3). and induced with isopropyl–thio-galactopyranoside (IPTG). After Ciprofibrate centrifugation, the bacterial pellet was resuspended and sonicated until a clear lysate was obtained. The target proteins were then purified by dialysis and stored at ?80C.(12) Table 1. Primer Sequences BL21 cells and induced with IPTG. After centrifugation, the bacterial pellet was resuspended and sonicated until a clear Ciprofibrate lysate was obtained. The target proteins were purified by dialysis and identified by SDS-PAGE (Fig. 1). The proteins were coated as antigen in ELISA assay for mapping epitope. Open in a separate windows FIG. 1. SDS-PAGE analysis of recombinant proteins SVCV-g-KG, SVCV-gA-KG (1C270?bp), SVCV-gB-KG (253C522?bp), SVCV-gC-KG (505C774?bp), SVCV-gD-KG Ciprofibrate (757C1077?bp). (A) Lane 1, protein marker; lane 2, bacilli precipitation of pGEX-KG; lane 3, bacilli precipitation of SVCV-g-KG; lane 4, bacilli precipitation of SVCV-gA-KG (1C270?bp); lane 5, bacilli precipitation of SVCV-gB-KG (253C522?bp); lane 6, bacilli precipitation of SVCV-gC-KG (505C774?bp); lane 7, bacilli precipitation of SVCV-gD-KG (757C1077?bp). (B) Construction of recombinant plasmids expressing Ciprofibrate full length or truncated forms of G protein. Based on the ELISA result, ten partially overlapping in length of 15 amino acids’ short peptides covering 757C1077?bp of G protein were synthesized. Further epitope mapping assay was investigated using these peptides as ELISA coating antigen. Results and Discussion G protein expression in BL21 cells and induced with 1 mmol/L IPTG at 37C for 3?h to express the recombinant protein. A protein band of 66?kDa was detected by SDS-PAGE Rabbit polyclonal to ERK1-2.ERK1 p42 MAP kinase plays a critical role in the regulation of cell growth and differentiation.Activated by a wide variety of extracellular signals including growth and neurotrophic factors, cytokines, hormones and neurotransmitters. and Western blot, which corresponded with the molecular weight of the fusion protein (Fig. 1). Generation of MAbs against G protein of SVCV During immunization, mice blood samples were collected and monitored by indirect ELISA; one showed high binding affinity was chosen for the last booster and further cell fusion. After three subclones by limiting dilution method, two MAbs 1H11 and 4B8 were finally isolated and expanded for further use. Subtype identification of MAbs against G protein of SVCV The subtype of the MAbs was identified by a rapid ELISA mouse MAb isotyping kit. The result showed that both 1H11 and 4B8 belong to the subtype IgG2b. The light chains Ciprofibrate of these MAbs were kappa (Table 2). Table 2. Detection and Characterization of Monoclonal Antibodies
ELISA titer409,600204,800MAb subclassIgG2aIgG2bLight chain Open in a separate window MAbs specifically recognize G protein of SVCV The specificity of MAbs was identified by IFA and Western blot assay. In IFA, both MAbs 1H11 and 4B8 showed positive reaction to SVCV-infected EPC cells but no fluorescence signals were observed in the unfavorable control cells (Fig. 2). Because of carbohydrate, a 80?kDa protein band was seen in the Western blot, which differs from the calculated size of 57.4?kDa(10) (Fig. 3). In conclusion, MAbs 1H11 and 4B8 were highly specific to SVCV. Open in a separate windows FIG. 2. Immunofluorescence staining (IFA) of SVCV-infected EPC cells with different MAbs. After 36?h post-infection, cells were washed three times and fixed with 100% paraformaldehyde for 10?min, then blocked with BSA for 30?min. After three washes, cells were reacted with 1:100 diluted MAbs for 1?h and incubated with Alexa Fluor 488 goat anti-mouse IgG for 30?min. Fluorescent images were examined with a fluorescent microscope. MAbs 1H11 and 4B8 against NS4B; uninfected EPC cells control (400). Open in a separate windows FIG. 3. Specificity of monoclonal antibodies against G protein analyzed by Western blot assay. Epitope mapping of MAbs against SVCV Recombinant proteins SVCV-gA-KG (1C270?bp), SVCV-gB-KG (253C522?bp), SVCV-gC-KG (505C774?bp), and SVCV-gD-KG (757C1077?bp) were coated as antigen separately. The ELISA result showed MAbs 1H11 and 4B8 reacted with SVCV-gD-KG (757C1077?bp) (Table 3). Then, a set of synthesized peptides was coated as antigen in ELISA for further epitope mapping. Finally, the amino acids 273C287 (DGTLVSGHRPGLDLI) were identified as the epitope of G protein (Table 4). This result may be useful for the development of diagnosis and detection methods of SVCV. Table 3. Epitope Screening of MAbs Against G Protein of SVCV by ELISA
SVCV-gA-KG (1C270?bp)0.06310.0946SVCV-gB-KG (253C522?bp)0.05380.0574SVCV-gC-KG (505C774?bp)0.15070.0886SVCV-gD-KG (757C1077?bp)2.15742.2298Negative control0.06830.0817 Open in a separate window Table 4. Epitope Screening of MAbs Against G Protein of SVCV by ELISA