ABSTRACT
The objective of this study was to investigate the function of vimentin in PRRSV infection. Vimentin gene from Marc-145 cells was amplified by RT-PCR, cloned into pET-28a vector and expressed in Escherichia coli BL21(DE3). The expressed vimentin was confirmed by Western blot and purified which was used to immunize BALB/c mice for the production of antibodies. Vimentin and antibodies were tested for blocking PRRSV infection of Marc-145 cells. The binding of vimentin to PRRSV N and GP5 proteins were tested by the ELISA. The results showed that vimentin gene was amplified successfully and expressed as identified by SDS-PAGE and Western blot. Mouse anti-vimentin antibodies were produced with the titer of 10(5). PRRSV infection of Marc-145 cells was blocked partially by vimentin while blocked completely by the antibobies. In addition, vimentin was bound N protein, but not GP5. These results provide additional information on PRRSV entry into Marc-145 cells.
Subject(s)
Animals , Female , Mice , Antibodies , Allergy and Immunology , Metabolism , Cell Line , Escherichia coli , Genetics , Metabolism , Genetic Vectors , Genetics , Mice, Inbred BALB C , Porcine Reproductive and Respiratory Syndrome , Genetics , Metabolism , Virology , Porcine respiratory and reproductive syndrome virus , Physiology , Protein Binding , Physiology , Recombinant Proteins , Genetics , Allergy and Immunology , Metabolism , Swine , Vimentin , Genetics , Allergy and Immunology , Metabolism , Viral Proteins , MetabolismABSTRACT
The spike (S) glycoprotein of HCoV-NL63 is a major target in the development of diagnostic assays and vaccines, but its antigenic and immunogenic properties remain unclear. Four fragments coding spike proteins (S1, S2, RL and RS) from HCoV-NL63 were amplified and cloned into the expression vector derived from vaccinia virus (Tiantan strain), and recombinant vaccinia viruses expressing four segments of spike proteins were generated (vJSC1175-S1; vJSC1175-S2; vJSC1175-RL; vJSC1175-RS), respectively. Their expression location in cell and level were characterized using indirect immune fluorescence assay (IFA) and Western-Blot, respectively. The expressions of four segments of spike proteins in recombinant vaccinia viruses were showed at appropriate level and with posttranslational modification (glycosylation), and S1, RL and RS were mainly distributed in the cell membrane, while the S2 was mainly distributed in the cytoplasm. Our results provide a basis for further exploring diagnostic role and vaccine development of different spike segments from HCoV-NL63.
Subject(s)
Humans , Base Sequence , Blotting, Western , Coronavirus NL63, Human , Chemistry , Fluorescent Antibody Technique, Indirect , Membrane Glycoproteins , Genetics , Molecular Sequence Data , Plasmids , Recombinant Proteins , Spike Glycoprotein, Coronavirus , Vaccinia virus , Genetics , Viral Envelope Proteins , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To rational design HCV DNA vaccine candidates and evaluate their specific We design to construct two DNA vaccine candidates, one consists of immunity to HCV in mice.</p><p><b>METHODS</b>We design to construct two DNA vaccine candidates, one consists of E2 (the envelope glycoprotein 2 of HCV) gene only, the second consists of E2-gAD (Globular Domain of Human Adiponectin) fusion gene via overlapping PCR. Confirm the expression of the DNA vaccines by Western blotting, and then vaccinated by injection of DNA vaccines with gene electrotransfer (GET) in BALB/c mice. The immune response was measured by IFN-gamma ELISPOT.</p><p><b>RESULTS</b>The DNA vaccine candidate consists of E2-gAD could effectively express in vitro , and it could induced a higher anti-HCV T cell response in mice than the one consists of E2 only.</p><p><b>CONCLUSION</b>The HCV DNA vaccine consists of E2-gAD fusion can increase the immunity of the E, to some extend, and the research paved a way to develop and optimize the novel HCV DNA vaccine.</p>