虎纹捕鸟蛛毒素-Ⅰ cDNA克隆及序列

 从中国珍稀毒蛛种虎纹捕鸟蛛 (Selenocosmia huwena)毒腺中分离纯化的虎纹捕鸟蛛毒素 - (Huwentoxin ,HWTX- ) ,其一级结构、二级结构和溶液构象均已阐明 ,生理功能实验已证实 HWTX- 是一种作用于突触前膜的神经毒素和高阈值钙通道抑制剂 .为深入开展对 HWTX- 的应用研究 ,根据 HWTX- 多肽氨基酸序列 ,设计其 N-端、C-端和中间段 3组简并引物 (引物 、引物 和引物 ) .从虎纹捕鸟蛛毒腺提取制备 m RNA,逆转录合成 c DNA.以引物 和引物 为引物 ,采用 PCR方法对虎纹捕鸟蛛毒腺总 c DNA进行简并引物扩增 ,得到两条相对特异性的 DNA片段 ,产物直接与 PCR克隆载体 p UC- T连接 .重组子经原引物再次 PCR扩增和同位素标记引物 进行 Southern分子杂交鉴定 .对 4个重组子测序结果表明 ,有 1个重组子所对应的氨基酸顺序与 HWTX- 一致 ,Gen Bank数据检索说明 HWTX- c DNA编码序列确实是一个从未报道的序列 .本研究结果为 HWTX- 在真核细胞表达 ,大量获取蜘蛛毒素组分奠定了基础 .     

虎纹捕鸟蛛毒素-Ⅵ的分离纯化与鉴定

通过离子交换及反相HPLC,从虎纹捕鸟蛛毒液中分离得到1种新的哺乳类神经毒素,命名为虎纹捕鸟蛛毒素-Ⅵ（Huwentoxin-Ⅵ,HWTX-Ⅵ）。MALDI-TOF质谱仪分析及氨基酸序列分析表明该多肽毒素分子量为4.44018kDa,序列为H2N-CIGEG VPCDE NDPRC CSGLV VLKKT LHGIW IKSSY CYKCK-COOH,其中6个Cys形成3对二硫键,小鼠脑内注射实验表明,HWTX-Ⅵ对神经系统具有明显的致瘫作用。这种致瘫作用又具有可逆性。     

虎纹捕鸟蛛毒素-III及其天然突变体的纯化与鉴定(英文)

虎纹捕鸟蛛毒素 III及其天然突变体是从虎纹捕鸟蛛粗毒中分离得到的两个毒素多肽。虎纹捕鸟蛛毒素 III含 33个氨基酸残基 ,其中包含 6个半胱氨酸残基 ;而其天然突变体只比虎纹捕鸟蛛毒素 III少了C端的色氨酸残基。MALDI TOF质谱测得虎纹捕鸟蛛毒素 III及其天然突变体的分子量分别为 385 3.35和 36 6 7.4 0。通过比较其理论分子量和质谱测定的分子量表明两个多肽的 6个半胱氨酸残基分别形成了三对二硫键。虎纹捕鸟蛛毒素 III与从同一种蜘蛛分离得到的凝集素 I具有 70 .5 %的序列相似性。生物学活性实验表明 ,虎纹捕鸟蛛毒素 III具有使美洲蜚蠊可逆的致瘫作用 ,其半有效剂量 (ED50 )为 (1 92 .95±1 2 0 .84 ) μg/g (P =0 .95 ) ,而且能加强由电刺激引起的大鼠输精管收缩 ;而其天然突变体却不具有上述生物学活性 ,表明C端色氨酸残基为虎纹捕鸟蛛毒素 III生物学活性相关残基 ;同时虎纹捕鸟蛛毒素 III及其天然突变体都不具有类似于凝集素 I对红细胞的凝集活性 ,表明虎纹捕鸟蛛毒素 III和凝集素 I两者氨基酸序列中不同氨基酸残基对于决定两者的生物学活性有着重要的作用     

虎纹捕鸟蛛毒素-Ⅰ-(HWTX-Ⅰ)28肽类似物的化学合成与活性鉴定

虎纹捕鸟蛛毒素-(huwentoxin-,HWTX-)是从我国虎纹捕鸟蛛毒素中分离纯化得到的一种多肽类神经毒素.该多肽分子由33个氨基酸残基组成,含三对二硫键.其一级结构和三级结构均已测定.弄清该毒素的活性部位,是研究其结构功能关系的基础.用固相Fmoc化学合成的方法,合成了虎纹捕鸟蛛-的28肽类似物.该突变体去掉了天然毒素分子N端的Alal和C端的Lys30-Trp31-Lys32-Leu33共5个残基.用氧化还原型谷胱甘肽法完成二硫键配对,并用HPLC进行纯化,所得突变体与天然HWTX-的紫外光谱相似.质谱鉴定确认合成产物正确,分子量为3124,浓度为1×10-5g/ml的突变体能可逆阻断小白鼠膈神经-膈肌的接头传递,阻断时间为60～70min.与天然毒素比较,活性有所下降.结果说明HWTX-的N端、C端残基对其生物活性有一定影响,但不是位于活性中心的重要残基.由结果推测,虎纹捕鸟蛛毒素-的活性中心位于该分子中连接β-折叠的回环区     

两种虎纹捕鸟蛛昆虫毒素的分离纯化及生物学活性鉴定

结合离子交换和反相高效液相色谱从虎纹捕鸟蛛粗毒分离纯化到 2种虎纹捕鸟蛛毒素 ,命名为虎纹捕鸟蛛毒素 Ⅶ和虎纹捕鸟蛛毒素 Ⅷ .经质谱测定这 2种毒素的分子量分别为 3981 0 2和 4 171 12 .氨基酸序列分析发现 ,这 2种虎纹捕鸟蛛毒素同源性非常高 ,只有 6个残基位点的不同 .虎纹捕鸟蛛毒素 Ⅶ和虎纹捕鸟蛛毒素 Ⅷ的生物学功能相似 ,都能对蝗虫起到麻痹作用 ;对小鼠的中枢神经作用高剂量能使小鼠产生致死 ,但低剂量虎纹捕鸟蛛毒素 Ⅷ能使小鼠产生惊厥反应 ,而低剂量虎纹捕鸟蛛毒素 Ⅶ不能使小鼠产生惊厥反应 ;这 2种毒素都能阻断小鼠离体膈神经膈肌的神经肌肉传递 ,且与虎纹捕鸟蛛毒素 I混合后都具协同作用     

虎纹捕鸟蛛毒素-Ⅰ突变体A1Y-HWTX-Ⅰ的固相合成和生物学活性测定

用芴甲氧羰基 (Fmoc)固相多肽合成的方法在自制自动蛋白质化学工作站上合成了用酪氨酸 (Y)替代虎纹捕鸟蛛毒素 - (HWTX- )第一位丙氨酸 (A1 )的突变体 A1 Y- HWTX- .合成的突变体用 Edman降解和电喷雾质谱法进行鉴定 .活性分析结果证明 ,合成的 A1 Y- HWTX- 在含有谷胱甘肽的缓冲体系中氧化折叠后显示出与天然 HWTX- 完全相同的生物学活性 ,提示 Y替代 HWTX- 的 A1后并不明显影响 HWTX- 的活性部位和空间构象 ;A1与 HWTX- 生物学活性无关 .此外 ,将 Y引入 HWTX- 分子有助于利用碘标记方法研究 HWTX- 的作用机制     

虎纹捕鸟蛛毒素HWTX—Ⅱ的^1H—NMR信号归属和二级结构分析

虎纹捕鸟蛛毒素HWTX-Ⅱ是从虎纹捕鸟蛛Selenocosmia huwena的毒液中分离出的一种新型杀虫肽。应用2D－NMR技术研究该毒素分子的溶液结构特点,通过分析水及重水DQF－COSY、COSY、TOCSY和NOESY等^1H－NMR谱,识别出HWTX-Ⅱ全部37个氨基酸残基自旋体系;通过NOESY谱中的dαN、dαδ、dβN和dNN联系完成了序列专一的谱峰归属,确认了所有主链质子和除了Lys侧链εNH2质子外的所有侧链质子的化学位移,为完全解析HWTX-Ⅱ的溶液三维象奠定了基础,并且通过核磁数据分析,确定HWTX-Ⅱ的二级结构特点是含有较多的伸展构象,尤其是C端有一个典型的双股反平行的β折叠（Trp27-Cys29和Cys34-Lys36),分子中缺乏螺旋结构,这些二级结构特点与已探明结构的其他蜘蛛毒素的基本相同。     

Thr28突变对虎纹捕鸟蛛毒素-Ⅳ钠通道活性的影响

虎纹捕鸟蛛毒素-Ⅳ(HWTX-Ⅳ)是从虎纹捕鸟蛛粗毒中分离纯化到的一种新型多肽类神经毒素,能明显抑制表达于大鼠背根神经节细胞的河豚毒素敏感型(TTX-S)钠通道.为了更好地研究该毒素的结构与功能之间的关系,采用芴甲氧羰基(Fmoc)固相多肽化学合成法合成了用谷氨酸(Glu)替代HWTX-Ⅳ第28位苏氨酸残基的突变体T28D-HWTX-Ⅳ,线性多肽合成产物经反相高效液相色谱(HPLC)分离纯化后进行谷胱甘肽氧化复性.复性产物采用基质辅助激光解析飞行时间质谱(MALDI-TOF/TOF MS)技术鉴定分子质量,通过全细胞膜片钳电生理技术测定其电压门控钠通道药理学活性.当第28位Thr残基被Glu取代后,突变体T28D-HWTX-Ⅳ对表达于大鼠DRG细胞膜上的TTX-S钠通道的IC50值约为362 nmol/L,对TTX-S钠通道的抑制活性比天然HWTX-Ⅳ(IC50值=30 nmol/L)下降了约12倍,显示第28位的Thr残基是HWTX-Ⅳ与TTX-S型钠通道相互作用的关键活性残基.目前的研究为进一步探索HWTX-Ⅳ的结构与功能关系及新型镇痛药物的研发奠定了基础.     

重组虎纹捕鸟蛛毒素Ⅰ在巴氏毕赤酵母中的表达及纯化

纹捕鸟蛛毒素Ⅰ是从虎纹捕鸟蛛粗毒中分离纯化,具有镇痛活性的肽类神经毒素。对巴氏毕赤酵母生产的重组HWTX-Ⅰ进行多步纯化,首先将分泌到培养上清的rHWTX-Ⅰ进行90％饱和度的（NH₄）₂SO₄沉淀,再用截留分子量3kD的滤膜脱盐,再用CM阳离子交换层析分离,最后用C₁₈反相层析脱盐纯化,真空干燥后得到的rHWTX-Ⅰ经Tricine SDS-PAGE,质谱鉴定,氨基酸组成分析,N-端序列测定及活性鉴定,证明已获得高纯度的重组HWTX-Ⅰ,摇瓶表达量约为80mg/L,约占总分泌量的23.6%,并对摇瓶发酵条件进行了优化,为利用基因工程方法生产HWTX－I的规模化生产及临床应用提供了证据。     

双向电泳和肽质量指纹谱技术鉴定支气管上皮细胞恶性转化相关蛋白质ANX1-human

采用 2 - D PAGE及质谱技术对α粒子照射诱发人支气管上皮恶性转化细胞的不同阶段进行了比较蛋白组分析与鉴定 .2 - D电泳后在分子量 1 4.4～ 94k D,等电点 3～ 1 0范围内分离出约 1 1 0 0个不同蛋白质斑点 .对等电点约 7,分子量约 40 k D的蛋白质点进行了质谱分析 .鉴定出分子量为38.58k D、等电点 6.64的蛋白质 ANX1 - human(脂皮质蛋白 ,lipocortin ) ,并且发现该蛋白质在BEP2 D细胞恶性转化过程的不同时期存在差异表达 .提示蛋白质 ANX1 - human参与了支气管上皮细胞恶性转化过程 ,与细胞恶性转化相关 .     

Indentification of venom proteins of spider S. huwena on two-dimensional electrophoresis gel by N-terminal microsequencing and mass spectrometric peptide mapping

Venom proteins of the spider Selenocosmia huwena were separated by two-dimensional gel electrophoresis, with the separation in the first dimension on a wide range of immobilized pH (3–10) gradients. Over 300 protein spots were presented on a silver-stained 2D gel. The protein spots with molecular weight > 10 kDa were analyzed, after electrotransferring to polyvinyldene difluoride (PVDF) membrane, by N-terminal microseqencing. Some of the silver-stained protein spots with molecular weight over 10 kDa were analyzed and identified by employing an improved procedure of mass spectrometric peptide mapping, including (1) in-gel reduction, alkylation, and enzymatic digestion; (2) extraction and desalting by using the pipette tip containing a small C18 microcolumn (Ziptip); and (3) direct MAIDI-TOF mass analysis and protein database searching. Several known toxins such as HWTX-I, HWTX-II, HWTX-IV, and SHL-I were identified and some new components were found among these protein spots.     

双向电泳结合质谱初步分析蛇岛蝮蛇毒蛋白质组

采用荧光染料(Cy5)标记中国辽宁蛇岛蝮(Gloydius shedaoensis shedaoensis,GSS)蛇毒(snake venom,SV,GSS-SV)蛋白质,获得了该蛇毒的双向十二烷基硫酸钠聚丙烯酰胺凝胶电泳(2D SDS-PAGE)图谱,经DeCyder软件分析,分辨出1000多个蛋白点,分子量范围在10～150ku间,等电点在4～7的蛋白质点占78.8%。凝胶后染色(post-staining)采用蛋白荧光染料Deep Purple,选取的5个蛋白点经胶内酶解,产生的肽段经高效液相色谱-电喷雾串联质谱(high performance liquid chromatography/HPLC-electro-spray ionization tandem mass spectrometry/ESI-MS/MS,HPLC-ESI-MS/MS)进行序列测定,质谱数据经Sequest Bioworks软件分析,为蛇毒L-氨基酸氧化酶、金属蛋白酶、类凝血酶、纤溶酶原激活物和磷脂酶A2的同源蛋白。本研究采用的荧光标记2DSDS-PAGE结合HPLC-ESI-MS/MS的技术适于高通量研究蛇毒蛋白组成。     

Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.     

Studies on structural proteins of the rat liver ribosomes. I. Molecular wights of the proteins of large and small subunits.

Structural proteins of active 60-S and 40-S subunits of rat liver ribosomes were analysed by two-dimensional polyacrylamide gel electrophoresis. 35 and 29 spots were shown on two-dimensional gel electrophoresis of proteins from large and small subunits, respectively. It was noted that the migration distances of stained proteins with Amido black 10B remained unchanged in the following sodium dodecyl sulfate-acrylamide gel electrophoresis, although some minor degradation and/or aggregation products were observed in the case of several ribosomal proteins, especially of those with high molecular weights. This finding made it possible to measure the molecular weight of each ribosomal protein in the spot on two-dimensional gel electrophoresis by following sodium dodecyl sulfate-acrylamide gel electrophoresis. The molecular weights of the protein components of two liver ribosomal subunits were determined by this 'three-dimensional' polyacrylamide gel electrophoresis. The molecular weights of proteins of 40-S subunits ranged from 10 000 to 38 000 and the number average molecular weight was 23 000. The molecular weights of proteins of 60-S subunits ranged from 10 000 to 60 000 and the number average molecular weight was 23 900.     

Lipocalin-type prostaglandin D synthase in milk: a new biomarker for bovine mastitis.

The whey protein pattern of milk from animals affected by mastitic inflammation was resolved by two-dimensional gel electrophoresis (2D-PAGE) and compared to milk from unaffected cows. Inflammation caused the appearance of four spots aligned at a molecular weight level of 26 kDa and over a pH-region of 5.0 to 6.4. The spots excised from 2D gels were treated with chymotrypsin and the resulting peptides analyzed by MALDI-TOF mass spectrometry and RP-HPLC. All four spots yielded highly similar chymotryptic peptide mass fingerprints as well as chromatographic peak patterns. A database search could identify the four spots as isoforms of the bovine prostaglandin D synthase (PGD-S). In one of the isoforms a defined cysteine residue was shown to be oxidized to a sulfonic acid.     

Possibility of shape conformers of the protein inhibitor of the cyclic adenosine monophosphate dependent protein kinase.

The heat-stable, protein inhibitor of the cyclic adenosine monophosphate (cAMP) dependent protein kinase [Walsh, D. A., Ashby, C. D., Gonzalez, C., Calkins, D., Fischer, E., & Krebs, E (1971a) J. Biol. Chem. 246, 1977-1985] has been purified to homogeneity from rabbit skeletal muscle by preparative electrophoresis. Employing a more sensitive assay system, we detected multiple charged forms of the inhibitor on diethylaminoethyl chromatography; the form that has been further characterized is the predominant species in skeletal muscle comprising greater than 70% of the total. The apparent molecular weight of the protein inhibitor, as determined by Sephadex G-75 gel exclusion chromatography, is 22 000 in initial cellular extracts and at all stages during the purification prior to the final purification step of preparative gel electrophoresis, after which the homogeneous protein exhibits a molecular weight of 11 000. These two forms are designated I and I', respectively. The I form migrates with an apparent molecular weight of 10 000 on nondenaturing gel electrophoresis and of 10 500-11 500 on sodium dodecyl sulfate (NaDodSO4) gel electrophoresis; the I' form migrates with an apparent molecular weight of 6500-8300 on NaDodSO4 electrophoresis and has a minimum molecular weight of 10 400 by amino acid analysis. Taking into account the anomalous behavior displayed by low molecular weight proteins with the various techniques employed, we suggest that the I and I' forms of the protein inhibitor may represent shape conformers.     

Evaluation of two-dimensional difference gel electrophoresis for protein profiling. Soluble proteins of the marine bacterium Pirellula sp. strain 1

Two-dimensional gel electrophoresis (2DE) is a central tool of proteome research, since it allows separation of complex protein mixtures at highest resolution. Quantification of gene expression at the protein level requires sensitive visualization of protein spots over a wide linear range. Two-dimensional difference gel electrophoresis (2D DIGE) is a new fluorescent technique for protein labeling in 2DE gels. Proteins are labeled prior to electrophoresis with fluorescent CyDyes trade mark and differently labeled samples are then co-separated on the same 2DE gel. We evaluated 2D DIGE for detection and quantification of proteins specific for glucose or N-acetylglucosamine metabolism in the marine bacterium Pirellula sp. strain 1. The experiment was based on 10 parallel 2DE gels. Detection and comparison of the protein spots were performed with the DeCyder trade mark software that uses an internal standard to quantify differences in protein abundance with high statistical confidence; 24 proteins differing in abundance by a factor of at least 1.5 (t test value <10(-9)) were identified. For comparison, another experiment was carried out with four SYPRO-Ruby-stained 2DE gels for each of the two growth conditions; image analysis was done with the ImageMaster trade mark 2D Elite software. Sensitivity of the CyDye fluors was evaluated by comparing Cy2, Cy3, Cy5, SYPRO Ruby, silver, and colloidal Coomassie staining. Three replicate gels, each loaded with 50 microg of protein, were run for each stain and the gels were analyzed with the ImageMaster software. Labeling with CyDyes allowed detection of almost as many protein spots as staining with silver or SYPRO Ruby.     

2D differential in-gel electrophoresis for the identification of esophageal scans cell cancer-specific protein markers

The reproducibility of conventional two-dimensional (2D) gel electrophoresis can be improved using differential in-gel electrophoresis (DIGE), a new emerging technology for proteomic analysis. In DIGE, two pools of proteins are labeled with 1-(5-carboxypentyl)-1'-propylindocarbocyanine halide (Cy3) N-hydroxy-succinimidyl ester and 1-(5-carboxypentyl)-1'-methylindodi-carbocyanine halide (Cy5) N-hydroxysuccinimidyl ester fluorescent dyes, respectively. The labeled proteins are mixed and separated in the same 2D gel. 2D DIGE was applied to quantify the differences in protein expression between laser capture microdissection-procured esophageal carcinoma cells and normal epithelial cells and to define cancer-specific and normal-specific protein markers. Analysis of the 2D images from protein lysates of approximately 250,000 cancer cells and normal cells identified 1038 protein spots in cancer cell lysates and 1088 protein spots in normal cell lysates. Of the detected proteins, 58 spots were up-regulated by >3-fold and 107 were down-regulated by >3-fold in cancer cells. In addition to previously identified down-regulated protein annexin I, tumor rejection antigen (gp96) was found up-regulated in esophageal squamous cell cancer. Global quantification of protein expression between laser capture-microdissected patient-matched cancer cells and normal cells using 2D DIGE in combination with mass spectrometry is a powerful tool for the molecular characterization of cancer progression and identification of cancer-specific protein markers.