Production And Characterization Of Porcine CD163Recombinant Proteins And The Antibodies

Porcine reproductive and respiratory syndrome (PRRS) is a highly contagious infectiousdisease caused by the infection of PRRS virus (PRRSV), a member of the familyArteriviridae. PRRS is characterized by disorder of reproduction and respiratory high deathrate of piglet, inhibition of immunity and persistence of infection. At present, PRRS is one ofthe most economically important diseases affecting the swine industry all over the world.The entry mechanism of PRRSV into the cell is receptor-mediated endocytosis. Untilnow, PRRSV cellular receptors on Marc-145cells are CD163and vimentin, and also maybeSn. CD163which belongs to the scavenger receptor cysteine-rich superfamily plays importantrole in virus uncoating and releasing of genomic RNA into cytoplasma. Another potentialPRRSV cellular receptor is non-myscle myosin II-A (NMHC II-A) which was purified fromPAM and Marc-145cells by an internal image monoclonal anti-idiotypic to PRRSV GP5antibodyusing co-immunoprecipitation. It has been documented that CD163has ninecysteine-rich domains (SRCR1~9), and SRCR5is the main functional domain for CD163mediating PRRSV infection through binding to GP2a and GP4protein of PRRSV. In addition,immune serum against CD163can partialy block PRRSV infection of the cells.Up to now, itis not fullyclear for the functions of SRCR1~9of CD163during PRRSV infection.The objectives of this study were to produce recombinant CD163SRCR1~4andSRCR5~6proteins and polyclonal and monoclonal antibodies against CD163and tocharacterize the CD163recombinant proteins and antibodies in PRRSV infection of Marc-145cells.This research prokaryotic expressed CD163-1and CD163-2recombinant proteins,producted the immune serum against CD163-1and CD163-2respectively, and producted themonoclonal antibody against CD163. Detected the function of the proteins and antibodiesthrough ELISA, Western-blot and IFA. The result showed that CD163-1and CD16-2recombinant proteins and immune sera against CD163-1and CD163-2can all interact withMarc-145cells, but only immune sera against CD163-2at the dilutions of1:20and1:40couldblock PRRSV infection of Marc-145cells,andall three monoclonal antibodies bound porcineCD163but not Marc-145cells.The results generated from this study demonstrated that recombinant CD163proteinswere produced and used to generate specific polyclonal and monoclonal antibodies. Immune serum against CD163SRCR5~6can partially block PRRSV infection of Marc-145cellsindicating that CD163SRCR5~6plays an important role in PRRSV infection. This studypaves way for future study of the roles of each of CD163SRCRs in PRRSV infection andinteraction with other PRRSV cellular receptors, especially its association with NMHC II-A.