Topology Structural Study Of Porcine Reproductive And Respiratory Syndrome Virus GP5Protein

Porcine Reproductive and Respiratory Syndrome (PRRS) is infectious disease characterized with sow abortions and piglets respiratory problems, caused by Porcine Reproductive and Respiratory Syndrome virus (PRRSV). Great damage has been brought to swine industry since PRRSV being recognized. Swine high fever has occurred in summer and autumn in China recently, which has been announced as high pathogenic PRRSV infection by Agriculture Department of China in2007. The envelope glycoprotein5(GP5) is one of the major structural protein for PRRSV, and also is the most important protein in inducing protective immune response. As everyone knows, protein structure determines its function. The membrane topology structure study of viral envelope protein is an elemental work for virus research. Topology structure study for PRRSV GP5is significant helpful not only in understanding the interaction between PRRSV and its host cell, but also the development of PRRSV epitope vaccine and diagnostic reagent.In this report, the topology structure for PRRSV GP5protein was determined with four methods. This paper includes:1. Analysis and prediction for GP5topology with softwareThe hydrophobic region for GP5was predicted with software Hmmtop, TMHMM-2.0, MemBrain and DAS, and results indicated that there are three hydrophobic regions in GP5located at Aa8-27, Aa63-82and Aa106-127. Among these hydrophobic regions, residues1-33constituted signal sequence at N-terminal, which will be cutted in the mature GP5protein. The "AB" epitope (Aa27-51) and C (Aa180-197) terminal contains the important antigen domain region for GP5protein. Usually, hydrophobic region is the transmembrane part for envelope protein. So, it is predicted that GP5protein contains two transmembrance regions that are Aa63-82and Aa106-127. The most two important antigen epitopes located within the N-terminus and C-terminus is outside envelope, while Aa84-105is located inside.2. Peptide specific antibody preparation for GP5protein hydrophobic region Together with a universal helper T cell epitope. four peptides were synthesized within the hydrophobic region according to software analysis for GP5protein. The first peptide was named IL-36, located at N terminal peptide signal of GP5protein; the secondary peptide was IG45, represented the first hydrophobic region; the third peptide was IY45, represented the second hydrophobic region, that inside virus envelope; the last peptide was IL35, which is located at the C terminal of GP5protein, represented the third hydrophobic region. After emulisificied with mineral oil, the synthesized peptides were injected to ICR mouse via intra muscle for three times at two weeks interval. Antibody titer and specificity was detected with enzyme-linked immunosorbent assay (ELISA). Lastly, mouse is executed to get as much as possible peptide specific antiserum.3. Topology analysis of GP5by Immunological methodsTopology structure for GP5protein was verified with indirect immunofluorescence with PRRSV infected Marc-145cells. Marc-145cells was infected with PRRSV JXA1strain, fixed with paraformaldehyde, and then permeabilized with tritonx-100or digitonin. On the other hand, PRRSV was purified with ultracentrifugation, treated with proteinase K and trypsogen. Western-blot was performed with GP5peptide specific antibody prepared in former chapter to determine the topology structure for GP5protein. Both immunofluorescence and Western-blot result indicated that N-terminus (residues Aa33-62) and C-terminus (residues127-200) are located inside luminal, which is consistent with the prediction with soft ware.4. Topology analysis of GP5by fusion proteinThe orientation N-terminus and C-terminus of GP5protein has been identified through Immunologic methods, while the other parts are not confirmed experimentally. HA-tag was inserted into four different sites of GP5protein with overlap extension Polymerase chain reaction (PCR). Four eukaryotic expression recombinant plasmids were constructed and transfected into HEK-293and Marc-145cell transported with liposome2000. Topology analysis for GP5protein was performed by HA localization through indirect immunofluorescence assay with monoclonal antibodies against HA. The result reveals that N-terminal signal sequence is processed after translation and the50HA and C-HA are on the luminal, but the95HA on the cytoplasm, which is also consistent with the prediction with soft ware and result from immunological method.