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

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.
Antibodies, Monoclonal/*immunology ; Antigens, Viral/chemistry/*immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Epitopes/immunology ; Nucleocapsid Proteins/chemistry/*immunology ; Rinderpest virus/*immunology

This article aimed to study the antigenicity of nucleocapsid proteins (NPs) in six pathogenic phleboviruses and to provide theoretical evidence for the development of serological diagnostic reagents. NPs of six pathogenic phleboviruses were expressed and purified using a prokaryotic expression system and rabbits were immunized with individual recombinant NPs. Cross-reactions among NPs and rabbit sera were determined by both indirect ELISA and Western blotting analyses, and the sera titer was determined by indirect ELISA. Furthermore, sera from SFTS patients were also detected by each recombinant NP as a coating antigen using indirect ELISA. The cross-reactions and the sera titer were subsequently determined. Both the concentration and purity of recombinant NPs of six pathogenic phleboviruses met the standards for immunization and detection. The results of indirect ELISA and Western blotting showed that each anti-phlebovirus NP rabbit immune serum had potential serological cross-reactivity with the other five virus NP antigens. Furthermore, the sera from SFTS patients also had cross-reactivity with the other five NP antigens to a certain extent. Our preliminary study evaluated the antigenicity and immune reactivity of six pathogenic phleboviruses NPs and laid the foundation for the development of diagnostic reagents.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Cross Reactions ; Humans ; Nucleocapsid Proteins ; genetics ; immunology ; Phlebotomus Fever ; diagnosis ; immunology ; virology ; Phlebovirus ; classification ; genetics ; immunology ; isolation & purification ; Rabbits

OBJECTIVETo investigate the virulence characteristics of two fixed strains (CTN and aG) and a street strain (HN10) of rabies viruses isolated in China.

METHODSICR mice of different age groups were inoculated with CTN, aG and HN10 rabies virus strains via the intracracerebral (i.c.) or intramuscular (i.m.) routes, and observed for 20 days.

RESULTSThe CTN strain was pathogenic to 7-day-old suckling mice that received i.c. inoculations and 3-day-old suckling mice that received i.m. inoculations. The aG strain was pathogenic to 4-week-old mice that received i.c. inoculations and 7-day-old suckling mice that received i.m. inoculations. The HN10 strain was pathogenic to mice of all age groups via both inoculation routes. In moribund mice, the viruses had spread to most regions of the brain. The CTN and HN10 strains had similar dissemination patterns in the brain; both viral antigens could be found in the dentate gyrus (DG), whereas few viral antigens were present in the DG from specimens that had been infected with the aG strain.

CONCLUSIONA comprehensive sequence analysis of the G protein suggested that differences in gene sequences may be responsible for producing strain-specific differences in pathogenicity and distribution in the brain.

Animals ; Antigens, Viral ; immunology ; Brain ; immunology ; virology ; China ; Mice ; Mice, Inbred ICR ; Nucleocapsid Proteins ; immunology ; Rabies ; virology ; Rabies virus ; immunology ; isolation & purification ; pathogenicity

OBJECTIVETo obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.

METHODSBALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.

RESULTSAfter cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.

CONCLUSIONThe anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Female ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; isolation & purification ; SARS Virus ; chemistry ; 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

OBJECTIVETo develop and evaluate a multiplex detection of IgM antibodies to pathogens caused viral hemorrhagic fever.

METHODSThe nucleocapsid proteins (NP) of HTN, SEO, Puu MBV, Lassa, RFV and HPS viruses expressed in prokaryotic cells and purified were coupled to 7 different xMAP fluorescent microbeads. The assay was evaluated and optimized when screened against a panel of reference sera collected from HFRS patients, and compared to commonly used MacELISA Kits.

RESULTSFor detection of anti-NP antibodies, the sensitivity and specificity of the assay were comparable to a commonly used MacELISA kit, but it could detect different antigen specific antibodies in one reaction simultaneously.

CONCLUSIONA robust, rapid and multiplex assay based on NPs could be developed via Luminex xMAP platform for laboratory diagnosis of viral hemorrhagic fever and seroepidemiological investigation.

Antibodies, Viral ; blood ; Fluorescence ; Hemorrhagic Fevers, Viral ; blood ; immunology ; virology ; Humans ; Immunoassay ; methods ; Immunoglobulin M ; blood ; Microspheres ; Nucleocapsid Proteins ; chemistry ; immunology ; Viruses ; immunology

To evaluate the effectiveness of rabies vaccination, we developed the SPA-ELISA method to detect the antibodies against rabies virus (RV) using the main antigenic determinant of nucleoprotein (RV N1) as antigen. The complete Nucleoprotein (N) gene and the partial N1 gene (1 000-1 353 bp) of RV Flury LEP strain were amplified using RT-PCR and PCR approaches. The two fragments were inserted into pGEX-6P-1 respectively. Then we transformed the recombinant plasmids into Escherichia coli BL21(DE3) strain and expressed them by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). SDS-PAGE analysis showed that both of the two recombinant proteins were presented as inclusion bodies. Compared with the complete nucleoprotein, the partial protein (RV N1) was expressed at a much higher level in E. coli BL21(DE3). The antigenic specificity of the partial N1 protein was confirmed by Western blotting. By coating the plates with purified RV N1 as an antigen, an SPA-ELISA method for the detection of the antibodies against RV was established. By optimizing this method, the optimal concentration of RV N1 coating the ELISA plate was 2 mg/L. The optimal concentration of serum samples and SPA-HRP was 1:100 and 1:4 000 respectively. Compared with a commercially available ELISA kit coating RV as antigen, the coincidence rate of SPA-ELISA was 94.1%. Our results show that the developed SPA-ELISA based on the RV N1 was useful for the detection of the antibodies against RV in the sera of dogs.
Animals ; Antibodies, Viral ; analysis ; immunology ; Dogs ; Enzyme-Linked Immunosorbent Assay ; methods ; Epitopes ; immunology ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Nucleocapsid Proteins ; immunology ; Rabies virus ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Staphylococcal Protein A

OBJECTIVETo prepare anti-recombinant protein antibody from immunized mice with recombinant nucleocapsid protein (NP) of human influenza A3 (IFV-A3) virus expressed in prokaryotic cell, and to explore the feasibility of utilizing anti-recombinant protein antibody to detect influenza A virus.

METHODSNP genes of human influenza A virus were analyzed with computer softwares of ClustalX, Antheprot, et al. to determine the antigenicity in conserved regions. Three different partial NP genes were harvested and cloned into pET-28(c) plasmid, the recombinant plasmids were induced to express partial NP segments in BL21 cells. The recombinant proteins were purified with Ni-agarose by affinity chromatography and immunized BALB/c mice. The polyclonal antisera harvested from mice were analyzed with Western Blot and immunohistochemistry assays to detect the reactions with IFV-A.

RESULTSThree recombinant plasmids were expressed with high yield in BL21 cells, about 15-20 mg/L. Western Blot results indicated that the three prepared antisera (1:2000) positively reacted with NP from IFV-A3-infected cells. And immunohistochemistry assays suggested that anti-NP1, anti-NP2, anti-NP3 antisera positively reacted with IFV-A3 or IFV-A1-infected MDCK cells, with titers of 1:640 to 1:1280.

CONCLUSIONThe recombinant NP of IFV-A3 would induce polyclonal antibodies with high titers in mice. The polyclonal antibodies would cross-react with IFV-A3 and IFV-A1. It is feasible to predict the antigenicity with systematical bioinformatics analyses and then induce anti-IFV antibodies with high dilutions, and it is possible to be utilized in the early detection and subtyping analyses of IFV-infections.

Animals ; Antibodies, Viral ; blood ; Antigens, Viral ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Influenza A virus ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Male ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; genetics ; immunology ; Recombinant Proteins ; genetics ; immunology

An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; Hepatitis Antigens ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Hepatitis Vaccines ; biosynthesis ; immunology