分泌蛋白质组研究进展

分泌蛋白质组是指组织、细胞等分泌的全部蛋白质。分泌蛋白主要分布于体液和细胞质胞浆中,参与许多重要的生命过程。分泌蛋白质组的研究主要涉及分泌蛋白的制备、多维色谱或二维凝胶电泳分离、质谱鉴定及生物信息学分析等。本文主要介绍了分泌蛋白的合成途径、蛋白质组技术在分泌蛋白组研究中的应用、分泌蛋白组研究现状及存在的问题等。     

牵牛光周期诱导过程中相关特异蛋白质出现时期的研究

本文研究了日本紫花牵牛子叶在短日照条件下诱导花芽分化的作用,运用双向电泳技术观察到在诱导后特定时期子叶中有一特异蛋白质的形成,不同时期去子叶对花芽分化有显著影响,核酸、蛋白质合成抑制剂处理子叶亦有抑制诱导的效应。结合三方面的实验结果,讨论了子叶中特异蛋白质合成的时间进程与诱导花芽分化的关系。     

氟化钠对雄性小鼠精子畸形的影响

为探讨氟化钠对雄性小鼠精子畸形的影响,选取50只成年雄性小鼠随机分为5组：氟化钠染毒高剂量组（34mg／kg）、中剂量组（17mg／kg）、低剂量组（8．5mg／kg）、环磷酰胺阳性对照组（50mg／kg）、生理盐水空白对照组,采用经口灌胃染毒,1次／d,连续5d,于首次染毒后的第35天颈椎脱臼将小鼠处死,取睾丸和附睾称质量,计算脏器系数;同时取附睾制片,观察精子形态,计数小鼠精子畸形率。结果表明,氟化钠染毒各组睾丸、附睾脏器系数与空白对照组比较,差别无统计学意义（P〉0．05）,染毒各组精子畸形率普遍高于空白对照组,差别有统计学意义（P〈0．001）,且随着染毒剂量的增加精子畸形率也有相应升高,呈现一定的剂量一反应关系（r=0．954,F=20．098,P〈0．05,R^2=0．909）。提示氟化钠能使雄性小鼠精子畸形率明显升高,具有一定的生殖毒性,能影响小鼠生殖功能,对小鼠生殖功能造成损害。     

Effect of Sodium Fluoride on the Rate of Sperm Deformity in Male Mice

为探讨氟化钠对雄性小鼠精子畸形的影响,选取50只成年雄性小鼠随机分为5组:氟化钠染毒高剂量组(34 mg/kg)、中剂量组(17 mg/kg)、低剂量组(8.5 mg/kg)、环磷酰胺阳性对照组( 50 mg/kg)、生理盐水空白对照组,采用经口灌胃染毒,1次/d,连续5d,于首次染毒后的第35天颈椎脱臼将小鼠处死,取睾丸和附睾称质量,计算脏器系数；同时取附睾制片,观察精子形态,计数小鼠精子畸形率.结果表明,氟化钠染毒各组睾丸、附睾脏器系数与空白对照组比较,差别无统计学意义(P＞0.05),染毒各组精子畸形率普遍高于空白对照组,差别有统计学意义(P＜0.001),且随着染毒剂量的增加精子畸形率也有相应升高,呈现一定的剂量-反应关系(r=0.954,F=20.098,P＜0.05,R2=0.909).提示氟化钠能使雄性小鼠精子畸形率明显升高,具有一定的生殖毒性,能影响小鼠生殖功能,对小鼠生殖功能造成损害.     

男性不育症与精液头发中微量元素的关系

微量元素与男性生殖功能的关系近年来已引起医务工作者的重视。在男性不育症中 ,原发性不育症约占一半以上 ,表现为精子数量及质量上的异常。传统疗法的疗效不佳与其病因及发病机理不很明了有关。现已知体内多种微量元素对机体代谢、生长和生殖有密切关系。研究表明Zn能影响精子代谢与精子质量、密度、活动度。Zn可影响脑垂体功能再间接影响性腺 ,当Zn不足时 ,促性腺激素分泌减少 ,使性腺发育不全 ;同时Zn还通过影响男性生殖系统及精子发生过程中一系列酶的活性 ,而使精子形成过程受到障碍和使精子活动环境发生改变。Cu抑制精子的氧化酵解…     

等电聚焦预分离用于肝癌细胞分泌蛋白质组的分级与鉴定

分泌蛋白质组(secretome)是指在特定的时空条件下,细胞、组织等分泌的全部蛋白质。分泌蛋白质组可能包含了大量的疾病诊断生物标志物,因此其相关研究越来越受到重视。分泌蛋白质组的组成高度复杂且浓度范围宽,这对分析方法提出了挑战。建立有效的蛋白质或肽段预分离策略,将有利于分泌蛋白质的高覆盖率鉴定。本研究以肝癌细胞系MHCC97L的无血清培养分泌蛋白质为研究对象,采用一种新型等电聚焦预分离(OFFGEL)系统,考察了肽段水平的分级对蛋白质鉴定结果的影响。结果表明,分离后各馏分中肽段的等电点分布与理论预测基本一致,每个馏分中单独鉴定的肽段比例接近80%,显示了该系统对肽段的高分辨分离能力。结合生物质谱技术,在肝癌细胞分泌系统中鉴定了2995个蛋白质,显示了该系统在复杂体系蛋白质组研究中的应用潜力。     

核酸溶液的物理化学性质

一、绪言从分子水平去了解生物学的特异性(如遗传、变异、免疫等),目前已成为科学上的一个重要问题。蛋白质与核酸是最重要的生物高分子,已知核酸在传递遗传信息及合成蛋白质上起着重要作用。蛋白质是包括约20种不同氨基酸的线型多肽链,这些氨基酸能够排列出各种不同的线型次序,这种特殊的次序引起链的一种特殊折迭,形成具有特殊作用的活性中心。所以可以这样说,生物体内最重要的作用,就是合成这些在顺序上具有特殊组合的蛋白质。根据目前比较成熟的有关蛋白质合成的模版学说,首先,经过特异酶活化以后的氨基酸,在胞浆中转移到可溶性核糖核酸分子上(分子量1万至4万)。可溶性核糖核酸有很多种,每种只能带有一种氨基酸。然后,可溶性核糖核     

叶绿体基因含有起源于核基因组的真核调控序列

用凝胶阻滞实验首次发现叶绿体psbA基因的上游调控序列可被核蛋白质因子特异结合,而且叶绿体中存在可与真核性质的CaMV35S启动子特异结合的蛋白质因子,认为这些实验结果表明某些叶绿体基因含有起源于核基因组的真核调控序列。     

基于3种非天然糖代谢标记的分泌蛋白质组分析性能对比

分泌蛋白质是调控细胞间信号转导的重要生物大分子。由于分泌蛋白的丰度相比于胞内蛋白以及培养基添加剂更低,因此分泌蛋白的高通量鉴定是目前蛋白质组学界研究的热点和难点。目前,基于生物质谱的分泌蛋白质组学分析一般均需要从无血清的条件培养基中获得分泌蛋白质,再对其进行富集和分析。该流程操作步骤繁琐,易造成分泌蛋白质的损失和降解。本工作采用基于生物正交化学生物学技术实现对分泌蛋白质的高选择性标记和高效富集。通过结合点击化学技术,综合评估了分泌蛋白质分析中用于代谢标记的不同糖类似物。采用3种最常用的商品化糖类似物,N-叠氮乙酰甘露糖胺(ManNAz)、N-叠氮乙酰半乳糖胺(GalNAz)和N-叠氮乙酰葡萄糖胺(GlcNAz)分别对HeLa细胞进行代谢标记,之后通过炔基生物素探针对条件培养基中的分泌蛋白进行富集,结合质谱分析来对比3种糖类似物对分泌蛋白的标记效率。最后通过无标定量蛋白质组学分析,系统评估了3种糖类似物用于分泌蛋白质组分析的性能。结果表明,基于ManNAz的分泌蛋白标记方法鉴定到了282个分泌蛋白、224个细胞质膜蛋白以及846个N-糖基化位点;对分泌蛋白的富集效率分别较GalNAz和GlcNAz提高了130%和67.2%;对细胞质膜蛋白的富集效率较GalNAz和GlcNAz分别提高了273.3%和148.7%,体现出了明显的优势。本研究的实验结果为分泌蛋白高选择性富集和系统分析提供了有益的对比分析和新技术策略。     

基于多肽的电化学传感器在蛋白质检测中应用的研究进展

生物体内的细胞通常会分泌各种各样的蛋白质,这些蛋白质在生物体中发挥着重要作用,尤其是可被用于诊断各种疾病的发生和发展。多肽具有良好选择性、空间适应能力和识别灵活的特点,可与不同类型的蛋白分子形成非共价键,用于蛋白质的生物检测。将多肽与电化学生物传感器结合用于蛋白质的广谱检测具有良好的发展前景。本文介绍了多肽修饰的电化学传感器在不同蛋白质检测方面的研究进展,分析了待测蛋白质的不同对多肽修饰的电化学传感器分类的影响及其优缺点,提出了基于多肽的电化学传感器在不同蛋白质检测中存在的问题,并展望了其未来发展。     

孔石莼质体蓝素的柱色谱纯化及其N-端氨基酸序列的分析测定

通过DEAE-Sepharose Fast Flow阴离子交换柱和Sephadex G-75凝胶过滤柱分离纯化得到了孔石莼(Ulva pertusa)的质体蓝素。其步骤为:将孔石莼样品以0.02 mol/L磷酸盐缓冲液(pH 7.2)进行匀浆,然后离心去除沉淀,将上清液用硫酸铵分级盐析获得饱和度为40%~80%的盐析蛋白;通过DEAE-Sepharose 柱色谱,在含有0~1.0 mol/L NaCl 的0.01 mol/L磷酸盐缓冲液线性梯度洗脱下,盐析蛋白有3个主要的洗脱峰,然后在Sephadex G-75凝胶过滤色谱柱中进一步纯化。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示,该蛋白质被纯化为单一条带。根据蛋白质电泳迁移率,纯化蛋白质的相对分子质量约为10000。该蛋白质不含糖。纯化的蛋白质经电转移至聚偏二氟乙烯（PVDF）膜后,以Edman降解法进行N-端氨基酸序列测定,前20个氨基酸残基序列为AAIVKLGPDDGSLAFVPSKI。通过对相关蛋白质数据库的检索,发现该序列与3种已报道的海藻的质体蓝素具有较高的序列同源性,其同源性分别为85%,85%和90%。据此,认为孔石莼的质体蓝素已获得纯化,其N-端20个氨基酸残基与已报道的海藻质体蓝素的氨基酸残基有较大的同源性,也存在着一定的变异。     

定点突变提高IL-4免疫毒素对淋巴瘤的选择性和杀伤活性

采用软件分析选择与IL-4分子结合与活性相关的重要位点13T,121R,通过定点突变得到IL-4突变基因cpIL4(13D121E),将其与绿脓杆菌外毒素突变基因PE38KDEL融合,成功地构建了编码免疫毒素cpIL4(13D121E)-PE38KDEL的融合基因.该基因在原核表达系统中得到了高效表达,表达量占细胞全蛋白的30%以上.表达产物经亲和色谱和阴离子交换色谱纯化后,进行细胞毒性实验,证明其对表达型IL-4受体的淋巴瘤细胞Daudi具有良好的细胞毒作用,活性是同类型IL-4免疫毒素的2倍,而对表达型IL-4受体的内皮细胞活性较低.     

Analysis of recombinant proteins by isoelectric focusing in immobilized pH gradients.

Isoelectric focusing in immobilized pH gradients (IEF-IPG) was used to analyze three different recombinant proteins. Recombinant leech hirudin (65 amino acids, three disulfide bonds) expressed in Saccharomyces cerevisiae as a secreted protein and purified by anion-exchange and reversed-phase chromatography proved to be homogeneous with regard to its isoelectric point (pI). In addition, the theoretical pI, calculated on the basis of the primary structure, corresponded precisely to the measured pI of 4.30. IEF-IPG was further employed to follow the stability of recombinant hirudin at pH 9, indicating that deamidation occurred under these conditions. A variant of recombinant human alpha 1-antitrypsin (AAT) (389 amino acids, one cysteine residue) expressed in Escherichia coli and purified by anion-exchange, metal chelate and hydrophobic-interaction chromatography appeared to be homogeneous by polyacrylamide gel electrophoresis under reducing and denaturing conditions as well as by various high performance liquid chromatography methods. However, some heterogeneity was detected by IEF-IPG between pH 5-6. The measured pI values of 5.43-5.58 were slightly lower than the calculated pI based on the primary structure (5.72). This indicated deamidations of Asn or Gln residues. A recombinant Schistosoma mansoni parasite antigen, p28 (210 amino acids, one cysteine residue) obtained after intracellular expression in Saccharomyces cerevisiae and affinity purification on glutathione agarose was analyzed by IEF-IPG in a pH 7.3-8.3 gradient. It appeared to be heterogeneous with regard to its pI, with the major component having a pI of 7.81 compared to the calculated value of 7.17.(ABSTRACT TRUNCATED AT 250 WORDS)     

梅花鹿茸多肽的化学结构及生物活性

在马鹿茸活性多肽结构与功能研究基础上, 从新鲜梅花鹿茸中分离纯化了活性单体多肽, 确定了其化学结构, 并与马鹿茸多肽进行结构与活性比较. 利用离子交换层析、 凝胶过滤层析及反相高效液相色谱层析等生物化学技术, 从梅花鹿茸中分离得到1个新多肽, SDS-PAGE电泳显示为一条带, HPLC图谱为单一峰, MALDI-TOF MS给出该多肽的精确分子量为3263.4, 其等电点pI=8.15. 一级结构研究表明, 该多肽是由32个氨基酸残基组成的直链多肽, 不含半胱氨酸, 富含缬氨酸、 赖氨酸、 亮氨酸和甘氨酸, 氨基酸序列为VLSATDKTNVLAAWGKVGGNAPAFGAEALERM. 生物活性检测结果表明, 该多肽可促进原代培养的表皮细胞和软骨细胞增殖, 也能刺激NIH3T3成纤维细胞株的分裂. 梅花鹿茸多肽与马鹿茸多肽在结构上均为32个氨基酸残基组成的直链多肽, 但第5, 8, 11和30位氨基酸残基不同. 2种多肽结构上的变化并未影响其促细胞增殖生物活性.     

Determination of the unusual amino acid hypusine at the lower picomole level by derivatization with 4-dimethylaminoazobenzene-4'-sulphonyl chloride and reversed-phase high-performance or medium-pressure liquid chromatography.

Hypusine, an unusual amino acid formed by post-translational modification of lysine, is normally determined by specific metabolic labelling followed by measurement of released radioactivity after protein hydrolysis. This paper describes a sensitive non-radioactive method for the determination of hypusine, involving complete protein hydrolysis and precolumn derivatization of the released amino acids with 4-dimethylaminoazobenzene-4'-sulphonyl chloride, followed by reversed-phase high-performance or medium-pressure liquid chromatography of the dabsylated derivatives. The detection limit of hypusine was about 500 fmol. Additionally, the hypusine-containing protein from the archaebacterium Sulfolobus acidocaldarius was purified. By applying the dabsylation method to the analysis of tryptic peptides derived from this protein, it was possible to determine the correct positioning of the hypusine residue in the amino acid sequence, which was not possible by the amino acid sequencing procedure alone.     

高效凝胶过滤色谱法分离测定豆薯种子蛋白

用高效凝胶蛋白往分离豆薯种子蛋白租提液,结合光电二极管阵列检测器对分离的蛋白峰进行紫外光谱扫描来确认蛋白的纯度,测定了3种蛋白的分子量,采用邻苯二甲醛（OPA）柱后衍生法测定了豆薯种子蛋白的氨基酸含量。     

Increased lysine N-methylation of a 23-kDa protein during hepatic regeneration

The methylation of a 23-kDa nuclear protein increased after partial hepatectomy and methylation returned to basal levels after the initial stage of regeneration. The methylating enzyme was partially purified from rat liver by ammonium sulfate precipitation, DEAE-anion exchange chromatography and Butyl-Sepharose chromatography. The 23-kDa protein was purified from a nuclear fraction of liver tissue with SP-Sepharose. When the 23-kDa protein was methylated with the partially purified methyltransferase and analyzed on C(18) high performance liquid chromatography (HPLC), the methylated acceptor amino acid was monomethyl lysine (MML). Previously, only arginine N-methylation of specific substrate proteins has been reported during liver regeneration. However, in this report, we found that lysine N-methylation increased during early hepatic regeneration, suggesting that lysine N-methylation of the 23-kDa nuclear protein may play a functional role in hepatic regeneration. The methyltransferase did not methylate other proteins such as histones, hnRNPA1, or cytochrome C, suggesting the enzyme is a 23-kDa nuclear protein- specific lysine N-methyltransferase.     

Cloning, overexpression, purification, and characterization of receptor-interacting protein 3 truncation inEscherichia coli

To facilitate structural studies of receptor-interacting protein 3 (RIP3), we developed a large-scale expression system of a glutathione-S-transferase (GST) fused with an 82 amino acid RIP3 protein inEscherichia coli. RIP3 truncation was subcloned into the pGEX-4T-1 vector and overexpressed in BL21(DE3)RIL cells. The soluble RIP3 protein was successfully purified to homogeneity using GST tag, an anion-exchange column, and gel filtration chromatography. The purity, identity, and conformation of the RIP3 protein were determined using sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, matrix-assisted laser desorption ionization mass spectrometry, circular dichroism, and fluorescence spectroscopic studies. RIP3 showed dominance of the α-helix structure and temperature-dependent conformational change.     

Linoleic Acid Isomerase from <Emphasis Type="Italic">Propionibacterium acnes</Emphasis>: Purification,Characterization, Molecular Cloning,and Heterologous Expression

Propionibacterium acnes strain ATCC 6919 catalyzes the isomerization of the double bond at the C9 position in linoleic acid (c9,c12, 18:2) to form t10,c12 conjugated linoleic acid (CLA, 18:2). CLA has significant health benefits in animal and human. The linoleic acid C9 isomerase was purified to an apparent homogeneity by successive chromatography on diethylaminoethyl (DEAE) anion exchange, hydrophobic interaction, and chromatofocusing columns. Two degenerated oligonucleotide primers were synthesized according to the N-terminal peptide sequence to clone, by polymerase chain reaction (PCR), a short nucleotide sequence (62 bp) of the isomerase gene. The linoleic acid isomerase gene (lai) was subsequently cloned by inverse PCR. The amino acid sequence deduced from the lai coding sequence predicts a protein of 424 amino acid residues (48 kDa), excluding the N-terminal methionine, which was absent in the polypeptide purified from the native host. The isomerase shares no significant sequence homology to other enzymes except a flavin-binding domain in the N-terminal region. The recombinant isomerase purified from Escherichia coli showed a typical ultraviolet spectrum for FAD-bound proteins. The recombinant enzyme produced a single isomer of t10,c12-CLA from linoleic acid, as demonstrated by gas chromatography and gas chromatography-mass spectrum analysis. The recombinant isomerase protein was expressed at high levels in E. coli, but it was almost totally sequestered in inclusion bodies. The level of active isomerase was increased 376-fold by medium and process optimization in bench-scale fermentors.     

A spider toxin binding protein from bovine brain: its purification and N-terminal amino acid sequence determination.

A 60kDa spider toxin binding protein from bovine brain was solubilized with digitonin and purified up to 5800-folds over starting crude homogenate. The purification procedure entailed DEAE-cellulose, concanavalin-A affinity, 1-naphthylacetyl spermine affinity and high performance liquid chromatography. The purified protein owned a very high affinity for ligand 125I-JSTX-3 binding Kd 15.6nM and Bmax 6.5nM. The amino acid composition of the protein was determined. The N-terminal amino acid sequence analysis yielded a unique sequence: NH2-X-Pro-X-Val-Tyr-Phe-Lys-Glu-Gln-Phe-Leu-Asp-Gly-Asp-X.