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1、Amino acids 1528 in the ectodomain of SARS coronavirus 3a protein induces neutralizing antibodies Sara A kerstro ma,1, Yee-Joo Tanb,*,1, Ali Mirazimia,* a Center for Microbiological Preparedness, Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden b Institute of Molecular and C
2、ell Biology, CAVR, 61 Biopolis Drive, Singapore 138673, Singapore Received 7 April 2006; revised 29 May 2006; accepted 1 June 2006 Available online 12 June 2006 Edited by Hans-Dieter Klenk AbstractA synthetic peptide corresponding to amino acids (aa) 1528 of the severe acute respiratory syndrome cor
3、onavirus (SARS-CoV) 3a protein was used to raise polyclonal antibodies in rabbits. This anti-3a N-terminal antibody could detect 3a pro- tein in infected cells, as did an anti-3a C-terminal antibody pre- viously described. The latter targeted the C-terminal cytoplasmic domain of 3a (aa 134274). The
4、anti-3a N-terminal antibody could detect intracellular 3a as well as 3a expressed on the cell surface. Interestingly, only the anti-3a N-terminal antibody can inhibit SARS-CoV propagation in Vero E6 culture although the binding affi nity of the anti-3a N-terminal antibody was lower than the anti-3a
5、C-terminal antibody. ? ? 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Severe acute respiratory syndrome; Coronavirus; Group-specifi c viral protein; 3a Protein; Neutralizing activities 1. Introduction The genome of the severe acute resp
6、iratory syndrome coro- navirus (SARS-CoV) encodes for eight group-specifi c proteins with no signifi cance sequence homology to viral proteins of other coronaviruses 13. The largest of these group-specifi c proteins is termed 3a and the 3a protein has also been shown to be expressed in SARS-CoV infe
7、cted cells 4,5 and could be detected in tissues obtained from SARS patients 57. Anti- bodies against 3a have also been detected in diff erent cohorts of SARS patients 811. The 3a protein consists of 274 amino acids (aa) and con- tains three putative transmembrane domains and it is ex- pressed on the
8、 cell surface 4,12. The topology of 3a on the cell surface was determined experimentally: its fi rst 34 aa, i.e. before the fi rst transmembrane domain, is facing the extracellular matrix and its C-terminal after the third transmembrane domain (i.e. aa 134274) is facing the cyto- plasm 4. As 3a is a
9、 novel coronavirus structural protein 12,13, its N-terminal ectodomain would be expected to pro- trude out of the virion. Interestingly, in two separate cohorts of SARS patients, one from Taiwan 14 and one from Hong Kong 15, B cells recognizing the N-terminal region of 3a were isolated from patients
10、. In addition, it was recently re- ported that the N terminal of 3a elicits strong and potentially protective humoral responses in infected patients 11. In this study, rabbit polyclonal antibodies targeted against the N-ter- minal ectodomain and the C-terminal cytoplasmic domain of the 3a protein we
11、re tested for their abilities to inhibit SARS- CoV propagation in Vero E6 culture. 2. Materials and methods 2.1. Cell-line and virus The Vero E6 cells and SARS-CoV isolate used in this study have been previously described 16. Culturing of 293T cells have been pre- viously described 4. 2.2. Synthesis
12、 of peptide and production of rabbit polyclonal antibodies A peptide (C)AQPVKIDNASPAST), which corresponds to amino acids 1528 of SARS-CoV 3a protein, was synthesized by BioGenes GmbH (Berlin, Germany). The peptide was conjugated to a carrier, Limulus Polyphemus Hemocyanine (LPH) from horseshoe crab
13、, and used to immunize two rabbits using standard protocols. All pro- cedures were performed by BioGenes GmbH. The immunization schedule is showed in Table 1. All the sera were tested by Western blot analysis. A rabbit polyclonal antibody raised against bacterially-expressed GST-3a (134-274aa) has b
14、een previously described 4. This antibody targets the C-terminal cytoplasmic domain of 3a and the 6th bleed was used in this study. A neutralizing antibody (rabbit anti-SD10) that targeted the SARS-CoV spike (S) protein was also used in the neutral- izing assays 17. The last bleed obtained after 16
15、immunizations was used. 2.3. Western blot analysis and immunofl uorescence experiments In order to express recombinant 3a protein in mammalian cells, Vero E6 cells were transfected with a cDNA construct (pXJ-3a) for expressing full-length 3a protein, as previously described 4. Transfected cells were
16、 then subjected to Western blot analysis and immunofl uorescence experiments as previously described 4. Briefl y, cells were grown to 80% confl uence in a 6 cm dish and transfected with 1 lg of the plasmid. The cells were harvested after ?16 h and washed with PBS and then lysed in 1 ml of lysis buff er (50 mM Tris, pH 8, 150 mM NaCl, 0.5% NP40, 0.5% deoxycholic acid, 0.005% SDS, 1 mM PMSF). After 6 rounds of alternate freezing and thawing, the cells suspension was centrifuged at 13000 rpm for 2
