Preparation And Characterization Of Monoclonal Antibodies Against Non-muscle Myosin Heavy Chain ⅡA And B

Porcine reproductive and respiratory syndrome (PRRS) is a severe disease caused byPRRS virus (PRRSV). It mainly results in reproductive disorder, such as premature, abortion,fetal death, mummy or weak generation. And it could induce porcine respiratory syndrome atvariety of ages with high mortality. PRRS has been prevailed in the major pig-raisingcountries and districts in the world, and caused great economic loss to the world’s hogindustry.The process PRRSV get into cells is receptors dependent. There are four receptorsidentified now. They are Heparin sulfate (HS), Sialoadhesin (Sn), CD163and Vimentinrespectively. HS adsorbs PRRSV to gather in cell surface, Sn mediates PRRSV endocytosis,and CD163may assist in this procedure. Beside, CD163mainly mediates PRRSV uncoatingand releasing viral genome into cell plasma. And Vimentin gather with other intermediatefilament proteins participate in virus replication and migration.Nonmuscle myosin II (NM-II) distributes ubiquitously throughout nature and plays arole in a variety of cellular activities including cell migration, adhesion, cytokinesis and celltransportation. Nonmusle myosin heavy chain II (NMHC-II) is devied into three types,NMHC II-A, NMHC II-B and NMHC II-C, whose amino acid homology is between60and80percent. However, there are significant differences between the amino terminus and thecarboxyl terminus among the three types, thus they could be recognized by specific antibodies.MAb2-5G2, an internal image generated against idiotypic antibody specific for GP5proteinof PRRSV, identified a soluble protein prepared from Marc-145cells. The protein wasidentified to be NMHC II-A. It has been found that, carboxyl terminus NMHC II-A reducesinfectivity of PRRSV to Marc-145cells, while carboxyl terminus NMHC II-B enhances thisprocess. It indicates that, NMHC II-A and NMHC II-B do play roles in PRRSV infection.The Monoclonal antibody (MAb) against carboxyl terminus NMHC II-A and NMHCII-B were prepared to study the role of them in PRRSV infection. Recombinant protein werepurified and immunized into BALB/c mice. Cell fusion and subclone were carried outsequentially. The hybridomas secreting MAb were established and detected by ELISA. TwoMAbs against carboxyl terminus NMHC II-A and four for carboxyl terminus NMHC II-Bwere got with IgG subtype. The MAbs reacted specifically with the corresponding antigen,and there were no cross reaction between the monoclonal antibodies. I-ELISA and westernblot showed that the epitope domain of MAbs against NMHC II-A located between1812and1894amino acid, while1759-1903AA for NMHC II-B. However, additive ELISA indicated these epitopes were similar or same. The MAbs were purified from ascites fluid for indirectimmunofluorescence assay (IFA). The IFA indicated all the MAbs can bind with Marc-145cells in a dose dependent manner, the more MAbs, the more fluorescences.Immunoprecipitation confirmed the binding of MAbs with biological antigens in cell lysate.The preparation of these MAbs lay the foundation for study the role of NMHC II-A andNMHC II-B in PRRSV infection.