共查询到20条相似文献,搜索用时 15 毫秒
1.
The C-terminal portion of the nucleocapsid protein demonstrates SARS-CoV antigenicity 总被引:3,自引:0,他引:3
Liu G Hu S Hu Y Chen P Yin J Wen J Wang J Lin L Liu J You B Yin Y Li S Wang H Ren Y Ji J Zhao X Sun Y Zhang X Fang J Wang J Liu S Yu J Zhu H Yang H 《基因组蛋白质组与生物信息学报(英文版)》2003,1(3):193-197
In order to develop clinical diagnostic tools for rapid detection of SARS-CoV (severe acute respiratory syndrome-associated coronavirus) and to identify candidate proteins for vaccine development, the C-terminal portion of the nucleocapsid (NC) gene was amplified using RT-PCR from the SARS-CoV genome, cloned into a yeast expression vector (pEGH), and expressed as a glutathione S-transferase (GST) and Hisx6 double-tagged fusion protein under the control of an inducible promoter. Western analysis on the purified protein confirmed the expression and purification of the NC fusion proteins from yeast. To determine its antigenicity, the fusion protein was challenged with serum samples from SARS patients and normal controls. The NC fusion protein demonstrated high antigenicity with high specificity, and therefore, it should have great potential in designing clinical diagnostic tools and provide useful information for vaccine development. 相似文献
2.
The epitope study on the SARS-CoV nucleocapsid protein 总被引:6,自引:0,他引:6
Li S Lin L Wang H Yin J Ren Y Zhao Z Wen J Zhou C Zhang X Li X Wang J Zhou Z Liu J Shao J Lei T Fang J Xu N Liu S 《基因组蛋白质组与生物信息学报(英文版)》2003,1(3):198-206
The nucleocapsid protein (N protein) has been found to be an antigenic protein in a number of coronaviruses. Whether the N protein in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is antigenic remains to be elucidated. Using Western blot and Enzyme-linked Immunosorbent Assay (ELISA), the recombinant N proteins and the synthesized peptides derived from the N protein were screened in sera from SARS patients. All patient sera in this study displayed strong positive immunoreactivities against the recombinant N proteins, whereas normal sera gave negative immunoresponses to these proteins, indicating that the N protein of SARS-CoV is an antigenic protein. Furthermore, the epitope sites in the N protein were determined by competition experiments, in which the recombinant proteins or the synthesized peptides competed against the SARS-CoV proteins to bind to the antibodies raised in SARS sera. One epitope site located at the C-terminus was confirmed as the most antigenic region in this prot 相似文献
3.
Ren Y Zhou Z Liu J Lin L Li S Wang H Xia J Zhao Z Wen J Zhou C Wang J Yin J Xu N Liu S 《基因组蛋白质组与生物信息学报(英文版)》2003,1(3):207-215
In the face of the worldwide threat of severe acute respiratory syndrome (SARS) to human life, some of the most urgent challenges are to develop fast and accurate analytical methods for early diagnosis of this disease as well as to create a safe anti-viral vaccine for prevention. To these ends, we investigated the antigenicity of the spike protein (S protein), a major structural protein in the SARS-coronavirus (SARS-CoV). Based upon the theoretical analysis for hydrophobicity of the S protein, 18 peptides were synthesized. Using Enzyme-Linked Immunosorbent Assay (ELISA),these peptides were screened in the sera from SARS patients. According to these results, two fragments of the S gene were amplified by PCR and cloned into pET-32a. Both S fragments were expressed in the BL-21 strain and further purified with an affinity chromatography. These recombinant S fragments were confirmed to have positive cross-reactions with SARS sera, either by Western blot or by ELISA. Our results demonstrated that the potenti 相似文献
4.
Hu Y Wen J Tang L Zhang H Zhang X Li Y Wang J Han Y Li G Shi J Tian X Jiang F Zhao X Wang J Liu S Zeng C Wang J Yang H 《基因组蛋白质组与生物信息学报(英文版)》2003,1(2):118-130
We studied structural and immunological properties of the SARS-CoV M (membrane) protein, based on comparative analyses of sequence features, phylogenetic investigation, and experimental results. The M protein is predicted to contain a triple-spanning transmembrane (TM) region, a single N-glycosylation site near its N-terminus that is in the exterior of the virion, and a long C-terminal region in the interior. The M protein harbors a higher substitution rate (0.6% correlated to its size) among viral open reading frames (ORFs) from published data. The four substitutions detected in the M protein, which cause non-synonymous changes, can be classified into three types. One of them results in changes of pI (isoelectric point) and charge, affecting antigenicity. The second changes hydrophobicity of the TM region, and the third one relates to hydrophilicity of the interior structure. Phylogenetic tree building based on the variations of the M protein appears to support the non-human origin of SARS-CoV. To inve 相似文献
5.
6.
Pedro Serrano Margaret A. Johnson Amarnath Chatterjee Bill Pedrini Kurt Wüthrich 《Biomolecular NMR assignments》2008,2(2):135-138
Sequence-specific NMR assignments of the globular core comprising the residues 1066–1181 within the non-structural protein
nsp3e from the SARS coronavirus have been obtained using triple-resonance NMR experiments with the uniformly [13C, 15N]-labeled protein. The backbone and side chain assignments are nearly complete, providing the basis for the ongoing NMR structure
determination. A preliminary identification of regular secondary structures has been derived from the 13C chemical shifts. 相似文献
7.
Expression, purification and sublocalization of SARS-CoV nucleocapsid protein in insect cells 总被引:2,自引:0,他引:2
Ren AX Xie YH Kong YY Yang GZ Zhang YZ Wang Y Wu XF 《Acta biochimica et biophysica Sinica》2004,36(11):754-758
The causative agent of severe acute respiratory syndrome (SARS) is a previously unidentified coronavirus, SARS-CoV. The nucleocapsid (N) protein of SARS-CoV is a major viral protein recognized by acute and early convalescent sera from SARS patients. To facilitate the studies on the function and structure of the N protein, this report describe the expression and purification of recombinant SARS-CoV N protein using the baculovirus 相似文献
8.
The E protein is a multifunctional membrane protein of SARS-CoV 总被引:1,自引:0,他引:1
Wu Q Zhang Y Lü H Wang J He X Liu Y Ye C Lin W Hu J Ji J Xu J Ye J Hu Y Chen W Li S Wang J Wang J Bi S Yang H 《基因组蛋白质组与生物信息学报(英文版)》2003,1(2):131-144
The E (envelope) protein is the smallest structural protein in all coronaviruses and is the only viral structural protein in which no variation has been detected. We conducted genome sequencing and phylogenetic analyses of SARS-CoV. Based on genome sequencing, we predicted the E protein is a transmembrane (TM) protein characterized by a TM region with strong hydrophobicity and α-helix conformation. We identified a segment (NH2-_L-Cys-A-Y-Cys-Cys-N_-COOH) in the carboxyl-terminal region of the E protein that appears to form three disulfide bonds with another segment of corresponding cysteines in the carboxyl-terminus of the S (spike) protein. These bonds point to a possible structural association between the E and S proteins. Our phylogenetic analyses of the E protein sequences in all published coronaviruses place SARS-CoV in an independent group in Coronaviridae and suggest a non-human animal origin. 相似文献
9.
10.
11.
12.
Yi Zhang Wei Wang Jin-rong Gao Li Ye Xiao-nan Fang Ying-chun Zeng Zheng-hui Wu Ying-long She Lin-bai Ye 《中国病毒学》2007,22(1):1-7
SARS-CoV is a newly discovery pathogen causing severe acute respiratory problems. It has been established that the S protein
in this pathogen plays an important rule in the adsorption and penetration of SARS-CoV into the host cell by interaction with
the ACE2 receptor. To determinant which functional motif of the S protein was involved in the interaction with ACE2, seven
truncated S proteins deleted from the N or C terminal were obtained by an E.coli expression system and purified by column
chromatography to homogeneity. Each truncated S protein was fixed on to the well of an ELISA plate and an interaction was
initiated with the ACE2 protein. The adsorption were quantified by ELISA, and the results indicated that amino acids from
388 to 496 of the S protein was responsible for the interaction with the ACE2 receptor, and the interaction could be completely
disrupted by an antibody specific to these amino acids. Deletions adjacent to this domain did not appear to have a significant
impact on the interaction with ACE2, suggesting that the S protein of SARS-CoV could be developed as a vaccine to prevent
the spread of SARS-CoV. 相似文献
13.
14.
15.
The dimeric interface of severe acute respiratory syndrome coronavirus main protease is a potential target for the anti-SARS drug development. We have generated C-terminal truncated mutants by serial truncations. The quaternary structure of the enzyme was analyzed using both sedimentation velocity and sedimentation equilibrium analytical ultracentrifugation. Global analysis of the combined results showed that truncation of C-terminus from 306 to 300 had no appreciable effect on the quaternary structure, and the enzyme remained catalytically active. However, further deletion of Gln-299 or Arg-298 drastically decreased the enzyme activity to 1-2% of wild type (WT), and the major form was a monomeric one. Detailed analysis of the point mutants of these two amino acid residues and their nearby hydrogen bond partner Ser-123 and Ser-139 revealed a strong correlation between the enzyme activity loss and dimer dissociation. 相似文献
16.
The SARS-CoV S glycoprotein: expression and functional characterization 总被引:36,自引:0,他引:36
Xiao X Chakraborti S Dimitrov AS Gramatikoff K Dimitrov DS 《Biochemical and biophysical research communications》2003,312(4):1159-1164
We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, S(e), were not cleaved to any significant degree. They ran at about 180-200kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17-537 and 272-537 but not 17-276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450-454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors. 相似文献
17.
18.
SARS-CoV是引起严重急性呼吸道综合症(SARS)的病原体.更多地了解SARS-CoV的基因组、蛋白结构以及它与其它冠状病毒的关系,将有助于SARS疾病的防治. 相似文献
19.
Shen S Lin PS Chao YC Zhang A Yang X Lim SG Hong W Tan YJ 《Biochemical and biophysical research communications》2005,330(1):286-292
The severe acute respiratory syndrome coronavirus (SARS-CoV) 3a protein is one of the opening reading frames in the viral genome with no homologue in other known coronaviruses. Expression of the 3a protein has been demonstrated during both in vitro and in vivo infection. Here we present biochemical data to show that 3a is a novel coronavirus structural protein. 3a was detected in virions purified from SARS-CoV infected Vero E6 cells although two truncated products were present predominantly instead of the full-length protein. In Vero E6 cells transiently transfected with a cDNA construct for expressing 3a, a similar cleavage was observed. Furthermore, co-expression of 3a, membrane and envelope proteins using the baculovirus system showed that both full-length and truncated 3a can be assembled into virus-like particles. This is the first report that demonstrated the incorporation of 3a into virion and showed that the SARS-CoV encodes a novel coronavirus structural protein. 相似文献
20.
SARS-Cov及其他冠状病毒基因组比较分析 总被引:7,自引:0,他引:7
摘要:对病毒种内和种间基因组的比较分析能获得很多关于病毒起源与演化的信息。对17株SARS-CoV的种内基因组变异分析发现共有137个变异位点,估算出SARS-CoV的突变率为8.04×10-3核苷酸替换/位点/年。变异位点在基因组上的分布不均匀,变异位点最多的是基因组中编码S1蛋白的区域,而在编码依赖于RNA的RNA聚合酶区域中几乎没有变异位点。核苷酸和氨基酸替换的偏性预示变异可能不仅仅是由随机漂变产生。对冠状病毒种间基因组结构比较分析发现,SARS-CoV的基因组结构与IBV很相似;而保守基因系统发育分析表明,SARS-CoV属于冠状病毒的一个新分支,并且与血清型第二组冠状病毒进化关系较近。对其他某些分子特征的分析发现,在不同的方面SARS-CoV和不同组冠状病毒有不同的相似点。进一步对基因组非保守开放阅读框(ORF)的基序(motif)和跨膜区分析发现,各组冠状病毒基因组中位于基因S-E间的非保守ORF可能是同源的,但不是绝对必要的;而IBV和SARS-CoV的基因组中位于基因M-N间ORF可能不是同源的。综合分析SARS-CoV与3组血清型冠状病毒进化关系、宿主分布,以及SARS-CoV和IBV的s2m的进化关系,可以推测SARS-CoV有可能来自禽类。
Abstract:The genome comparison of inter-species and intra-species can give us much information about the origin and evolution of viruses.There are 137 mutation sites in the 17 genomes of SARS-CoV,and the mutation rate is about 8.04×10-3 substitution/site/year.The distribution of the segregating sites is not steady,the most variable region appears in S1 protein,and the nucleotide sequence of RNA-dependent RNA polymerase has very few mutation sites.The substitution bias of nucleotide acids and amino acids indicates the non-random drift products.The comparison of genome structures of SARS-CoV and other coronaviruses shows that SARS-CoV and IBV share the same genome structure.Phylogenetic analyses of conserved genes of coronaviruses indicate that SARS-CoV is a new branch of coronaviruses and appears more close to the group II coronaviruses.Interestingly,SARS-CoV shares some different features with different groups of coronaviruses.Additional analyses show that the first ORFs between S and E genes of some coronaviruses are transmembrane proteins and share the common motif,indicating the possible common ancestor.From the host distribution of different groups of coronaviruses and the phylogeny of s2m,we can deduce that avian is the probable natural host of SARS-CoV. 相似文献