Escherichia coli ; genetics ; Humans ; Recombinant Fusion Proteins ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

OBJECTIVETo clone 1b type of HCV NS3-4b Gene and express in HEK 293 cells, lay the foundation for further study of the HCV NS3-4b recombinant adeno-associated virus vaccine and its dendritic cell vaccine.

METHODSHCV 1b patients' serum was collected, and full length NS3-4b segment was amplified by RT-PCR and cloned into adeno-associated virus' expression vector pAAV. CMV. EGFP in order to express in HEK 293 cells. At last, it was validated whether express or not by Western Blot.

RESULTSThe 1b type gene NS3-4b were amplified and consistent to the expected size (2838 bp), the recombinant plasmid has been confirmed its successful restructured by double enzyme and sequencing, at last, Western Blot map can see objective protein expression after it transfect HEK 293 cells.

CONCLUSIONThe adeno-associsted virus recombination HCV NS3-4b plasmid have successfully constructed and it can express in eukaryotic cells.

Dependovirus ; genetics ; Genetic Vectors ; HEK293 Cells ; Hepacivirus ; genetics ; Humans ; Plasmids ; Vaccines, Synthetic ; immunology ; Viral Nonstructural Proteins ; genetics ; Viral Vaccines ; immunology

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

Cloning, Molecular ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

Porcine reproductive and respiratory syndrome virus (PRRSV) non-structural protein 7 (Nsp7) plays an important role in the induction of host humoral immune response and could serve as an ideal antigen for serological genotyping assay for PRRSV based on the significant difference in immunoreactivities of North American (NA) and European (EU) PRRSV Nsp7. In this study, Nsp7 of NA and EU PRRSVwas separately expressed and purified using prokaryotic expression system. The purified recombinant Nsp7 proteins reacted with serum antibodies against corresponding genotype PRRSV in Western blotting. However, nonspecific reaction of whole recombinant Nsp7 with antibodies against another genotype PRRSV was observed, indicating that whole NA PRRSV Nsp7 and EU PRRSV Nsp7 have similar antigenic epitopes and recombinant proteins could not be used for genotyping of antibodies against PRRSV. Based on the analysis of similar antigenic epitopes at the hydrophilic region of NA PRRSV Nsp7 and EU PRRSV Nsp7 by bioinformatics assessment, partial Nsp7 gene region deleted sequences encoding similar antigenic epitopes was constructed by fusion PCR. The recombinant truncated Nsp7 (NA-deltaNsp7 and EU-deltaNsp7, about 43 kDa) was expressed and the molecular weight was about 43 kDa. The results of Western blotting showed that NA-deltaNSP7 and EU-deltaNSP7 could be specifically recognized by positive serum to NA or EU PRRSV individually and nonspecific reaction was eliminated. This study provided a basis for further development of serological genotyping assay for North American and European genotype PRRSV infection.
Animals ; Genotype ; Porcine respiratory and reproductive syndrome virus ; classification ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; immunology ; Swine ; Viral Nonstructural Proteins ; biosynthesis ; immunology

OBJECTIVEOf this study was to prepare high sensitivity and high specificity of highly pathogenic H5N1 subtype avian influenza virus NS1 protein antibody and a preliminary assessment of its potency.

METHODSConstruct pET-28a (+) recombinant vector containing the H5N1 subtype of avian influenza virus NS1 sequences of E. coli BL21 (DE3), induced expression of NS1 protein, NS1 recombinant protein was obtained by Ni-NTA column purified by affinity chromatography, and SDS-PAGE and Western Blot analysis. Purified protein antigen to immunize New Zealand white rabbits, obtained rabbit anti-NS1 serum, affinity-purified polyclonal antibodies. Using ELISA and Western Blot analysis of purified antibody titer and specificity.

RESULTSNS1 fusion protein was highly expressed in a purity of greater than 90%, with the fusion protein was used to immunize New Zealand white rabbits anti-NS1 polyclonal antibody titer of 1:80 000, and specific recognition of the H5N1 subtype of avian influenza virus NS1 protein.

CONCLUSIONSNS1 polyclonal antibodies to NS1 recombinant protein purified antigen, with better potency and specificity, and to prepare the conditions for the development of the H5N1 subtype of avian influenza virus detection kit.

Animals ; Antibodies, Viral ; biosynthesis ; immunology ; Escherichia coli ; genetics ; Influenza A Virus, H5N1 Subtype ; immunology ; Rabbits ; Recombinant Fusion Proteins ; immunology ; isolation & purification ; Viral Nonstructural Proteins ; genetics ; immunology

NS2 and NS3 are two post-transcriptional gene silencing suppressors that are encoded by Rice stripe virus. Gene silencing suppressors are always related to the pathogenicity of viruses. In this study, the cDNA of NS2 and NS3 were recombined by overlapping PCR assays, ligated to the RNAi vector, and inserted into the PXQ expression vector using Pst I; the expressed vector was transferred into calluses induced from seeds of the japonica rice cultivar, 'Nipponbare', using an Agrobacterium-mediated method. Thirty-one T0 transgenic plants were selected by G418 screening. PCR and southern blot analyses confirmed that the target gene was transformed into transgenic rice successfully, and different transgenic plants contained various copies of the gene. The disease resistance assay revealed that T0 transgenic rice had a delayed onset of RSV for approximately 10-20 d, and the accumulation of virus in the transgenic plants was reduced by 30%-50%. This was related to the delayed onset of disease.
Disease Resistance ; Oryza ; genetics ; immunology ; virology ; Plant Diseases ; genetics ; immunology ; virology ; Plants, Genetically Modified ; genetics ; immunology ; virology ; RNA Interference ; Tenuivirus ; genetics ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

PrME and NS1 gene were the two main immuneprotect proteins of Japanese encephalitis virus (JEV), and they were also N-linked glycosylation proteins. To clear the effect of N-glycosylation on JEV immunity, the N-glycosylation site of prME and NS1 gene were eliminated by site-directed mutant PCR, subtituting the N to Q. And the the mutant genes were subcloned into eukaryotic expression plasmid. Four-weeks female mice were immuned with the wildtype and mutant gene by twice. The antibodies against prME were detected by ELISA and the neutralization antibodies were tested by viral neutralizing assay. The immunoprotection were determined by attack with JEV virulent strain. Compare with the wild-type gene immuned-groups, one N-glycan eliminated prME gene could induce a little higher ELISA antibody, neutralization antibody and immunoprotection, but the immunity of gene with both N-glycan absence was decreased. The similar status were observed in the wildtype and mutant NS1 groups. Thus these results show that the N-linked glycosylation in the prME and NS1 gene were correlated with the immunity, one glycan absent would enhance the immunity but both two loss would impair it.
Animals ; Antibodies, Viral ; immunology ; Encephalitis Virus, Japanese ; genetics ; immunology ; metabolism ; Encephalitis, Japanese ; immunology ; virology ; Female ; Glycosylation ; Humans ; Mice ; Mice, Inbred BALB C ; Viral Nonstructural Proteins ; genetics ; immunology ; metabolism

Dengue virus (DENV) is a re-emerging disease transmitted by the Aedes mosquitoes and has become a major public health problem in southern China. Currently, no antiviral drug or effective vaccine exist to control this disease. The chimeric DENV structural protein vaccine cannot elicit balanced levels of protective immunity to each of the four viral serotypes; therefore, non-structural protein components may be required to construct an effective DENV vaccine. The Dengue virus non-structural 1 (DENV NS1) protein plays a critical role in viral pathogenesis and protective immunity. Therefore, immunity to Dengue 1-4 NS1 subtypes may be crucial for the prevention of severe disease. This review attempts to provide an overview about the structure and function of DENV NS1.
Animals ; Dengue ; immunology ; prevention & control ; virology ; Dengue Vaccines ; chemistry ; genetics ; immunology ; Dengue Virus ; chemistry ; genetics ; immunology ; Humans ; Viral Nonstructural Proteins ; chemistry ; genetics ; immunology

In this study, we cloned the NS3 gene from bovine viral diarrhea virus (BVDV) VEDEVAC strain. The result showed that the average P-distance of Pestivirus NS3 amino acid sequence was 0.07 and the VEDEVAC strain was classified to BVDV type 1. Using pET-30a(+) as vector and Escherichia coli Rosetta (DE3) as host, we obtained purified recombinant NS3 protein by Ni-NTA affinity chromatography. Western blotting analysis demonstrated that both BVDV positive serum and classical swine fever virus (CSFV) positive serum were able to recognize the recombinant NS3 protein. Indirect-ELISA assay indicated that the protein could be used as detection antigen.
Animals ; Cattle ; Cloning, Molecular ; Diarrhea Viruses, Bovine Viral ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Peptide Hydrolases ; genetics ; immunology ; Phylogeny ; RNA Helicases ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Sequence Analysis, Protein ; Viral Nonstructural Proteins ; genetics ; immunology