呼肠病毒BYD株吸附蛋白σ1的原核表达及其抗原性鉴定

将呼肠病毒BYD株S1片段编码的细胞吸附蛋白基因连接至pET-28a( ),构建表达载体pET-28a( )-S1,转化表达宿主菌E.coli BL21(DE3),分析表达载体能否表达预期大小的重组σ1,并进一步鉴定重组σ1的抗原性。SDS-PAGE实验表明,经IPTG诱导后,表达载体能表达53kD左右蛋白质,与重组σ1的预期大小相符;免疫印迹分析表明重组σ1有较好的抗原性和特异性,可用于呼肠病毒诊断试剂的制备。     

新型鸭呼肠孤病毒的分离与鉴定

本研究从临床表现为出血性坏死性肝炎的病死鸭肝脾中分离到病毒。病原特性鉴定显示,分离毒能致死番鸭胚和鸡胚;人工感染1日龄雏番鸭、雏半番鸭均能复制出与临床自然发病鸭相同的临床症状和病理变化,并能回收到病毒。分离毒能在MDEF等多种细胞中增殖并产生细胞病变。电镜下病毒在细胞浆中呈大量散在、成堆和晶格状排列,病毒粒子呈球形、无囊膜、双层衣壳、直径70nm左右。在SDS-PAGE中具有禽呼肠孤病毒10个RNA片段的特征,但M1-3和S1-4片段的迁移率明显不同于番鸭呼肠孤病毒(MDRV)。分离毒S3基因全序列与禽呼肠孤病毒(ARV)、火鸡呼肠孤病毒(TRV)和MDRV的核苷酸同源性分别为60%～60.2%,61.9%,62.3%～62.7%,氨基酸同源性分别为68.2%～69%,68.2%,69.3%～70.1%;S3基因编码的σB蛋白属于单独的进化分支,提示分离毒S3基因具有不同于ARV和MDRV的特征。结果表明鸭出血性坏死性肝炎的病原是一种属于呼肠孤病毒科正呼肠孤病毒属新型鸭呼肠孤病毒。     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析。结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%。RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定。利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp prote-ase proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性。     

水稻瘤矮病毒基因组S9片段的基因结构特征

应用RT-PCR技术克隆了水稻瘤矮病毒(RGDV)中国广东信宜分离物(RGDV-C)的基因组S9片段,测定了全序列并进行了生物信息学分析.结果表明,RGDV-C S9片段全长共有1202bp(登录号AY556483),含有一个长的开放阅读框,这一开放阅读框编码一个由323个氨基酸残基组成的多肽,推测分子量约35.6kDa,与泰国分离物(RGDV-T)的全序列相比,它们的核苷酸长度相等,核苷酸同源性为98.1%,氨基酸同源性为98.5%.RGDV S9片段编码的Pns9蛋白在植物呼肠孤病毒属内未发现同源蛋白,其功能尚待确定.利用NCBI的BLAST查找与比较,发现Pns9与伯氏疏螺旋体(Borrelia burgdorferi)ATP依赖的Clp蛋白水解酶组分[ATP-dependent Clp protease proteolytic component(clpP-1)]有21.8%的氨基酸序列同源性.     

水稻矮缩病毒第一号组份基因和编码蛋白的序列分析

水稻矮缩病毒（RiceDwarfVirus,简称RDV）是我国南方水稻病毒病的重要病原,属植物呼肠孤病毒。从中国福建分离物中克隆了基因组第一号片段（S1）的全长cDNA并对其进行全序列分析,结果表明RDV福建分离物S1克隆片段全长4422bp,含有一个长4332bp的开放阅读框架,编码一个由1444个氨基酸组成的多肽（P1）,分子量为164kD．根据基因序列,对推测的P1氨基酸序列分析表明,序列中含有依赖于RNA的RNA聚合酶（RNA-dependentpolymerase-RDRP）保守序列：motifＩ（DXXXXD）、motifⅡ（SGXXXTXXXN）和motifⅢ（GDD）,除此之外,在模式Ⅲ后还存在一个很保守的区域EXXKXY。由此说明RDVS1编码的蛋白P1可能是病毒的一种RDRP。将RDV福建分离物引核苷酸和编码蛋白氨基酸序列与日本流行株系相比,同源性分别为95％和97％。RDV福建分离物S1序列已被DenBank接受,号码为U73201。     

草鱼呼肠孤病毒HZ08株S4基因序列分析

草鱼呼肠孤病毒HZ08株是本实验室从患出血病草鱼体内分离到的一个新毒株,已完成部分基因序列的分析,其氨基酸序列的同源性和873株相比,仅为20%~30%之间.因序列差异较大,无法通过设计特异性引物来扩增和分析其基因序列,采用单引物扩增技术,对HZ08株S4基因进行序列分析表明:S4全长为2263 bp,最大的ORF编码717个氨基酸,推导出其表达的蛋白约为79 kDa.正如其他基因节段,基因末端也含有保守碱基序列5′(GUAAUUU…UUCAUC),3′.S4基因推导的氨基酸序列与同宿主的其他呼肠孤病毒的非结构蛋白NS1同源性最大,其次是和哺乳动物正呼肠孤病毒的非结构蛋白mu-NS以及禽呼肠孤病毒非结构蛋白NS1同源性较大,表明S4可能表达细胞骨架相关蛋白.基于S4推导出的氨基酸序列构建的系统进化树HZ08株单独作为一个分支,与同宿主的其他呼肠孤病毒亲缘关系比较近,而与其他呼肠孤病毒则相对较远.这提示HZ08株可能是多个毒株的遗传信息经长期的遗传进化而得,综合其它已知序列信息,推测HZ08株可能为呼肠孤病毒的一个新成员.     

马尾松毛虫质型多角体病毒S4的cDNA克隆及序列分析

从马尾松毛虫质型多角体病毒湖南株中提取病毒核酸,回收、纯化第四片段S4,经RT-PCR扩增得到了S4的cDNA克隆并测定了其全序列。结果表明,S4全长由3262个碱基组成,包含一个编码1058个氨基酸的完整开放阅读框架。B1ast同源分析显示,DpCPV S4与LdCPVS4、BmCPVS4和RRSV S2编码蛋白氨基酸的同源性分别为94％、92％和22％。另外,氨基酸序列部分区域与Methanosarcina mazei Goel的甲基化转移酶有同源性,且序列中含有鸟苷酸转移酶活性位点。     

兔出血症病毒中国株衣壳蛋白基因的克隆和序列分析

应用RT—PCR技术,从兔出血症病毒中国分离株WX84中成功扩增出预期大小为1．7kb的特异性条带,将扩增产物提纯后克隆入pGEM^R—T载体,经转化、筛选及酶切鉴定后,获得了该株病毒衣壳蛋白基因的克隆,序列分析表明扩增的中国株BHD衣壳蛋白基因片段长度为1740bp,共编码580个氨基酸。该核酸序列与其它国家报道的多株BHDV序列相互间同源性高达98．2％一99．0％,其推导的氨基酸序列同源性也达98．3％--99．1％,为极度保守片段。     

单引物法扩增马尾松毛虫CPV基因组第8片段及其序列分析

本文利用T4 RNA连接酶将5'－磷酸,3'-氨基修饰的引物1连接到马尾松毛虫质型多角体病毒第8片段dsRNA的3'-OH端,经逆转录,退火,补齐形成全长双链cDNA。使用单一的互补引物2进行PCR 增,扩增产物克隆在pMD18-T载体上,对重组子进行限制性内切酶分析及序列测定。结果表明,克隆片段全长330bp,S'端具有CPV－1型末端保守序列AGTAAA'端具有保守序列GTTAGCC。起始密码子从ATG位于38－40残基,终止密码子TAA位于1208－1210残基。推测S8片段编码390年氨基酸多肽,分子量为44kDa。与舞毒蛾质多角体病（LdCPV)第8片段相比较,核苷酸和氨基酸同源性分别为97％和98％。与家蚕质型多角体病毒（BmCPV）第8片段相比较,核苷酸和氨基酸的同源性分别为83％和85％。与人的呼肠孤病毒第8片段比较没有明显的同源性。     

Symmetric primary and tertiary structure mutations within a symmetric superfold: a solution, not a constraint, to achieve a foldable polypeptide

In previous studies designed to increase the primary structure symmetry within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) a combination of five mutations were accommodated, resulting in structure, stability and folding kinetic properties similar to wild-type (despite the symmetric constraint upon the set of core residues). A sixth mutation in the core, involving a highly conserved Met residue at position 67, appeared intolerant to substitution. Structural analysis suggested that the local packing environment of position 67 involved two regions of apparent insertions that distorted the tertiary structure symmetry inherent in the beta-trefoil architecture. It was postulated that a symmetric constraint upon the primary structure within the core could only be achieved after these insertions had been deleted (concomitantly increasing the tertiary structure symmetry). The deletion of these insertions is now shown to permit mutation of position 67, thereby increasing the primary structure symmetry relationship within the core. Furthermore, despite the imposed symmetric constraint upon both the primary and tertiary structure, the resulting mutant form of FGF-1 is substantially more stable. The apparent inserted regions are shown to be associated with heparin-binding functionality; however, despite a marked reduction in heparin-binding affinity the mutant form of FGF-1 is surprisingly approximately 70 times more potent in 3T3 fibroblast mitogenic assays. The results support the hypothesis that primary structure symmetry within a symmetric protein superfold represents a possible solution, rather than a constraint, to achieving a foldable polypeptide.     

Integrating statistical pair potentials into protein complex prediction

The biophysical study of protein-protein interactions and docking has important implications in our understanding of most complex cellular signaling processes. Most computational approaches to protein docking involve a tradeoff between the level of detail incorporated into the model and computational power required to properly handle that level of detail. In this work, we seek to optimize that balance by showing that we can reduce the complexity of model representation and thus make the computation tractable with minimal loss of predictive performance. We also introduce a pair-wise statistical potential suitable for docking that builds on previous work and show that this potential can be incorporated into our fast fourier transform-based docking algorithm ZDOCK. We use the Protein Docking Benchmark to illustrate the improved performance of this potential compared with less detailed other scoring functions. Furthermore, we show that the new potential performs well on antibody-antigen complexes, with most predictions clustering around the Complementarity Determining Regions of antibodies without any manual intervention.     

Protein microarrays as a discovery tool for studying protein–protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein–protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein–protein interaction studies.     

The heat capacity of proteins

The heat capacity plays a major role in the determination of the energetics of protein folding and molecular recognition. As such, a better understanding of this thermodynamic parameter and its structural origin will provide new insights for the development of better molecular design strategies. In this paper we have analyzed the absolute heat capacity of proteins in different conformations. The results of these studies indicate that three major terms account for the absolute heat capacity of a protein: (1) one term that depends only on the primary or covalent structure of a protein and contains contributions from vibrational frequencies arising from the stretching and bending modes of each valence bond and internal rotations; (2) a term that contains the contributions of noncovalent interactions arising from secondary and tertiary structure; and (3) a term that contains the contributions of hydration. For a typical globular protein in solution the bulk of the heat capacity at 25°C is given by the covalent structure term (close to 85% of the total). The hydration term contributes about 15 and 40% to the total heat capacity of the native and unfolded states, respectively. The contribution of non-covalent structure to the total heat capacity of the native state is positive but very small and does not amount to more than 3% at 25°C. The change in heat capacity upon unfolding is primarily given by the increase in the hydration term (about 95%) and to a much lesser extent by the loss of noncovalent interactions (up to ～5%). It is demonstrated that a single universal mathematical function can be used to represent the partial molar heat capacity of the native and unfolded states of proteins in solution. This function can be experimentally written in terms of the molecular weight, the polar and apolar solvent accessible surface areas, and the total area buried from the solvent. This unique function accurately predicts the different magnitude and temperature dependences of the heat capacity of both the native and unfolded states, and therefore of the heat capacity changes associated with folding/unfolding transitions. © 1995 Wiley-Liss, Inc.     

A solubility-enhancement tag (SET) for NMR studies of poorly behaving proteins

Protein-fusion constructs have been used with great success for enhancing expression of soluble recombinant protein and as tags for affinity purification. Unfortunately the most popular tags, such as GST and MBP, are large, which hinders direct NMR studies of the fusion proteins. Cleavage of the fusion proteins often re-introduces problems with solubility and stability. Here we describe the use of N-terminally fused protein G (B1 domain) as a non-cleavable solubility-enhancement tag (SET) for structure determination of a dimeric protein complex. The SET enhances the solubility and stability of the fusion product dramatically while not interacting directly with the protein of interest. This approach can be used for structural characterization of poorly behaving protein systems, and would be especially useful for structural genomics studies.     

Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.     

A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins

A previously developed computer program for protein design, RosettaDesign, was used to predict low free energy sequences for nine naturally occurring protein backbones. RosettaDesign had no knowledge of the naturally occurring sequences and on average 65% of the residues in the designed sequences differ from wild-type. Synthetic genes for ten completely redesigned proteins were generated, and the proteins were expressed, purified, and then characterized using circular dichroism, chemical and temperature denaturation and NMR experiments. Although high-resolution structures have not yet been determined, eight of these proteins appear to be folded and their circular dichroism spectra are similar to those of their wild-type counterparts. Six of the proteins have stabilities equal to or up to 7kcal/mol greater than their wild-type counterparts, and four of the proteins have NMR spectra consistent with a well-packed, rigid structure. These encouraging results indicate that the computational protein design methods can, with significant reliability, identify amino acid sequences compatible with a target protein backbone.     

Lethal mutations in the major homology region and their suppressors act by modulating the dimerization of the rous sarcoma virus capsid protein C‐terminal domain

An infective retrovirus requires a mature capsid shell around the viral replication complex. This shell is formed by about 1500 capsid protein monomers, organized into hexamer and pentamer rings that are linked to each other by the dimerization of the C‐terminal domain (CTD). The major homology region (MHR), the most highly conserved protein sequence across retroviral genomes, is part of the CTD. Several mutations in the MHR appear to block infectivity by preventing capsid formation. Suppressor mutations have been identified that are distant in sequence and structure from the MHR and restore capsid formation. The effects of two lethal and two suppressor mutations on the stability and function of the CTD were examined. No correlation with infectivity was found for the stability of the lethal mutations (D155Y‐CTD, F167Y‐CTD) and suppressor mutations (R185W‐CTD, I190V‐CTD). The stabilities of three double mutant proteins (D155Y/R185W‐CTD, F167Y/R185W‐CTD, and F167Y/I190V‐CTD) were additive. However, the dimerization affinity of the mutant proteins correlated strongly with biological function. The CTD proteins with lethal mutations did not dimerize, while those with suppressor mutations had greater dimerization affinity than WT‐CTD. The suppressor mutations were able to partially correct the dimerization defect caused by the lethal MHR mutations in double mutant proteins. Despite their dramatic effects on dimerization, none of these residues participate directly in the proposed dimerization interface in a mature capsid. These findings suggest that the conserved sequence of the MHR has critical roles in the conformation(s) of the CTD that are required for dimerization and correct capsid maturation. Proteins 2013. © 2012 Wiley Periodicals, Inc.