In this research the gene was cloned from JL01 (serovar 1) and expressed as a glutathione-BL21(DE3). TonB2 ELISA were 95% and 88%, respectively. The TonB2 ELISA Ercalcidiol provides an alternative method for rapid serologic diagnosis of infection through antibody screening, which would be especially useful when the infection status or serovar is unknown. Rsum Dans la prsente tude le gne a t clon partir dJL01 (srovar 1) et exprim en tant que protine de fusion de la glutathione-par analyse par immunobuvardage. Le complexe GST-TonB2 a t valu pour sa capacit protger les souris BALB/c contre une infection par Les souris ont t Ercalcidiol Ercalcidiol vaccines avec GST-TonB2 par voie sous-cutane et inocules par voie intra-pritonale avec ~4,0 105 units formatrices de colonies (UFC) ou ~1,0 106 UFC d4074. Elles ont t examines quotidiennement pendant 7 j aprs linfection dfi. Le taux de survie des souris TonB2 vaccines tait significativement plus lev que celui des souris qui avaient re?u uniquement la GST recombinante ou ladjuvant. Les rsultats ont dmontr que TonB2 dest immunogne chez les souris et devrait tre valu de manire plus approfondie comme candidat potentiel pour un vaccin contre linfection par laide dun vaccin vivant attnu. Lorsque compar une preuve dhmagglutiantion indirecte, la sensibilit et la spcificit de lELISA TonB2 taient respectivement de 95 % et 88 %. LELISA TonB2 fournit une mthode alternative rapide pour le diagnostic srologique dinfection par au moyen dune mthode de tamisage des anticorps, ce qui serait spcialement utile lorsque le statut de linfection ou le srovar infectant sont inconnus. (Traduit par Docteur Serge Messier) Introduction is the causative agent of porcine contagious pleuropneumonia (PCP), a highly contagious and often fatal disease that causes great economic losses in industrialized pig production worldwide (1). Vaccination is potentially an effective tool for the prevention of PCP. Exploration of potential immunogens is usually a primary step in developing effective vaccines. Previous studies of immunogens were focused on surface-exposed proteins such as RTX toxins (2), lipopolysaccharides (3), outer membrane lipoprotein A (OmlA) (4), transferrin-binding protein A (5), and outer membrane proteins (6). TonB2, the periplasm protein of the 2nd system that functions in iron acquisition by transporting protons from the cytoplasmic membrane to outer membrane receptors, has been found in and (7C11). This system was first reported in by Beddek et al (12). The system is important for bacterial growth in vitro and in vivo and plays an important role in virulence (12). In the current study the gene of was cloned and expressed Rabbit polyclonal to PCDHB11. in The immunogenicity of the recombinant protein was tested in a murine vaccination/challenge model. An indirect enzyme-linked immunosorbent assay (ELISA) based on this protein was developed. This ELISA can be used for surveillance of antibodies against strains were cultured in tryptic soy broth or tryptic soy agar (Becton, Dickinson and Company, Baltimore, USA) supplemented with nicotinamide adenine dinucleotide (NAD, Sigma-Aldrich, St. Louis, Missouri, USA), 10 g/mL. The strains were cultured in LuriaCBertani broth supplemented with ampicillin (50 g/mL) as required. Table I Bacterial strains, primers, and plasmids used in this study, in which the gene was cloned from JL01 (serovar 1) and expressed as a glutathione-BL21(DE3) Cloning of JL01 (serovar 1) was performed as described previously (16). The open reading frame (ORF) was cloned from the genomic DNA by polymerase chain reaction (PCR) with the use of primers P1 and P2 (Table I), synthesized by Sangon Biotech, Shanghai, China. The PCR product was cloned into the A/T cloning vector pMD18-T (Takara, Dalian, China) to form pMD-tonB2, which was transformed into DH5. Plasmids were extracted by alkaline lysis and sequenced in both directions with the use of primers M13C47 and RV-M (synthesized by Sangon Biotech, Shanghai, China). Multisequence alignment with published TonB2 sequences (4074, JL03, L20, and AP76) was done by means of clustalW. The antigenic property of TonB2 was predicted with the bioinformatics method of EMBOSS explorer (http://emboss.bioinformatics.nl/cgi-bin/emboss/antigenic) and expressed as an antigenic site, defined as the occurrence of hydrophobic residues Cys, Leu, and Val on the surface of a protein. Expression of TonB2 in BL21(DE3) (Takara) made up of the recombinant plasmid pKG-TonB2. As a negative control, GST was also produced in BL21(DE3) made up of the vacant plasmid pGEX-KG. After induction with isopropyl–D-thiogalactoside for 3 h, the bacteria made up of the plasmids were collected and disrupted by sonication. The 2 2 proteins were purified by means of a glutathioneCSepharose 4B column (Amersham.