SARS冠状病毒的起源和进化初探

发展了一种研究基因序列进化的随机替代模型,根据一组从某个最近共同祖先演化而来的若干序列,可以预测此共同祖先序列,并计算随机替代概率矩阵,确定了从祖先序列的各个碱基到进化序列的各个碱基之间的替代概率。将这一模型应用于分析包括SARS冠状病毒在内的4种冠状病毒全基因组的演化规律,确定了在若干较保守的编码蛋白基因序列中同义替代位点的最近共同祖先序列以及分歧进化后的累计同义替代数目。结果表明,SARS病毒和其他几种已知的冠状病毒具有相当的进化历程,但存在不同的进化途径,支持了SARS病毒在造成此次大规模感染人体之前已经历了较长的进化历程的猜测。     

SARS-CoVS基因表达及纯化

严重急性呼吸道综合症是由一种新的冠状病毒SARS-CoY引起的．作者通过PCR扩增得到了S蛋白的6个编码片段，并利用表达载体pET28a( )在E．coli BL21中进行了原核表达．通过亲和层析纯化了包含大部分ACE2结合区域的S蛋白片段(S4)．ELISA分析结果表明S4与SARS病人恢复期血清具有良好的反应能力．     

Infection of SARS-CoV on juvenile and adult Brandt’s vole Microtus brandtii

We studied the infectious effect of SARS-CoV virus on juvenile and adult Brandt‘s Vole (Microtus brandtii) by nasal cavity spraying method (CCIDso is 10^5.7). SARS virus caused serious deaths in adults. The death adults demonstrated hemorrhage from mouth, nasal cavity and intestine, hemorrhageious interstitial pneumonia and gore in liver, spleen and kidney. The survival adults demonstrated local hemorrhagic spot in lung and emphysema, but the other organs showed no pathological abnormality. SARS virus caused no deaths in juveniles, but locomotion of infected juveniles became slower. In the early stage, there was local pneumonia in lung and SARS viruses were isolated from the pathological tissue. Only one control juvenile lived and the infected juvenile showed local pneumonia in lung. The results demonstrated that SARS-CoV infected Brandt‘s vole seriously and adults were more susceptive to SARS-CoV than juveniles. The Brandt‘s vole may be a potential animal model for SARS research.     

Preparation, characterizationand preliminary in vivo studies of inactivated SARS-CoV vaccine

A large quantity of SARS-CoV virus was proliferated in Veto cells, inactivated with β-ptopiolactone, then purified by Sepharose 4FF column chromatography to prepare inactivated vaccine. The vaccine was identified by Western blot, mass spectrographic analysis, ELISA and electton microscopy. The vaccine with or without aluminum hydtoxide adjuvant was inoculated into female BALB/c mice at different dosages. The result showed that the antibodies to SARS-CoV were induced in the mice. The antibody levels induced by the vaccine with aluminum hydroxide were higher than those without aluminum hydroxide.     

SARS病毒E基因的克隆及原核表达

为了得到纯的SARS-CoV E蛋白以进一步研究其功能,通过Touch-down PCR程序,从SARS-CoV WHU cDNA(pMD18-T载体)文库中扩增了E基因,构建了重组质粒pGEX-E,并在Ecoli DH5α中进行了原核表达。SDS/PAGE及Western blot结果表明SARS病毒E蛋白的分子量约5 kDa,较预测的略小,并进一步分析了这种现象产生的可能原因。     

SARS冠状病毒N蛋白的表达及二级结构预测分析

通过RT-PCR获得SARS冠状病毒N蛋白基因，分别克隆到原核表达载体pET21a，pET32a和pGEX-4T-1中，将3种重组质粒pET2la-N，pET32a-N和pGEX-4T-1-N分别转化大肠杆菌BL21(DE3)，经IPTG诱导，细菌中分别表达出约46kD的重组N蛋白、约60kD的6xHis-N融合蛋白和约70kD的GST-N融合蛋白，表达量分别达总蛋白的45％、40％和30％．进一步的分析表明：6xHis-N融合蛋白在大肠杆菌中为可溶性表达，该可溶性组分占细菌裂解液的70％左右，且能被6xHis抗体所识别．用蛋白分析软件对N蛋白进行了序列分析和二级结构预测．SARS冠状病毒N蛋白在大肠杆菌中的高效可溶性表达，有助于进一步结晶后进行X射线晶体衍射分析其结构与功能．     

天然产物活性化合物抗SARS-CoV研究

针对系统综合征冠状病毒 2 (SARS-CoV-2),在全球范围大流行并呈指数级传播,迫切需要有 效的抗病毒药物和疫苗来控制和预防 SARS-CoV-2 疫情,寻找与开发有效、价格低廉的治疗 SARS-CoV-2 药物,是全世界面临的挑战;结合国内外最新文献,介绍了冠状病毒的起源、种类、人类感染的危害与应对措 施,发现药用植物与其它天然产物,其活性化合物抗 SARS-CoV 和增强免疫力廉价、可行,中医临床也证实 天然药物治疗 SARS-CoV 的有效性;天然产物包括药用植物、真菌和海洋生物的活性成分,可能成为 SARS- CoV-2 抑制剂研究与开发的新前沿。     

Identification of encoding proteins related to SARS-CoV

By sampling I00 encoding proteins from SARS-coronavirus (SARS-CoV, NC 004718) and other six coronaviruses and selecting 23 variables through stepwise multiple regression (SMR) from 172 variables, the multiple linear regression (MLR) model was established with good results of the quantitative modelling correlation coefficient R^2=0.645 and the cross-validation correlation coefficient Rcv^2=0.375. After removing 4 outliers, the quantitative modelling and cross-validation correlation coefficients were R^2=0.743 and Rcv^2=0.543, respectively.     

A Coordination Model of Gene Sequences for SARS-CoV and its Receptor

IntroductionFrom the emergence of SARS CoV, manystudies havebeen done on its biological medicine aspects, such aspathogeny characteristics, mechanisms of causing disease,clinic diagnosis and treatment, bacterin development andthe spreading rules. At the level of molecule biology,studies have been done on its genome sequences characteristics, structures and functions of the translatedproteins, and the evolution relationships in sequences[1 5].The caus…     

Identification of protein superfamily from structurebased sequence motif

The structure-based sequence motif of the distant proteins in evolution, protein tyrosine phosphatases (PTP) Ⅰ and Ⅱ superfamilies, as an example, has been defined by the structural comparison, structure-based sequence alignment and analyses on substitution patterns of residues in common sequence conserved regions. And the phosphatases Ⅰ and Ⅱ can be correctly identified together by the structure-based PTP sequence motif from SWISS-PROT and TrEBML databases. The results show that the correct rates of identification are over 98%. This is the first time to identify PTP Ⅰ and Ⅱ together by this motif.     

Molecular phylogeny of coronaviruses including human SARS-CoV

The outbreak of SARS sets an urgent task to reveal the origin of human SARS-CoV, i.e. its relation to other known species of coronavirus, and to trace the genetic variation in the spreading process of SARS. Partial answer to the problem may be obtained from phylogenetic analy-sis of available genomes. We call a phylogenetic tree of different species of coronavirus including the human SARS-Cov a CoV Tree and that of different isolates of SARS-CoV a SARS Tree. CoV trees have been c…     

The longest ultraconserved sequences and evolution of vertebrate mitochondrial genomes

ULTRACONSERVED SEQUENCES (ULTRACONSERVED, 100% IDENTITY WITH NO INSERTIONS OR DELETIONS) AND SMALL RNA IN GENOMES OFTEN PLAY A SPECIAL ROLE IN LIFE AND EVOLU- TION OF MANY SPECIES[1-4]. WE COMPARED 753 GENOMESOF BACTERIA, ARCHAEA, AND MITOCHONDRIA (MORE T…     

Genome sequence variation analysis of two SARS coronavirus isolates after passage in Vero cell culture

SARS coronavirus is an RNA virus whose replication is error-prone, which provides possibility for escape of host defenses, and even leads to evolution of new viral strains during the passage or the transmission. Lots of variations have been detected among different SARS-CoV strains. And a study on these variations is helpful for development of efficient vaccine. Moreover, the test of nucleic acid characterization and genetic stability of SARS-CoV is important in the research of inactivated vaccine. The whole genome sequences of two SARS coronavirus strains after passage in Vero cell culture were determined and were compared with those of early passages, respectively. Results showed that both SAPS coronavirus strains have high genetic stability, although nearly 10 generations were passed. Four nucleotide variations were observed between the second passage and the llth passage of Sinol strain for identification of SARS inactivated vaccine. Moreover, only one nucleotide was different between the third passage and the 10th passage of Sino3 strain for SARS inactivated vaccine. Therefore, this study suggested it was possible to develop inactivated vaccine against SARS-CoV in the future.     

Identification of true EST alignments and exon regions of gene sequences

Expressed sequence tags (ESTs), which have piled up considerably so far, provide a valuable resource for finding new genes, disease-relevant genes, and for recognizing alternative splicing variants, SNP sites, etc. The prerequisite for carrying out these researches is to correctly ascertain the gene-sequence-related ESTs. Based on analysis of the alignment results between some known gene sequences and ESTs in public database, several measures including Identity Check, Gap Check, Inclusion Check and Length Check have been introduced to judge whether an EST alignment is related to a gene sequence or not. A computational program EDSAcl.0 has been developed to identify true EST alignments and exon regions of query gene sequences. When tested with human gene sequences in the standard dataset HMR195 and evaluated with the standard measures of gene prediction performance, EDSAel.0 can identify proteincoding regions with specificity of 0.997 and sensitivity of 0.88 at the nucleotide level, which outperform that of the counterpart TAP. A web server of EDSAcl.0 is available at http://infosci.hust.edu.cn.     

Endocasts—the direct evidence and recent advances in the study of human brain evolution

Brain evolution is one of the most important aspects of human evolution,usually studied through endocasts.Analysis of fossil hominid endocasts allows inferences on functional anatomy,physiology,and phylogeny.In this paper,we describe the general features of endocast studies and review some of the major topics in paleoneurology.These are:absolute and relative brain size evolution;brain shape variation;brain asymmetry and lateralization;middle meningeal vessels and venous sinuses;application of computed tomography and virtual imaging;the history of Chinese brain endocast studies.In particular,this review emphasizes endocast studies on Chinese hominin fossils.     

New strategies of protein engineering——directed evolution of enzyme in vitro

New and highly effective strategies for directed enzyme evolution in vitro have been developed in the protein engineering field. They allow engineering all kinds of enzymes in vitro so that new ones with novel functions and features can be obtained by the methods of error-prone PCR, DNA shuffling (exon shuffling), hybrid enzyme, random-priming in vitro recombination (RPR), stagger extension process (StEP), random-directed mutagenesis in vitro, etc., even with little knowlodge of spatial structure and catalytic mechanism of enzymes in advance. The process that would take millions of years in nature can in principle be accomplished in the test within several years.     

mRNA序列与蛋白序列对比

生物序列的对比是计算生物学中的一个基本问题．目前已有许多算法对DNA序列或蛋白序列之间进行对比,多是对同种生物序列进行对比．为得到mRNA序列和蛋白序列之间的对比,采用动态规划算法,提供了寻求mRNA序列和蛋白序列的局部对比和全局对比,解决了核酸与氨基酸之间的对比问题．算法的时间复杂度为O(nm)．     

Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 1 5-mer phage-displayed library

Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study, we cloned, expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then, the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly, all of the 89 clones from monoclonal ELISA were positive （S/N〉2.1） and the result was further confirmed experimentally once again. Six N protein-binding peptides, designated separately as SNA1, SNA2, SNA4, SNA5, SNA9 and SNG11, were selected for sequencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4, SNA2, SNA9 and SNA1. In addition, the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also, the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program, and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur subunits （UCRI or UQCR）, otherwise known as the Rieske iron-sulfur proteins （RISP）. Notablely, in the [2Fe-2S] redox centre of UCRI, there were 6 residues [GGW（Y）F（Y）CP] compatible to the residues （position 2→7, GGWFCP7） of the NH2-terminal of the 15-mer peptide, which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here, the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.