Many studies have reported certain immune features of outer membrane proteins from different bacterial species. The E. coli OmpX, which can induce a Th1/Th2 mixed humoral response, is considered to be an immunogenic protein (Maisnier-Patin et al., 2003). The OmpA of many bacteria exhibits immune properties, and it has been shown to activate dendritic cells and macrophages, produce cytokines, and

elicit strong humoral responses (Torres et al., 2006; Pore et al., 2011). Porins are a class of outer membrane learn more proteins that are vital for Gram-negative bacteria, and often act as diffusion channels responsible for transport of certain hydrophilic nutrients (Benz, 1988). Porins are the main passage for many antibiotics and altered expression of these proteins is related to antibiotic resistance of bacteria (Pages et al., 2008). Porins are also involved in interactions with the host immune system (Massari et al., 2003). Two major porins, OmpC and OmpF from Salmonella enterica serovar Typhi, are immunogenic (Kumar et al., 2009). From the perspective

of structure and function, the OmpF of E. coli has putative antigenic epitopes located on several loops (Klebba et al., 1990; Fourel et al., 1993), indicating that it may have some immune properties. In the present study, the immunogenicity of OmpC and OmpF of porcine ExPEC was systemically evaluated and identified. Together with phylogenetic analysis, OmpC

Nutlin-3a chemical structure was inferred to be a novel protective antigen of porcine ExPEC and may serve as a promising vaccine candidate against ExPEC infection. Nine ExPEC strains (listed in Table 1) were isolated from diseased pigs in Hubei province, China. All isolated strains together with E. coli strains DH5α and BL21 (DE3) were grown in Luria–Bertani (LB) medium and plated on LB medium containing Amylase 1.5% agar (w/v) at 37 °C. The following primer pairs were designed based on the consensus sequences of genes ompC and ompF from previously sequenced E. coli genomes: ompC-fw (5′-ATCGGGATCCATGAAAGTTAAAGTACTGTCCCTCC-3′), ompC-rev (5′-GGCGCTCGAGTTAGAACTGGTAAACCAGRCCMA-3′); ompF-fw (5′-ATCGGGATCCATGATGAAGCGCAATATTCT-3′), ompF-rev (5′-GGCGCTCGAGTTAGAACTGGTAAACGATAC-3′). The complete open reading frames of genes ompC and ompF were PCR-amplified using the boiled ExPEC strains as a template. PCR was carried out at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 1 min, and 72 °C for 7 min. The positive products were purified and cloned into an expression vector pET-28a with His-tag (Novagen, Shanghai, China) using the BamHI and XhoI sites. After transformation, the positive clone was sequenced with an ABI 3730 DNA sequencer (Applied Biosystems, Foster City, CA). Two recombinant proteins, OmpC and OmpF, from ExPEC strain EcWH001 were overexpressed in BL21. Bacterial cells were grown to mid-log phase at OD600 nm= 0.