海芋凝集素cDNA的分子克隆及其性质预测

用cDNA末端快速扩增-聚合酶链式反应（RACE-PCR）方法克隆了海芋（Alocasia macrorrhiza）凝集素的全长cDNA（GenBank检索号DQ340864）,并用多种生物信息学工具对其性质进行了预测。根据来源于天南星科其他植物的凝集素和类似蛋白的保守区的DNA序列,设计了几个海芋凝集素基因aml特异引物（GSP）。用RNeasy试剂盒从海芋块茎中提取出总RNA,并以此为模板,用SMART^TM RACE cDNA扩增试剂盒提供的经特殊设计的通用引物以及不同的基因特异引物,分别获得海芋凝集素5′-和3′-RACE-PCR扩增片段。这些PCR产物经0．8％琼脂糖凝胶纯化后,分别与T克隆载体pMD 18-T相连,筛选获得阳性克隆并提取质粒,经双酶切和特异引物的PCR验证无误后,进行序列分析。从5′-和3′-RACE-PCR测序结果拼接出全长海芋凝集素cDNA序列,并用新设计自5′-RACE-PCR 5′末端的引物GSP7进行全长3′-RACE-PCR反应,获得全长海芋凝集素cDNA克隆并再次测序验证。这一新克隆的海芋凝集素cDNA的长度为1124核苷酸,分析表明它是一个编码270个氨基酸残基的蛋白质,其等电点为pH 5．7,相对分子量为29．7kD。同源性分析结果表明,海芋凝集素与其他来源于天南星科的甘露糖凝集素以及相似蛋白具有高度同源性。在海芋凝集素序列中发现了2个B型凝集素功能区域和3个甘露糖的结合位点。综合上述信息,认为这一新克隆的海芋凝集素cDNA是一个编码甘露糖识别凝集素的基因序列。     

蛇毒C型凝集素类蛋白cDNA与基因组DNA序列分析显示特别的转录后加工(英文)

为研究蛇毒C型凝集素类蛋白的快速进化机制和结构功能关系 ,使用PCR技术扩增了若干编码C型凝集素类蛋白 β链的cDNA分子以及agkisasinβ的基因组DNA ,并将这些扩增产物进行克隆和测序 .对测序结果与试验过程中的具体条件进行了因果关系分析 ,并且进行点阵图比较和多序列比对 .结果表明 ,可能存在“转录后同源重组”等转录后的事件 ,在蛇毒C型凝集素类蛋白的多样性上起着重要的作用 .对于解释基因数目与蛋白质数目的差异这一后基因组时代的重要问题 ,具有一定的参考价值 .首次报告蛇毒C型凝集素类蛋白的基因组DNA序列 ,其中未发现有内含子     

马铃薯Alfin1-like锌指蛋白基因克隆与序列分析

利用紫花苜蓿盐胁迫相关锌指结构蛋白Alfin1基因cDNA序列,通过电子克隆在GenBank中对马铃薯同源EST序列进行查询比较和拼接,获得了1个含有完整编码区的cDNA序列,并通过RT-PCR成功获得了该序列.获得的全长cDNA序列中包含1个747 bp的最大读码框,编码248个氨基酸,将其命名为Stfin1.氨基酸序列分析表明存在典型的Cys4-His-Cys3锌指结构,与Alfin1一致性达93.09%.从结构分析结果推测Stfin1与Alfin1在功能上具有一定的相关性.     

小麦NBS类抗病基因同源cDNA序列的克隆与特征分析

根据已克隆植物抗病(R)基因NBS保守结构域设计简并引物,采用RT-PCR和cDNA末端快速扩增技术(RACE),在小麦抗叶锈病近等基因系材料TcLr19中进行抗病同源基因cDNA全长的扩增。获得了1个通读的NBS类抗病同源基因S11A11cDNA序列,该序列全长2923bp,编码878个氨基酸序列。生物信息学分析结果表明,该片段含有NB-ARC保守结构域和多个LRR结构域。聚类分析表明,S11A11编码的蛋白与小麦抗叶锈病基因Lr1编码的蛋白亲缘关系较近,而与Lr10亲缘关系较远。半定量RT-PCR分析表明,该基因在小麦叶片中为低丰度组成型表达。本研究在TcLr19小麦中成功获得了抗病基因同源序列,为最终克隆小麦抗叶锈病目的基因奠定了基础。     

一种新的甘露糖结合凝集素--朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列.该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517 bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白.成熟蛋白由109个氨基酸残基组成,分子量为11.9kD.成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4%、85.3%、80.7%和83.5%的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY).     

白桂木凝集素基因cDNA克隆及其性质预测

为了探索白桂木凝集素在分子水平的作用机制,本研究采用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法首次克隆了白桂木凝集素(Atrocarpus hypargyreus lectin,AHL)cDNA序列的全长。序列分析与预测结果表明,AHL cDNA序列全长933 bp,含有一个编码40个氨基酸的信号肽和1个编码235个氨基酸的708 bp开放阅读框(ORF);AHL分子量为25 579.2 Da。同源性分析表明AHL与木菠萝家族相关凝集素氨基酸序列有很高的同源性。     

人参euFUL基因的克隆与生物信息学分析

旨在研究人参(Panax ginseng C.A.Meyer)花的形成,根据GenBank数据库中基因片段设计特异引物,利用cDNA快速末端扩增方法(RACE),克隆了人参花中一个5'端部分缺失euFUL(命名为PgFu L)的编码区序列。获得的Pg FUL基因由867个核苷酸组成,编码212个氨基酸。生物信息学分析结果显示,PgFUL编码蛋白分子质量为24.64 k D,等电点p I5.80,不含信号肽,无跨膜区。二级结构中α-螺旋结构占60.19%、延伸链占5.21%、无规则卷曲占34.60%。序列比对和系统进化分析表明,PgFUL人参PgMADS protein3(BAK20018.1)的序列同源性为100%,与加拿大蝙蝠葛(AGX01589.1)相似度达95%,属于A功能基因中的eu FUL进化系。     

日本白鲫IGF-1基因全长cDNA克隆及组织表达分析

通过已获得的多个物种IGF-1基因的cDNA序列,设计合成简并引物,用日本白鲫的肝脏总RNA反转录获得的cDNA做模板,克隆获得了IGF-1中间序列.在此基础上,通过SMART RACE,获得了IGF-1全长cDNA序列.经过序列对比分析,所得到的序列是日本白鲫(Carassius cuvieri)IGF-1 cDNA全长,其中开放阅读框为486 bp,编码含161个氨基酸的蛋白质,该蛋白质含有保守的信号肽、B、C、A、D和E结构域和6个半胱氨酸残基.同时,系统进化分析表明,在进化过程中IGF-1基因在鱼类中保持着高度保守的进化特征.IGF-1在日本白鲫中广泛表达于多个组织,尤其在肝脏中表达量最高.在垂体、心脏、肾脏和肌肉中有较高的表达.而在脑、脾脏、精巢和卵巢中的表达量较低.     

一种新的甘露糖结合凝集素——朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列。该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白。成熟蛋白由109个氨基酸残基组成,分子量为11.9kD。成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4％、85.3％、80.7％和83.5％的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY)。     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

Production and characterization of the B chains of mistletoe toxic lectins

Mistletoe toxic lectins consist of two polypeptide chains: an enzymatically active A chain, which is a toxic component, and a disulfide-bonded B chain, which confers the lectin properties on the total molecule. Mistletoe leaves contain three toxic lectins encoded by three genes. The B chains of these lectins were overproduced in Escherichia coli in a soluble form. The recombinant proteins bound with asialofetuin, but had substantially lower affinity for simple sugars D-galactose and N-acetyl-D-galactosamine as compared with the natural proteins. The functional properties of the B chains strongly depended on the storage conditions (salt concentration and the presence of galactose); the dependence was explained by structural instability of nonglycosylated recombinant proteins. The lectin activity of one of the recombinant B chains was close to that of the native protein, which was attributed to the lack of N-glycosylation sites in the latter.     

Obtaining and characterization of the B-chains of mistletoe toxic lectins

Mistletoe toxic lectins consist of two polypeptide chains: an enzymatic A chain, the toxic component, is joined by disulfide bond to a B chain conferring the lectin properties to the complete molecules. Mistletoe leaves contain three of toxic lectins encoded by three genes. The three B chains were produced in Escherichia coli in a soluble form. The recombinant proteins were found to bind to asialofetuin but in contrast to native proteins could be competed to a less extent by simple sugars D-galactose and N-acetyl-D-galactosamine. The functional properties of the proteins were strongly influenced by storage conditions such as salt concentration and simple sugar presence thus indicating an unstable folding. The lectin activity of one of the recombinant B chains was most close to the native protein that possibly results from the absence of N-glycosylation.     

Expression and functional properties of genetically engineered ricin B chain lacking galactose-binding activity.

Ricin is a potent plant toxin consisting of two disulfide-bonded subunits. The A chain of ricin is an N-glycosidase which inactivates 28 S RNA and inhibits protein synthesis. The B chain is a galactose-specific lectin with two galactose-binding sites. The genes encoding preproricin and its A and B chains have been cloned and expressed. In addition, X-ray crystallographic studies have identified the galactose-contact residues in both the high- and low-affinity galactose-binding sites of the B chain. In this study, the high-affinity galactose-contact residue of the B chain was changed from Asn-255 to Ala-255 by oligonucleotide-directed mutagenesis. The resulting mutant was sequenced to confirm the presence of a single mutation and was expressed in Cos-M6 cells. Both wild-type and mutant recombinant B chain could be immunoprecipitated with a heterologous anti-B chain antibody and both could form A-B heterodimers. However, as compared to the wild-type, the mutant B chain lacked more than 99% of its lectin activity and cytotoxicity as an A-B dimer. In conclusion, altering the contact residue of the high-affinity galactose-binding site of ricin B chain from Asn-255 to Ala-255 abrogates more than 99% of its lectin activity and the cytotoxicity of the A-B heterodimer to ricin-sensitive cells.     

The major elderberry (Sambucus nigra) fruit protein is a lectin derived from a truncated type 2 ribosome-inactivating protein

The major protein of elderberry ( Sambucus nigra L.) fruits is a lectin, called Sambucus nigra agglutinin IVf or SNAIVf. This lectin is composed of subunits that strongly resemble the B chain of the type 2 ribosome-inactivating protein (RIP), called SNAVf, present in the same tissue. To corroborate the possible relationship between both proteins their corresponding cDNAs were cloned and compared. Alignment of the deduced amino acid sequences revealed that the cDNA encoding SNAIVf is almost identical to that of SNAVf except that its A chain is truncated. Northern blot analysis confirmed that the mRNA encoding SNAIVf is about 500 nucleotides shorter than the SNAVf mRNA. In addition, the occurrence of a truncated type 2 RIP gene was unambiguously demonstrated by the analysis of PCR amplified genomic sequences. These results not only demonstrate for the first time that a plant lectin is encoded by a truncated type 2 RIP gene but also address important questions with respect to the molecular evolution of RIP and lectins.     

Complete structure determination of N‐acetyl‐D‐galactosamine‐binding mistletoe lectin‐3 from Viscum album L. album

The primary structure of the B chain of the N‐acetyl‐D ‐galactosamine‐recognizing mistletoe lectin‐3 (ML‐3B) has been deduced from proteolytic digest peptides of the purified glycoprotein, their HPLC‐separation and Edman degradation and confirmation of the peptide sequences by MALDI‐MS. ML‐3B consists of 262 amino acid residues including 10 cysteine moieties. The structure and linkage of the carbohydrate side chains, connected to two N‐glycosylation sites at positions Asn⁹⁵ and Asn¹³⁵ of the lectin, were determined by a combination of glycosidase treatment and MALDI‐MS of corresponding glycopeptide fragments. The sequence alignment reveals a high homology with other B chains of type‐II RIPs, although there are remarkable differences in the D ‐galactose‐specific mistletoe lectin‐1B chain. The recently published primary structure of the mistletoe lectin‐3A chain 1 and the now available primary sequence of the 3B chain allowed the construction of a preliminary homology model of ML‐3. The model demonstrates, unequivocally, that ML‐3 is a member of the type‐II RIP family with rigid conservation of the enzymatic active site of the A chain and an identical overall protein fold. Specific amino acid residue exchanges and the different glycosylation pattern in comparison with ML‐1 are discussed and related to the properties of the two glycoproteins. The knowledge of the complete primary structure of mistletoe lectin‐3 is a major contribution towards more insight into the mechanism of the biological activity of commercial mistletoe preparations. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

HIV-1 Gag P17m的融合表达、纯化及NMT有效底物

 利用PCR技术扩增编码HIV 1GagP17m的DNA片段 ,进而构建了T7启动子控制下的C端His Tag融合表达质粒pMF P17mHT .SDS PAGE分析结果表明 ,融合蛋白 (约 2 1kD)在大肠杆菌BL2 1(DE3)中得到高表达 ,表达产物约占全菌蛋白 2 0 % .该融合蛋白主要以可溶性形式存在 ,通过金属离子 (Ni2 +)螯合亲和层析予以纯化 ,纯度在 90 %以上 .体外标记结果表明 ,融合表达的P17mHT能被人NMT有效地N端豆蔻酰化 .这些结果为深入研究筛选HIV 1GagP17或P55的豆蔻酰化专一性抑制剂 ,奠定了良好的基础     

Characterization of a novel collagen chain in human placenta and its relation to AB collagen.

A novel collagen chain, termed alpha C, has been isolated from human placenta by limited pepsin digestion. The collagen containing the alpha C chain copurifies with placental AB collagen during selective salt precipitation but is virtually absent from fetal birth membranes, which contain relatively larger amounts of AB. Both native AB and alpha C-containing collagens are resistant to human skin collagenase under conditions that support cleavage of type I by greater than 90%. The alpha C chain was separated from alpha B by phosphocellulose chromatography and subsequently from alpha P by chromatography on CM-cellulose. Its amino acid composition is distinct from alpha A and alha B although all three chains posses compositional features in common; the carbohydrate content of the alpha C chain was intermediate between those of alpha A and alpha B. Analysis by NaDodSO4-polyacrylamide gel electrophoresis of peptides produced by CNBr cleavage and by limited digestion with the enzyme mast cell protease indicated different and unique products for the alpha A, alpha B, and alpha C chains. The data support the existence of another collagen chain which is related to the alpha A and alpha B chains but which is structurally unique. The proteins containing these chains may in turn comprise a subfamily of collagen isotypes which represents a divergence from and/or specialization of the type IV basement membrane collagens.     

Sequence comparison and phylogenetic analysis by the Maximum Likelihood method of ribosome-inactivating proteins from angiosperms

Ribosome-inactivating proteins (RIPs) from angiosperms are rRNA N-glycosidases that have been proposed as defence proteins against virus and fungi. They have been classified as type 1 RIPs, consisting of single-chain proteins, and type 2 RIPs, consisting of an A chain with RIP properties covalently linked to a B chain with lectin properties. In this work we have carried out a broad search of RIP sequence data banks from angiosperms in order to study their main structural characteristics and phylogenetic evolution. The comparison of the sequences revealed the presence, outside of the active site, of a novel structure that might be involved in the internal protein dynamics linked to enzyme catalysis. Also the B-chains presented another conserved structure that might function either supporting the beta-trefoil structure or in the communication between both sugar-binding sites. A systematic phylogenetic analysis of RIP sequences revealed that the most primitive type 1 RIPs were similar to that of the actual monocots (Poaceae and Asparagaceae). The primitive RIPs evolved to the dicot type 1 related RIPs (like those from Caryophyllales, Lamiales and Euphorbiales). The gene of a type 1 RIP related with the actual Euphorbiaceae type 1 RIPs fused with a double beta trefoil lectin gene similar to the actual Cucurbitaceae lectins to generate the type 2 RIPs and finally this gene underwent deletions rendering either type 1 RIPs (like those from Cucurbitaceae, Rosaceae and Iridaceae) or lectins without A chain (like those from Adoxaceae).     

Characterization of recombinant human antithrombin III synthesized in Chinese hamster ovary cells

Biochemical and physiochemical properties of recombinant human antithrombin III were examined. This protein, produced in Chinese hamster ovary cells, showed a conformation apparently identical with the natural product isolated from human plasma when examined by circular dichroism, UV absorbance, and fluorescence spectroscopy. Comparison of the NH2-terminal sequences of recombinant and human plasma-derived antithrombin III showed that on synthesis and secretion of the recombinant protein from Chinese hamster ovary cells the signal peptide is correctly cleaved by the corresponding endoplasmic signal peptidase. The recombinant antithrombin III has identical properties in heparin binding and biological activities as determined in vitro by two-dimensional immunoelectrophoresis, progressive inhibitor, and heparin cofactor assays. Analysis of the carbohydrate portion of recombinant antithrombin III synthesized in Chinese hamster ovary cells revealed glycosylation of the complex type. Characterization of the oligosaccharide chains present in the recombinant protein reveals three major fractions, A (20%), B (60%), and C (20%). Fraction A contains tri- and tetraantennary complex-type oligosaccharides, fraction B contains biantennary oligosaccharides, and fraction C partially truncated biantennary structures. Pharmacokinetic studies with recombinant and plasma-derived antithrombin III in rabbits showed that the clearance behavior of both proteins is very similar and can be described by a double exponential decrease with almost identical kinetic parameters.     

原核增强子样序列3A的克隆及其结构与功能的研究

 从大肠杆菌 C60 0株染色体基因组中筛选到一个原核增强子样序列 3A,其正、反向增强活性分别能提高 β-半乳糖苷酶活性 7.1 1和 2 .93倍 .采用体内转录 ,RNA斑点印迹杂交的方法 ,证明3A序列对于基因表达的调控发生在转录水平上 . 3A功能区的研究表明 ,3A增强活性主要位于距其正向克隆 5′端 (增强活性较强的方向称为正向 ,反之为反向 ) 30 0～ 540 bp,长约 2 4 0 bp的区段内 ,并证明 3A片段增强活性至少由两个增强活性位点组成