Non-specific antibody binding sites were blocked by immersion of the membrane in 5% skimmed milk in PBS overnight at 4C. and functions of the prM protein and may also be developed for potential clinical applications. == Introduction == West Nile computer virus (WNV) is usually a zoonoticmosquito-transmitted arbovirus belonging to the genus Flavivirus in the ITD-1 family Flaviviridae and is widely prevalent in Africa, Southern Europe, Russia, the Middle East, India, Australia, and North America.(1,2)Since its first large outbreak in Romania in 1996,(3)WNV has become a major public health and veterinary ITD-1 concern. In 2012, the US experienced a WNV outbreak, with 5387 cases of WNV disease reported, including 243 deaths.(4) WNV was a single-stranded (positive sense) RNA virus with a genome of approximately 11 kb that encodes three structural proteins (C, prM/M, and E) and seven non-structural proteins (NS1, MCH6 NS2A, NS2B, NS3, NS4A, NS4B, and NS5).(5,6)The prM protein comprises 167 amino acids, with a molecular weight of ITD-1 20 to 26 kDa. The prM protein, which acts as a chaperone that assists in the maturation of the E protein, interacts with E as a prM/E heterodimer, which is usually important for the formation of immature virions.(7,8)The pr peptide is contained within the amino terminal region of the prM protein and is cleaved from prM by the protease furin in the trans-Golgi apparatus during release of the virus from the cell. The proteolytic cleavage of prM to generate the M protein results in the formation of a fusion qualified particle.(9,10)However, the functional role of prM in replication, computer virus infectivity, and induction of neutralizing activity(11)is not fully understood. In this study we generated a hybridoma cell line secreting a monoclonal antibody (MAb) specific for the pr region of the WNV prM protein. We subsequently characterized the MAb produced, which may be a useful tool for further investigations of the characteristics and functions of the prM protein, and as a potential candidate for clinical applications in the diagnosis and therapy of WNV. == Materials and Methods == == Cell lines, computer virus, and other reagents == Baby hamster kidney (BHK-21) cells and SP2/0 myeloma cells were cultured in RPMI-1640 medium (Hyclone, Beijing, China) supplemented with 10% fetal calf serum (PAA, Somerset, United Kingdom) and antibiotics (100 IU/mL penicillin and 0.1 mg/mL streptomycin). All cells were maintained in a humidified 5% CO2atmosphere at 37C. Inactivated WNV (strain Chin-01) cell cultures and rabbit anti-WNV sera were kindly provided by Dr. Cheng-Feng Qin (Beijing Institute of Microbiology and Epidemiology, Beijing, China). == Expression and purification of prM protein == The recombinant plasmids were constructed and confirmed ITD-1 in our laboratory prior to transformation ofE. coliER2523 (NEB Express, Ipswich, MA) cells for protein expression. Recombinant protein was expressed according to a previously described procedure.(12)Briefly, overnight cultures of the transformed cells were diluted 1:100 in 50 mL Luria-Bertani (LB) broth containing 100 g/mL ampicillin at 37C. When the OD600reached 0.6, isopropyl-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO) was added to the broth at a final concentration of 0.3 mM to induce protein expression and the cells were incubated at 16C overnight. Subsequently, bacterial cells were removed from the growth medium by centrifugation at 5000gfor 15 min and lysed by sonication in cold phosphate-buffered saline (PBS, pH 7.4). Predominant expression of the recombinant prM protein in the maltose-binding protein (MBP) fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 12%) and Western blotting. The fusion protein was affinity-purified for use as an immunogen with an amylose resin column. == Generation, selection, and purification ITD-1 of monoclonal antibodies against prM == The purified recombinant protein was used as an immunogen in mice. Hybridomas secreting anti-prM antibodies were generated according to a previously described method.(13)Female BALB/c mice (aged 5 weeks) were immunized subcutaneously with recombinant protein emulsified with Freund’s complete adjuvant (Sigma, St. Louis, MO). Two booster injections.