人巨细胞病毒UL149蛋白结合多肽的氨基酸序列分析

目的 初步确定与UL149蛋白具有较强结合能力的多肽,并对这些多肽氨基酸序列的特点进行分析.方法 应用随机多肽文库分别筛选与UL1493种不同基因型克隆表达的UL149蛋白结合的克隆,并测序,对相应的多肽氨基酸序列进行同源性比较,并与蛋白数据库中已知的蛋白序列进行比较.结果 与3种不同基因型的UL149蛋白结合的多肽序列之间具有较高的同源性,氨基酸序列中存在较为集中的区域,即W/A/F/V-D/E-D/E-G-W/F/I/L.与已知人类蛋白数据库进行比较,发现与人免疫球蛋白重链可变区关系密切,并与SH3、WD结构域、丝氨酸/苏氨酸蛋白激酶、补体因子H、锌脂蛋白、MHC Ⅰ类分子、真核细胞转录起始因子、核因子NF等存在结合关系.结论 人巨细胞病毒UL149蛋白可能通过多条途径来干扰机体对人巨细胞病毒的免疫应答反应.     

基因重组人巨细胞病毒PPUL32蛋白在诊断中的应用

为组建可在诊断中应用的高纯度、高效价基因工程抗原,我们在同一克隆中同时表达了人巨细胞病毒(Human cytomegalovirus HCMV)PPUL32蛋白的两个抗原决定簇基因,在诊断中取得满意的结果。 材料和方法 1.克隆 A:表达HCMV PPUL32蛋白位于UL32区第1783～1842核苷酸的抗原决定簇基因。 2.克隆 B:表达HCMV PPUL32蛋白位于UL32区第3070～3144核苷酸的抗原决定族基因;克隆C和D分别表达此基因的二个和三个拷贝。     

7株TT病毒部分核苷酸及氨基酸序列分析

目的 了解TT病毒（Transfusion transmitted virus,TTV）在我国的流行情况,研究我国TTV株序列特点。方法 采用半巢式聚合酶链反应（PCR）扩增TTVDNA,PCR阳性扩增产物直接测序。结果 我国7例TTVDNA株部分序列与日本株相比,核苷酸及氨基酸同源性分别为64．7％－98．4％及62．7％－96．4％;7株间的核苷酸及氨基酸同源性分别为63．5％－98．4％及60．2％－96．4％,分别属于两种型及其中一型中的两种亚型。结论 我国存在TTV并可能存在多种TTV基因型。     

人巨细胞病毒PP150蛋白C端多肽与gp52蛋白片段的融合表达

目的：将人巨细胞病毒PP150蛋白C端多肽与gp52蛋白片段融合,构建表达该融合蛋白的工程菌。方法：合成PP150蛋白C端25个氨基酸的基因片段,并选有细菌偏爱的密码子。用PCR方法扩增gp52蛋白基因片段。将这2个基因片段分别克隆至同一质粒pET28a( )内的NcoI/BamHI和BamHI/EcoRI位点,使两者串联在一起,并且翻译框架一致,可表达1个融合蛋白。将重组质粒转化大肠杆菌,用酶切及SDS－PAGE等方法筛选表达融合蛋白的工程菌。结果：构建成功了高效表达PP150C端多肽与pg52蛋白片段的融合蛋白工程菌,表达量为35％左右。初步纯化获得了表达的融合蛋白。结论:初步纯化的融合蛋白能与已知的抗gp52-IgM阳性血清和抗PP150－IgM阳性血清反应,说明融合蛋白C端的gp52蛋白保持较好的抗原性,融合蛋白N端的PP150C端多肽也显示有抗原性。     

人巨细胞病毒pp65蛋白基因的克隆及表达

目的 构建人巨细胞病毒pp65蛋白全长基因的原核表达载体，并对表达产物进行纯化。方法 PCR扩增pp65的全长基因。将其插入到原核表达载体pRSET，在工程菌BDPS中进行IPTG诱导表达，采用Westen blot进行鉴定并通过亲和层析对表达产物进行纯化。结果 成功构建了全长HCMVpp65的原核表达载体并诱导其高效表达。用镍离子柱亲和层析获取纯度达90%以上的表达蛋白。结论 我们获取的HCMVpp65原核表达蛋白纯度较高，可将其应用于免疫治疗和临床检测的进一步研究。     

人巨细胞病毒蛋白pp65诱导特异性免疫应答的研究

目的研究人巨细胞病毒(human cytomegalovirus,HCMV)蛋白pp65(HCMV pp65)蛋白对特异性免疫应答的诱导作用。方法采用T细胞体外激活试验检测重组HCMVpp65蛋白对人外周血单个核细胞(perpheral blood mononuclear cells,PBMC)的刺激作用,并对激活的淋巴细胞进行传代培养,观察细胞的增殖;收集传代增殖的淋巴细胞,检测其对受HCMV感染的人胚肺成纤维细胞(human embryo lung fibro-blasts,HEL)的杀伤作用和对受感染后不同时段的HEL的杀伤功能。结果重组HCMV pp65蛋白在体外能激活PBMC,并使激活的细胞增殖;增殖的淋巴细胞可特异性地杀伤受HCMV感染的HEL细胞,但对未受感染的HEL细胞无杀伤作用,且其对受染2h后的HEL即有杀伤作用。结论重组HCMV pp65蛋白可诱导CD8 T细胞的激活与增殖,对受HCMV感染细胞产生特异性的杀伤作用,此作用不需要病毒转录和蛋白合成,对于HCMV的免疫治疗将有重要的作用。     

应用外部引导序列切割人巨细胞病毒UL148 RNA的研究CSCD

目的 研究人巨细胞病毒（Human cytomegalovirus,HCMV）临床病毒株UL/b＆#39;区域UL148基因功能及抗HCMV治疗新策略,应用DNA性质外部引导序列（External guide sequences,EGS）在体外抑制UL148 RNA的表达.方法 应用RNA structure生物软件模拟UL148 RNA二级结构,选择二级结构相对简单的区域设计EGS,并合成EGS核苷酸序列.应用PCR方法分别扩增UL148及M1RNA基因,克隆至T7启动子下游,在T7 RNA聚合酶的作用下,体外转录UL148 RNA及M1RNA,其中UL148以32p标记并进行体外转录.混合EGS、M1RNA及UL148 RNA于切割反应液中,反应1h后,进行尿素变性聚丙烯酰胺凝胶电泳分离,Typhoon扫描仪观察结果.结果 根据UL148 RNA二级结构选取第58 ～72位碱基作为EGS结合区域,其切割位点位于57位,针对此区域设计EGS57,并在体外进行EGS57与UL148 RNA结合的二级结构的模拟,可形成M1RNA天然作用底物类似的茎环结构.在T7 RNA聚合酶的作用下,分别在体外转录M1RNA及UL148 RNA,其大小均与预期相符.经切割反应,可获得与预期切割位点相符的切割条带.结论 EGS57可切割UL148RNA,其切割位点准确,与预期相符.该研究为进一步探讨UL148基因功能提供有效基因沉默工具.     

p62蛋白氨基酸和cDNA序列测定和分析

目的:分析p62^dok氨基酸和cDNA序列,探讨p62^dok酪氨酸磷酸化及其与p21^ras鸟苷三磷酸酶激活蛋白(p21^rasGAP)的关系。方法:从过渡表达人类胰岛素受体的中国仓鼠卵巢细胞(CHO/IR细胞)中提取、纯化p62^dok,进行微肽序列分析,设计相应引物PCR、克隆cDNA并测序。利用免疫沉淀和蛋白免疫印迹检测p62^dok是否存在酪氨酸磷酸化及与p21^rasGAP的关系。结果:p62^dokcDNA由1863bp组成,编码481个氨基酸,含15个酪氨酸残基,富PXXP基序,并有一个pleckstrin同源区。当p62^dok酪氨酸磷酸化后,可与p21^rasGAP相结合。结论:p62^dok可能是一种新的信息分子,并通过p21^rasGAP参与胰岛素信号转导系统中RAS/MAPK径路的调节。     

北京新发现的人偏肺病毒N蛋白编码基因的序列分析

目的　了解新近从北京地区发现的人类偏肺病毒 (humanmetapneumovirus ,HMPV)N蛋白编码基因的特征。方法　从经RT PCR检测HMPV阳性的临床鼻咽洗液标本中进行HMPVN蛋白全基因扩增、克隆至pUCm T载体中并进行测序 ,与GenBank中的多株HMPVN蛋白基因序列进行比较和进化树分析。结果　经扩增并测序 ,标本BJ1816和BJ1887N蛋白基因全长为 1185bp ,编码 394个氨基酸。BJ1816N蛋白基因与国际上第一株HMPV分离株NDL0 0 1和GenBank中其它的多株HMPV的核苷酸和氨基酸同源性分别为 86 .2 %～ 99.0 %和 96 .2 %～ 99.7%。BJ1887N蛋白基因与以上进行比较的各株HMPV的核苷酸和氨基酸同源性分别为 86 .6 %～ 97.0 %和 96 .4 %～ 99.5 %。BJ1816和BJ1887之间N基因的核苷酸和氨基酸同源性分别为 86 .6 %和 96 .4 %。提示BJ1816和BJ1887分属于HMPV的两个不同的基因进化簇。结论　从基因水平进一步证明新近发现的婴幼儿肺炎的新病原确为HMPV ,提示北京地区婴幼儿中同时存在两个不同进化簇 (即基因型 )的HMPV感染。     

副黏病毒F蛋白融合活性位点中病毒特异性氨基酸基因突变分析

目的 了解副黏病毒融合蛋白(F)融合活性位点中的病毒特异性氨基酸在细胞融合中的作用.方法 以新城疫病毒(NDV)和人副流感病毒(hPIV)为例,在已确定的F蛋白融合活性位点中对病毒特异性氨基酸进行定点突变,然后将突变体F基因与同源或异源的血凝素.神经氨酸酶(HN)基因共转染BHK21细胞后,在真核细胞中表达.Giemsa染色和指示基因法检测细胞融合功能,荧光强度分析(FACS)检测F蛋白的表达效率.结果 在NDV F的突变体中,N150D-L152D的融合功能达到野毒株的46.31%;而N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E的融合活性却几乎消失,分别只有野毒株的1.25%、3.14%和2.23%;N296D-N297D的融合功能是野毒株的97.68%.在hPIV F的突变体中,D143A-E145A的融合功能达到野毒株的32.63%;E223Q-K224A几乎不能形成合胞体,其融合活性只有野毒株的1.91%;K263A-R265A、D268A-D270A和R475A-R476A的融合功能分别是野毒株的14.63%、19.52%和28.95%.FACS结果表明,NDV F的N257D-N258D-Q259E、G271D-N272D和Q279E-Q281E突变体及hPIVF的E223Q-K224A突变体F蛋白在细胞表面几乎没有表达;其余所有突变体F蛋白的表达效率与野毒株相比,基本不变.结论 对于NDV F来说,N257、N258、Q259、G271、N272、Q279、Q281对NDV F的特异性细胞融合功能起重要作用;N150和L152也起一定的作用,但是N296和N297却没有作用.对于hPIV F来说,E223和K224对hPIV F的特异性细胞融合功能起非常重要的作用;D143、E145、K263、R265、D268、D270、R475、R476的作用也很重要.     

哇巴因结合肽氨基酸序列的测定

目的:确定能与哇巴因特异性结合的多肽氨基酸及基因序列,为实现阻断或拮抗哇巴因与钠泵的作用奠定实验基础。方法: 采用噬菌体随机肽库表面呈现技术筛选出哇巴因特异性结合肽,通过测序,获得氨基酸序列,进行同源性分析、合成筛选出的12肽,人工合成哇巴因结合肽,并检测其与哇巴因的结合能力。 结果: 从噬菌体12肽库中筛选出3种多肽。肽A(12肽)的筛选一致率达到66.7%(8/12);肽B(8肽) 筛选一致率为16.7%(2/12);肽C(12肽)为8.3%(1/12);仅1例未见插入的多肽片段。 GenBank中蛋白质同源性分析结果:肽A、B、C均未见同源蛋白。获得特异性哇巴因结合肽的氨基酸序列为:Leu-Leu-Ala-Asp-Thr-Thr-His- His-Arg-Pro-Trp-Thr。结论: 哇巴因特异性结合肽氨基酸及DNA序列的获得,为哇巴因的研究提供了实验基础;同时,本研究所采用的方法也为使用噬菌体随机肽库进行甾体类激素特异性受体(配体)的筛选提供了可靠的实验依据。     

The effect of peptides containing the arginine-glycine-aspartic acid sequence on the adsorption of foot-and-mouth disease virus to tissue culture cells

Sequencing of the VP1 of a large number of subtypes of foot-and-mouth disease virus (FMDV) has revealed the presence of a conserved arginine-glycine-aspartic acid (RGD) sequence located in a highly exposed region. This sequence has been shown to be essential for the interaction of certain extracellular matrix and adhesion proteins with a superfamily of cell-surface receptors called integrins. We have examined the effects of synthetic peptides containing the RGD sequence on the binding of eight different subtypes of FMDV to tissue culture cells. The results showed that such peptides inhibited viral adsorption by 50–80%. The inhibition was dose dependent but not as great as that achieved by using a saturating amount of virus as an inhibitor. Substitution of other amino acids for any of the three main residues lowered the inhibitory properties of the peptides. These results suggest that the RGD sequence in FMDV VP1 appears to be important for the interaction of virus with cellular receptor sites.     

Conservation of structure detected in two trypanosome surface glycoproteins by amino acid sequence alignment

The predominant molecule exposed to antibody on the surface of Trypanosoma brucei is a glycoprotein of about 60 000 molecular weight which varies in amino acid sequence. The complete sequences of two such variable surface glycoproteins (VSGs) from randomly isolated, different antigenic types of trypanosomes were compared by amino acid sequence alignment. Homologous sequences were found distributed over various regions of the VSGs. Particularly good homology was observed between residues 16-34, 91-115, 177-194 and 254-345 from the N-terminus, in addition to the known conserved region close to the C-terminus. Homology was also demonstrated in the corresponding regions of the cDNA sequences by matrix analysis.     

The shikimate pathway: Review of amino acid sequence,function and three-dimensional structures of the enzymes

Abstract

The aromatic compounds such as aromatic amino acids, vitamin K and ubiquinone are important prerequisites for the metabolism of an organism. All organisms can synthesize these aromatic metabolites through shikimate pathway, except for mammals which are dependent on their diet for these compounds. The pathway converts phosphoenolpyruvate and erythrose 4-phosphate to chorismate through seven enzymatically catalyzed steps and chorismate serves as a precursor for the synthesis of variety of aromatic compounds. These enzymes have shown to play a vital role for the viability of microorganisms and thus are suggested to present attractive molecular targets for the design of novel antimicrobial drugs. This review focuses on the seven enzymes of the shikimate pathway, highlighting their primary sequences, functions and three-dimensional structures. The understanding of their active site amino acid maps, functions and three-dimensional structures will provide a framework on which the rational design of antimicrobial drugs would be based. Comparing the full length amino acid sequences and the X-ray crystal structures of these enzymes from bacteria, fungi and plant sources would contribute in designing a specific drug and/or in developing broad-spectrum compounds with efficacy against a variety of pathogens.     

Augmentation of the bactericidal activities of human cathelicidin CAP18/LL-37-derived antimicrobial peptides by amino acid substitutions

Objective: Mammalian myeloid and epithelial cells express various peptide antibiotics (such as defensins and cathelicidins) that contribute to the innate host defense against invading micro-organisms. Among these, human cathelicidin CAP18/LL-37 (L¹-S³⁷) possesses potent antibacterial activities against Gram-positive and Gram-negative bacteria. In this study, to develop peptide derivatives with improved bactericidal actions, we utilized the amphipathic 18-mer peptide (K¹⁵–V³²) of LL-37 as a template, and evaluated the activities of modified peptides.Methods: Antibacterial activities of the peptides (0.022 ~ 4.4 M corresponding to 0.1 ~ 10 g/ml) were assessed by alamarBlue^TM assay using Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa as target organisms. Furthermore, the membrane-permeabilization activities of the peptides were examined by using E. coli ML-35p as a target.Results: By substituting E¹⁶ and K²⁵ with two L residues, the hydrophobicity of the peptide (18-mer LL) was increased, and by further substituting Q²², D²⁶ and N³⁰ with three K residues, the cationicity of the peptide (18-mer LLKKK) was enhanced. Among peptide derivatives, 18-mer LLKKK exhibited the most potent antibacterial actions against S. aureus (methicillin-resistant and -sensitive), S. pneumoniae, S. pyogenes, E. coli and P. aeruginosa, and possessed the most powerful membrane-permeabilizing activities against E. coli ML-35p at the effective concentrations (p < 0.05, 18-mer LLKKK vs. 18-mer LL, 18-mer K¹⁵-V³² and LL-37).Conclusions: Bactericidal activities of the amphipathic human CAP18/LL-37-derived 18-mer peptide can be augmented by modifying its hydrophobicity and cationicity, and 18-mer LLKKK is the most potent among peptide derivatives with therapeutic potential for Gram-positive and Gram-negative bacterial infections.Received 1 September 2004; returned for revision 1 October 2004; accepted by M. Katori 23 October 2004     

AAQUANT: A computer program for quantitative amino acid analysis of proteins and peptides

Quantitative amino acid analysis is an important tool used in the characterization and structural determination of peptides and proteins. A new computer program, AAQUANT, has been developed specifically to aid researchers in analyzing amino acid composition data. AAQUANT calculates amino acid recoveries, including 95% confidence intervals, following acid hydrolysis of peptides and proteins, and also includes useful routines to locate regions of a specified amino acid composition in known protein sequences, compute amino acid composition reports of known protein sequences, generate proteolytic digestion maps of proteins, and create and edit protein sequence data files. This report describes the AAQUANT routines, and demonstrates the use of the program.     

The first and second order Markov chain analysis on amino acids sequence of human haemoglobin α-chain and its three variants with low O2 affinity

The first and second order Markov chain was used to analyse the amino acids sequences in human haemoglobin α-chain and its three variants with low O₂ affinity. In general, in the human haemoglobin α-chain, 300 of 400 (75%) possible two-amino acid sequences do not exist, about 30 two-amino acid sequences appear more than once; 7863 of 8000 (98.29%) possible three-amino acid sequences do not exist, two three-amino acid sequences appear more than once. The transition probabilities with respect to repeated sequences were calculated.     

心钠素结构中氨基酸改变对免疫结合的影响

作者应用放射免疫测定检测心钠素单克隆抗体(McAb)与人工固相合成的心钠素(CN)Ⅲ(24AA),α-hANP(28AA)及其衍生物A1,A2,A2(87),A4,A4(87)与生长激素释放因子的结合反应。CNⅢ与α-hANP的免疫结合反应相当好;其衍生物由于CN结构中个别氨基酸改变,McAb识别能力下降,交叉反应强度依次为;A2(87)>A2>A1>A4>A4(87)。生长激素释放因子无交叉反应性。本文结果提示:CN分子结构中与免疫活性有关的基团主要与其环状结构(二硫键)及C-末端结构有关,环内氨基酸改变的影响大于环外结构。环内环外某一氨基酸同时发生改变,免疫活性便完全丧失。     

Amino acid sequence studies of human properdin—N-terminal sequence analysis and alignment of the fragments produced by limited proteolysis with trypsin and the peptides produced by cyanogen bromide treatment

Treatment of human properdin with cyanogen bromide gave six major peptides. The yields, apparent mol. wts and N-terminal amino acid sequences of these peptides indicate that, in dissociating conditions, properdin is composed of only one type of polypeptide chain of approximately 56,500 mol. wt. The finding, by Minta & Lepow (1974), that glutamic acid, proline, glycine and half-cystine account for approximately 46% of the total amino acid composition of properdin has been confirmed. This unusual amino acid composition appears to be quite evenly distributed throughout the molecule but there is no evidence for the presence of any repeating type of amino acid sequence from the data given (which account for 225 residues out of an estimated total of approximately 480 residues). Limited proteolysis of properdin by trypsin, at 25°C, resulted in the cleavage at only one lysyl bond in each of the four 56,500 mol. wt polypeptide chains present in a molecule of properdin. The trypsin-treated properdin had the same mol. wt as untreated properdin when examined, without the reduction of disulphide bonds, in dissociating or nondissociating conditions. Reduction and alkylation of the trypsin-treated properdin yielded two fragments, of 36,500 and 20,000 apparent mol. wts, which were separated by gel-filtration. N-terminal amino acid sequence analysis and cyanogen bromide treatment of the two tryptic fragments have allowed an alignment of the major cyanogen bromide peptides derived from properdin to be made.