OBJECTIVETo develop the monoclonal antibody against N protein of SARS virus and study its applicability.

METHODSBALB/c mice were immunized with recombinant N protein. Spleen cells were collected and infused with SP2/0 cell. The infused cells were screened for anti-N protein antibody with ELISA. The positive cells were cloned and injected into abdominal cavity. The antibodies were purified from ascites. The affinities of those purified antibodies were analyzed with ELISA. The ELISA for detection of SARS virus antigen was developed by using antibody with the highest affinity. Its sensitivity and specificity were also evaluated primarily.

RESULTSEleven monoclonal cells secreting antibody have been developed. Three of the 11 purified monoclonal antibodies had very high affinity to N protein, while 4 purified McAbs showed very weak reaction to N protein, the affinities of remaining 4 McAbs were in between. The ELISA for detection of SARS virus antigen was developed with McAb 7. Its sensitivity was about 31 PFU/ml and had no cross reaction with other respiratory viruses.

CONCLUSIONThe monoclonal antibody has good specificity and may be used to detect SARS virus antigen. However, its sensitivity is to be evaluated further with clinical samples from SARS patients, especially at acute phase.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibodies, Viral ; biosynthesis ; Antigens, Viral ; analysis ; Humans ; Hybridomas ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; SARS Virus ; immunology ; Sensitivity and Specificity

In order to research immunogenicity of the recombinant rVP2-IL-2 fusion protein, we obtained the rVP2-IL-2 fusion protein using Pichia pastoris expression system, and then evaluated its potential to induce immune responses in chicken. The effect was determined in the form of protective anti-IBDV VP2 titers, antibodies (IgG1 and IgG2a), lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and challenge experiment. Antibody titers and proliferation lymphocyte level suggested that the fusion protein could elicit specific humoral immune and cellular immune responses, antibody sub-type results indicated that the rVP2-IL-2 fusion protein induced secretion both of IgG1 and IgG2a. The seem result elicited from cytokines ELISA test, secretion of both of Th1 (gamma-IFN) and Th2 (IL-4) were induced by the rVP2-IL-2 fusion protein. Challenge experiment result shown that chicken immunized the rVP2-IL-2 fusion protein obtained 85% protection. These results confirm that the fusion protein enhances the protection against IBDV through both humoral and cell-mediated immunity, and thus could serve as a candidate for the development of IBDV subunit vaccine.
Animals ; Antibodies, Viral ; biosynthesis ; blood ; Chickens ; immunology ; Immunoglobulin G ; blood ; Interleukin-2 ; biosynthesis ; genetics ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Th1 Cells ; immunology ; Th2 Cells ; immunology ; Vaccines, Subunit ; immunology ; Viral Structural Proteins ; biosynthesis ; genetics ; Viral Vaccines ; immunology

Animals ; Antibodies, Neutralizing/biosynthesis* ; Antibodies, Viral/biosynthesis* ; Body Weight/immunology* ; COVID-19/virology* ; Disease Models, Animal ; Disease Progression ; Humans ; Immunohistochemistry ; Lung/virology* ; Male ; Mesocricetus ; Nasal Cavity/virology* ; RNA, Viral/immunology* ; SARS-CoV-2/pathogenicity* ; Severity of Illness Index ; Viral Load

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

Citrus tristeza virus (CTV) was purified from a citrus sample by a modified protocol, and the yield was about 1 mg from 100 g citrus tissues. Polyclonal antibody was prepared by immunizing rabbits with the purified CTV preparation with a titer 1:25600 in indirect ELISA test. Eighteen hybridoma-cell lines secreting monoclonal antibodies (MAbs) against CTV were screened after the fusion of mouse myeloma cells (SP2/0) with spleen cells from BALB/c immunized with the virus preparation. Four hybridoma-cell lines were selected randomly for later analysis. The results indicated that the titers of ascetic fluids against these hybridoma cell lines ranged from 1:51200 to 1:204800 in indirect ELISA, and their isotypes and subclasses were IgG2a for 2G and 3H and IgG2b for IE and 4H. These four Mabs were used to detect CTV in citrus samples in different sources. Results showed that TAS-ELISA with polyclonal antibody as trapping antibody and monoclonal antibody as testing antibody had a higher specificity and sensitivity than PAS-ELISA. Four Mabs showed different intensities of serological reaction with different CTV isolates. However, much work remains for realizing the characteristics and the serological relationships among these isolates.
Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Blotting, Western ; Citrus ; virology ; Closterovirus ; immunology ; isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Mice ; Mice, Inbred BALB C ; Rabbits

BACKGROUNDTo determine the antigenicity of HGV NS5 recombinant proteins expressed in E.coli.

METHODSHGV NS5a,NS5b and core/NS5b fusion genes were cloned into pThioC vector. Three expression plasmids were transformed into JM109(DE3) competent cells then expressed with induction by IPTG. Western blot and ELISA were used to determine the antigenicity after the three recombinant proteins were purified.

RESULTSAfter identification by restriction enzyme and sequencing, it was confirmed that the expressed was target proteins espected. Purified expression proteins were found strongly immunoreactive among anti HGV positive sera by Western blot and ELISA. Compared with mixed recombinant antigen (including core, NS5a synthetic peptide and NS3 recombinant proteins), in the 22 positive sera detected with mixed antigen, 68%(15/22), 90%(20/22) and 73%(16/22) were positive by P5a,P5b and Pc?5b antigens; In the 70 negative samples with mixed antigen, 7%(5/70), 1%(1/70) and 6%(4/70) were positive by P5a, P5b and Pc?5b antigens. The positive alone was found among RTPCR positive specimen using these recombinant antigens.

CONCLUSIONSNS5 gene expressed in E.coli?which couldn't be covered with other regions of antigens was one of the essential epitopes to HGV immunologic diagnosis.

Antibodies, Viral ; blood ; Antigens, Viral ; blood ; Epitopes ; immunology ; GB virus C ; genetics ; immunology ; Humans ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

BACKGROUNDTo express recombinant non-structural protein 3 of hepatitis C virus (HCV) in E. coli.

METHODSThe non-structural 3 (NS3) region DNA fragment of HCV was amplified by polymerase chain reaction (PCR) and inserted into inducible proeukaryotic expressive vector pET 30C(+)at Bam H1/EcoR1 sites. The competent BL21 (DE3) E.coli was transformed, and then cultured and induced with IPTG. The expressed HCV NS3 protein was confirmed with ELISA and dot blot hybridization using HCV NS3-specific single chain Fv (ScFv) antibody.

RESULTS1 893 bp DNA fragment of HCV NS3 coding region was amplified by PCR technique. HCV NS3 expressive vector pET-NS3 was constructed. After transformation with pET-NS3 and induction with IPTG, recombinant HCV NS3 protein was expressed and confirmed by specific ELISA and dot blot hybridization.

CONCLUSIONSThe recombinant HCV NS3 can be expressed in E. coli.

Escherichia coli ; genetics ; Gene Expression ; Hepatitis C Antibodies ; biosynthesis ; genetics ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; Viral Nonstructural Proteins ; biosynthesis ; genetics

Six recombinant plasmids co-expressing the wild-type GP5 gene or the codon-optimized GP5 gene (containing pan-DR epitope) of porcine reproductive and respiratory syndrome virus (PRRSV) and the E2 gene of classical swine fever virus (CSFV) or the E2 fused with the UL49 of pseudorabies virus (PrV) were constructed based on the suicidal DNA vaccine pSFV1CS-E2 described previously. Expression of GP5 and E2 was confirmed by indirect immunofluorescence assay. The immunogenicity of six plasmids was evaluated in BALB/c mouse model. For the six plasmids, low-level of E2 and GP5 protein specific antibodies could be detected in the sera of the immunized mice. Specific lymphoproliferative responses to the PRRSV or CSFV stimulation were induced in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. Antigen specific IFN-gamma and L-4 secretion was detected in the splenocytes of some immunized mice by cytokine ELSIA. Fusion with the PrV UL49 in the suicidal vaccines induced significantly higher lymphoproliferative responses and cytokine secretion. Taken together, the suicidal DNA vaccines co-expressing GP5 and E2 could induce PRRSV and CSFV specific humoral and cell-mediated immune responses.
Animals ; Antibodies, Viral ; blood ; Antibody Formation ; Cytokines ; blood ; Female ; Immunity, Cellular ; Lymphocytes ; immunology ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Vaccines, DNA ; biosynthesis ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; Viral Structural Proteins ; genetics ; immunology ; Viral Vaccines ; biosynthesis ; immunology

OBJECTIVETo obtain the monoclonal antibody against hexon protein of human adenovirus.

METHODSBALB/c mice were immunized with purified recombinant hexon protein, and the spleen cells of the mice were isolated and fused with myloma cells. Four hybridoma cell strains were screened by indirect ELISA and cultured, and the sensitivity, specificity and virus neutralizing activity were analyzed with ELISA, Western blotting and neutralizing test.

RESULTSThe mouse ascites produced by these hybridoma cells contained specific monoclonal antibodies against hexon protein of human adenovirus as identified by ELISA and Western blot, and the antibody generated by 4C6 strain showed human adenovirus type 3-neutralizing activity.

CONCLUSIONThe monoclonal antibodies against hexon protein with high specificity have been successfully obtained, and these antibodies can be useful in developing assays for early diagnosis of HAdV3 infection and also in study of therapeutic drugs of the infection.

Adenoviruses, Human ; chemistry ; immunology ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Blotting, Western ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology

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