The full length cDNA of SARS coronavirus nucleocapsid (N) protein was amplified by PCR and cloned into yeast expression vector pPIC3.5K to generate expression vector pPIC3.5K-SCoVN. The plasmid was linearized and then transformed into P. pastoris (His- Mut+) by electroporation method. His+ Mut+ recombinant strains were screened on G418-RDB and MM/MD plates, and further confirmed by PCR. The influence of various inducing media, dissolved oxygen(DO) and the different final concentration of methanol was subsequently investigated. The results showed that the FBS medium was optimal for recombinant N protein expression and growth of the recombinant strain. The optimal final concentration of methanol is 1% (V/V), and the DO has a significant effect on recombinant N protein expression and growth of recombinant strain. The recombinant N protein expressed was about 6% of the total cell proteins, 410 mg/L of recombinant N protein and 45 OD600 were achieved in shake flask. Western-blot showed that the recombinant N protein had high specificity against mouse-anti-N protein-mAb and SARS positive sera, but had no cross-reaction with normal human sera. The result of scale-up culture in fermemtator demonstrated that 2.5g/L of recombinant N protein and the maximum cell 345 OD600 of were achieved, which was 6.1 times and 7.7 times higher than that in shake flask. So this study provide a basis for further researches on the early diagnosis of SARS and the virus reproduction and pathology reaction of SARS coronavirus.
Cloning, Molecular ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; SARS Virus ; genetics

A novel human ScFv H12 against SARS-CoV has been selected from a SARS immune library. In order to produce a large amount of ScFv H12, pET28a-H12 expression vector was constructed and ScFv H12 was expressed at yield about 30% of total proteins in E. coli . Here two different refolding procedures were used to refold ScFv H12 from inclusion body: gel filtration chromatography and dilution. The results showed that ScFv H12 could be efficiently refolded by both procedures. However, the refolding via gel filtration was 1.5 time more effective than that of dilution. The affinity of ScFv H12 to SARS-CoV virion was detected as Kd = 73.5 nmol/mL.
Antibodies, Monoclonal ; biosynthesis ; genetics ; Antibodies, Viral ; immunology ; Escherichia coli ; genetics ; metabolism ; Humans ; Immunoglobulin Fragments ; biosynthesis ; genetics ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Inclusion Bodies ; genetics ; immunology ; Protein Renaturation ; Recombinant Proteins ; biosynthesis ; chemistry ; immunology ; SARS Virus ; immunology

The aim of this study is to construct a SARS-CoV S protein-specific phage displayed antigen library for the epitope characterization of anti-S monoclonal antibodies (mAbs). First, the full-length gene of SARS-S protein was PCR amplified, purified and then digested with DNase I to obtain DNA fragments in the size range of 50-500 bp. The resulting fragments were blunt-end ligated to the modified phage display vector pComb3XSS. The reactions were electrotransformed into XL1-Blue and infected with VCSM13 helper phage. The SARS-CoV S protein-specific phage displayed antigen library was biopanned and screened against two anti-S mAbs, S-M1 and S-M2. The results showed that we successfully constructed the phage displayed antigen library with a size of 5.7 x 10(6). After three-rounds of biopanning, 14 positive phage clones for S-M1 and 15 for S-M2 were respectively identified. Sequence analyses revealed the possible epitopes of two mAbs. Therefore, the S protein-specific phage displayed antigen library provides a crucial platform for the epitope characterization of anti-S antibodies and it is highly valuable for development of SARS vaccines and diagnostics.
Antibodies, Viral ; immunology ; Bacteriophages ; genetics ; metabolism ; Epitopes ; genetics ; immunology ; Humans ; Membrane Glycoproteins ; genetics ; immunology ; Peptide Library ; SARS Virus ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Spike Glycoprotein, Coronavirus ; Viral Envelope Proteins ; genetics ; immunology

Severe acute respiratory syndrome (SARS) is a life-threatening disease for which accurate diagnosis is essential. Although many tools have been developed for the diagnosis of SARS, false-positive reactions in negative sera may occur because of cross-reactivity with other coronaviruses. We have raised polyclonal and monoclonal antibodies (Abs) using a recombinant form of the SARS virus nucleocapsid protein. Cross-reactivity of these anti-SARS Abs against human coronavirus (HCoV) 229E and HCoV OC43 were determined by Western blotting. The Abs produced reacted with recombinant SARS virus nucleocapsid protein, but not with HCoV 229E or HCoV OC43.
Antibodies, Viral/*immunology ; Blotting, Western ; Coronavirus 229E, Human/*immunology ; Coronavirus OC43, Human/*immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins/genetics/*immunology ; Recombinant Proteins/immunology ; SARS Virus/genetics/*immunology ; Severe Acute Respiratory Syndrome/diagnosis/*immunology

BACKGROUNDSevere acute respiratory syndrome (SARS) is an infectious disease caused by SARS-CoV. There are no effective antiviral drugs for SARS although the epidemic of SARS was controlled. The aim of this study was to develop an RNAi (RNA interference) approach that specifically targeted the N gene sequence of severe acute respiratory syndrome associated coronavirus (SARS-CoV) by synthesizing short hairpin RNA (shRNA) in vivo, and to assess the inhibitory effect of this shRNA on SARS-CoV N antigen expression.

METHODSThe eukaryotic expression plasmid pEGFP-C1-N, containing SARS-CoV N gene, was co-transfected into 293 cells with either the RNAi plasmid pshRNA-N or unrelated control plasmid pshRNA-HBV-C4. At 24, 48 and 72 hours post transfection, the green fluorescence was observed through a fluorescence microscope. The RNA levels of SARS-CoV N were determined by reverse transcription polymerase chain reaction (RT-PCR). The expression of Green Fluorescent Protein (GFP) and protein N were detected using Western blot.

RESULTSThe vector, pshRNA-N expressing shRNA which targeted the N gene of SARS-CoV, was successfully constructed. The introduction of RNAi plasmid efficiently and specifically inhibited the synthesis of protein N. RT-PCR showed that RNAs of N gene were clearly reduced when the pEGFP-C1-N was cotransfected with pshRNA-N, whereas the control vector did not exhibit inhibitory effect on N gene transcription.

CONCLUSIONSOur results demonstrate that RNAi mediated silencing of SARS-CoV gene could effectively inhibit expression of SARS-CoV antigen, hence RNAi based strategy should be further explored as a more efficacious antiviral therapy of SARS-CoV infection.

Antigens, Viral ; genetics ; Cells, Cultured ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Nucleocapsid Proteins ; antagonists & inhibitors ; genetics ; RNA Interference ; SARS Virus ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; therapy

OBJECTIVEAngiotensin-converting enzyme 2 (ACE-2) has been identified as a functional receptor of severe acute respiratory syndrome coronavirus (SARS-CoV), so its gene was cloned and eukaryotic expressed for further insight into mechanisms in SARS-CoV entry and pathogenesis, as well as development of a safe and reliable neutralization assay for SARS-CoV.

METHODSTotal RNA was extracted from right atrial tissue of a patient with right heart failure resected during a valvular replacement surgery by Trizol one-step method, and the full-length ACE-2 encoding gene was acquired by RT-nested-PCR. The ACE-2 encoding gene was then cloned into pcDNA4/HisMax-TOPO eukaryotic expression vector to construct the recombinant plasmid pcDNA4/ ACE-2, which was then transfected into 293 T cell and ACE-2 eukaryotic transient expression was detected by Western Blot. Syncytia inhibition assay was established to detect SARS-CoV neutralizing antibody, and compared parallelly with SARS pseudovirus neutralization assay.

RESULTSThe recombinant plasmid pcDNA4/ ACE-2 could express ACE-2 protein in eukaryotic cells and induce cell-cell fusion between S protein- and ACE2-expressing cells. This cell-cell fusion assay could be used to detect SARS-CoV neutralizing antibody.

CONCLUSIONSARS-CoV receptor ACE-2 gene was successfully cloned and eukaryotic expressed, and used to establish syncytia inhibition assay for SARS-CoV neutralizing antibody assay.

Animals ; Blotting, Western ; Cell Line ; Cercopithecus aethiops ; Cloning, Molecular ; Gene Expression ; Humans ; Neutralization Tests ; Peptidyl-Dipeptidase A ; genetics ; immunology ; metabolism ; Plasmids ; genetics ; Receptors, Virus ; genetics ; immunology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; SARS Virus ; immunology ; metabolism ; Transfection ; Vero Cells

In order to develop clinical diagnostic tools for rapid detection of the 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.
Antigens, Viral ; immunology ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Genetic Vectors ; Genome, Viral ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism ; SARS Virus ; genetics ; immunology ; Yeasts ; genetics

Orf 9b was amplified by PCR from SARS-CoV genome and cloned into the Nco I and Bam HI sites of the pET32c expression vector, and then recombinant plasmid pET32c-Orf 9b was constructed. The recombinant fusion protein Orf 9b was expressed by IPTG induction and purifed. After being cleaved by rEK, Orf 9b protein with MW 11 kD was separated and collected. It was demonstrated by ELISA that the purified Orf 9b protein could react with sera of SARS rehabilitaion patients but not with sera from healthy donors. CD and Infrared spectroscopy were used to predict the secondary structure of the purified Orf 9b protein. The distribution percentages for the the secondary structures of alpha-helix, beta-sheet, and random coil in the Orf 9b protein estimated by CD were 12.5%, 40%, and 47.5%, respectively, while the same parameters estimated by Infrared spectroscopy were 13.7%, 47.5%, and 37.9%, respectively. The results obtained by the two methods were substantially identical and showed that the structure of the Orf 9b protein consisted mostly of beta-sheet, and comprised only few alpha-helix. The acquisition of purified protein and the structural information presented herein may provide foundation for further functional study.
Antibodies, Viral ; immunology ; Cloning, Molecular ; Gene Expression ; Humans ; Open Reading Frames ; Protein Structure, Secondary ; SARS Virus ; chemistry ; genetics ; immunology ; Severe Acute Respiratory Syndrome ; immunology ; virology ; Viral Proteins ; chemistry ; genetics ; immunology

A recombinant vaccinia virus (rWR-SARS-S)expressing spike protein of severe acute respiratory syndrome coronavirus was constructed. The expression of full length recombinant SARS spike protein (rSS) in HeLa cells possessing specific reaction ability to chicken anti-sera was confirmed by SDS-PAGE and Western-blot (190 kD). HeLa cells infected with rWR-SARS-S also showed high sensitivity in detecting specific serum antibody by indirect immunofluoresence assay (IFA). The results above indicated that the availability of such a faithful model system offers particular advantages for the study of SARS in that it reduces the need for direct manipulation of an exotic pathogen. In the absence of infectious SARS, we may safely carry out detailed biochemical and genetic manipulations to investigate features of viral replication and gene function, as well as explore new avenue for vaccine development.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; immunology ; Blotting, Western ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Fluorescent Antibody Technique, Indirect ; Gene Expression ; HeLa Cells ; Humans ; Immune Sera ; immunology ; Immunization ; methods ; Membrane Glycoproteins ; genetics ; immunology ; Mice ; Recombinant Proteins ; immunology ; SARS Virus ; genetics ; immunology ; Spike Glycoprotein, Coronavirus ; Vaccinia virus ; genetics ; Viral Envelope Proteins ; genetics ; immunology

OBJECTIVETo investigate the exact mechanism of SARS-CoV pathogenesis at the protein level.

METHODUnder the condition of the establishment of dendrtic cells (DC) culture method, we used recombinant adenovirus to infect mature DC to make clear the development changes in mRNA levels and secreted protein levels of proinflammatory cytokines of IL-1beta, IL-6, IL-8 and TNF-alpha by using RT-PCR and ELISA.

RESULTWe found that mRNA levels and secreted protein levels of IL-6 and TNF-alpha in DC had increased gradually after rAd-N infection during first 24 h compared with the control DC infected by rAd-LacZ (P < 0.05 or P < 0.01).

CONCLUSIONIt is suggested that N protein may be related to the excessive secretion of proinflammatory cytokines during SARS-CoV infection at the acute phase.

Adenoviridae ; genetics ; immunology ; Animals ; Cercopithecus aethiops ; Cytokines ; genetics ; secretion ; Dendritic Cells ; metabolism ; virology ; Enzyme-Linked Immunosorbent Assay ; Interleukin-6 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; SARS Virus ; genetics ; immunology ; metabolism ; Severe Acute Respiratory Syndrome ; metabolism ; Tumor Necrosis Factor-alpha ; genetics ; metabolism