass results for some differentially expressed proteins. The results indicated that at least 19 protein spots were differentially expressed between URPL and normal placentas (P < 0.05), and twelve of them were successfully identified. While only two proteins were downregulated (calumenin and enolase 1), the remaining ten spots (actin gamma 1 propeptide, cathepsin D prepropeptide, heat shock protein gp96, tubulin beta, tubulin alpha 1, glutathione S-transferase, vitamin D binding protein, prohibitin, actin beta, apolipoprotein A-I) showed increased expression in URPL cases in comparison with normal placentas. Real-time PCR also confirmed the downregulation of calumenin and upregulation of prohibitin and apolipoprotein A-I at the mRNA levels. In conclusion, the results of the present study showed that alteration in the expression of proteins involved in proliferation and migration of endothelial cells as well as control of coagulation by these cells might play an important role in the pathogenesis of URPL.     

Differential protein profiles in duck meat during the early postmortem storage period

The present study was designed to investigate proteomic differences in duck breast muscle during the early postmortem storage period. The meat quality was evaluated at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, black Muscovy ducks and Mule ducks. Differentially expressed proteins were detected by two‐dimensional gel electrophoresis (2‐DE) and matrix‐assisted laser desorption ionization‐time‐of‐flight mass spectrometry (MALDI‐TOF/TOF MS) at 0 hr and 24 hr postmortem in the three duck breeds. The results showed that 53 proteins spots were differentially expressed at 0 hr and 24 hr postmortem at 4°C in Pekin ducks, 75 spots in black Muscovy ducks, and 72 spots in Mule ducks. A total of 30 (10 spots for each breed) were selected for identification by mass spectrometry. Seven proteins were identified in Pekin ducks, eight in black Muscovy ducks and seven in Mule ducks. Moreover, the above results obtained by 2‐DE and MALDI‐TOF/TOF MS were confirmed by western blotting. To our knowledge, this study is the first to provide insights into the protein profiles of ducks during postmortem storage and provides a better understanding of the biochemical processes that contribute to duck meat quality.     

Proteomic analysis of mammary tissues from healthy cows and clinical mastitic cows for identification of disease-related proteins

To investigate the disease-related proteins and understand molecular mechanism of mastitis at the protein level, this project presents the protein changes in the mammary gland between healthy cows and clinical mastitic cows using two-dimensional gel electrophoresis (2-DE), after stained with colloidal Coomassie Bright Blue, six spots of differentially expressed protein were detected by PDQuest software and subjected to ion trap mass spectrometer equipped with a HPLC system, and five proteins were identified. Hemoglobin beta, kappa-casein and tryptophanyl-tRNA-synthetase (TrpRS) in healthy dairy cows, while hemoglobin beta, cytochrome C oxidase and annexin V in clinical mastitic cows were identified, they were involved in binding, transport and catalytic activity. The results may provide valuable information for the investigating of the host mammary immune system response to defense against pathogens at the protein level and potential protein targets for treatment.     

Proteomic analysis of plasma from cows affected with milk fever using two-dimensional differential in-gel electrophoresis and mass spectrometry

Milk fever is an important metabolic disorder of dairy cows after calving, and is characterized by hypocalcemia, tetany, lateral recumbency, and eventual coma. To date, there have been many reports about the pathogenesis and pathophysiology of milk fever, but the plasma protein profile in milk fever has not been reported. The aim of our study was to investigate novel pathophysiological changes in the plasma proteome of cows affected with milk fever. Plasma samples were collected from eight Holstein cows with milk fever (T), and eight control Holstein cows without milk fever (C), at an intensive Holstein dairy farm in Heilongjiang province, China. Samples were analyzed by fluorescence two-dimensional (2D) differential in-gel electrophoresis (DIGE), followed by in-gel digestion, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) for peptide mass fingerprinting of selected protein spots. Eight of the 23 differential protein spots in the plasma of T and C cows were isolated and identified by 2D-DIGE and MALDI-TOF-MS. The protein spots represented five unique proteins, and had significant alterations in spot volume as determined by DeCyder differential in-gel analysis (DIA) software. The upregulated proteins were identified as serpin peptidase inhibitor (angiotensin), which regulates blood pressure and maintains fluid and electrolyte homeostasis, and endopin 2B which is involved in neural regulation. The downregulated proteins were serum albumin, which acts as a transport protein, fibrinogen beta chain which is involved in blood coagulation, and IgG heavy-chain C-region (IgG-C(H)) which participates in the immune response. In conclusion, we were able to use proteomic technologies to identify several novel plasma proteins in cows affected with milk fever. These findings may reveal new pathophysiological changes that occur in cows with milk fever.     

沟叶结缕草应答高温和低温胁迫的蛋白质表达差异分析

为阐明沟叶结缕草(Zoysia matrella (L.) Merr.)相对耐高温而不耐低温的分子机理,于2008年5月应用差异蛋白质组学方法分析了沟叶结缕草在低温(5℃)和高温(40℃)胁迫下的叶片蛋白质组差异表达,共检测到35个差异蛋白质点,其中26个得到鉴定。分析表明:沟叶结缕草在低温胁迫下,一方面加强了胞内信号转导、活性氧的清除、蛋白质防御以提高其低温适应性;另一方面,由于一些蛋白质的下调表达,使得其低温下植物体呼吸作用表现为先增强后减弱、光合作用下降、蛋白降解、细胞的转运能力减弱,因而抗低温性能下降。在高温胁迫下,则通过一些蛋白质的上调表达,加强了体内信号识别和转导、光合作用、呼吸作用、次生代谢作用、活性氧的清除、蛋白质防御及合成等功能,沟叶结缕草表现出相对较强的高温抗逆性。     

二化性家蚕卵低温和常温催青的胚胎期蛋白质表达差异分析

家蚕二化性品种的滞育性受上代胚胎期环境条件调控,查找家蚕胚胎期滞育关联蛋白,可为最终阐明家蚕滞育的分子机制提供实验依据。以家蚕二化性品种秋丰的蚕卵为材料,分别在25℃常温和18℃低温条件下催青,提取胚胎不同发育时期蚕卵的易溶性和难溶性蛋白,采用蛋白质双向电泳(2-DE)和图像分析技术,研究蚕卵在胚胎不同发育时期的蛋白质差异表达谱。在易溶性和难溶性蛋白图谱中分别检测到5个和7个差异显著的蛋白点。对这些差异蛋白点进行质谱鉴定,有8个蛋白点得到可信的最佳匹配蛋白报告,共鉴定出卵特异蛋白、卵黄原蛋白、表皮蛋白和丙酮酸脱氢酶激酶4种蛋白。这些差异表达蛋白可能导致蚕卵胚胎中物质代谢和能量代谢的不同,据此推测低温和常温催青可能在蚕卵胚胎中诱导了不同的生理生化反应。     

太子参连作障碍蛋白差异表达分析

为探明太子参连作障碍分子机理,本研究采用差异蛋白质组学方法研究了不同种植制度下(连作、轮作)太子参叶片蛋白质的差异表达。结果表明,不同种植制度下太子参叶片中共有27个差异表达蛋白质点,经生物信息学查询到24个蛋白质。差异蛋白质功能分析结果表明,连作导致太子参叶片中与植物衰老或病害相关的5个蛋白表达量全部上调;与植物抗性相关的5个蛋白(磷脂氢谷胱苷肽过氧化物酶、植物性丝氨酸-苏氨酸蛋白激酶、钙依赖蛋白激酶、查尔酮合成酶和硫氧还相关蛋白)表达量上调,4个蛋白(细胞壁富含甘氨酸蛋白、SKP1同源蛋白、锌指蛋白和成熟酶K)表达量下调;与光合作用相关的2个蛋白(Rubisco大亚基和细胞色素b₆)表达量下调,2个蛋白(核酮糖二磷酸碳酸酵素小链c和细胞色素b₆/f复合体亚基VI)表达量上调;与能量代谢相关的蔗糖-UDP葡萄糖基转移酶和与细胞分裂相关的NF-Y5表达量均下调,而蛋白酶1型β亚基、ATP合成酶β亚基、细胞分裂素组氨酸磷光体转移蛋白4和NAD依赖-3-磷酸甘油醛脱氢酶表达量均上调。这说明太子参连作导致叶片致病性或衰老相关蛋白表达量上调,是太子参连作病害频发和提前退黄的主要原因,同时连作导致抗性相关蛋白以及能量代谢和细胞分裂相关蛋白紊乱(或降低或上调),是太子参连作障碍的结果在蛋白质水平的表现,连作还导致重要的光合相关蛋白表达量下调,是连作太子参光合作用能力下降的分子基础。