, 2007; Aranda et al., 2008; Baums et al., 2009; Tan et al., 2009). Recently, several immunogenic cell wall or extracellular S. suis proteins were identified using genomic and immunoproteomic approaches
(Geng et al., 2008; Zhang et al., 2008; Liu et al., 2009). When combined with effective adjuvants, enolase and the proteins HP0197 and HP0272 showed good protection in mice and/or pigs against S. suis challenge (Zhang et al., 2009a, b; Chen et al., 2010). In a previous study, we identified S. suis genes preferentially expressed in vivo. Several genes encoding cell wall-associated proteins were significantly upregulated in the brains and lungs of infected pigs (Li et al., 2010), one of which was hp0245 (SSU05_0245). In this study, we confirmed that the in vivo-induced protein HP0245 is an immunogenic surface protein of SS2. Immunization of mice with the extracellular peptide of this protein (HP0245EC) provided even better
Selleckchem GSK-3 inhibitor protection than autogenous SS2 bacterin against the selleck chemical homologous SS2 challenge. HP0245 can be recommended as a vaccine candidate for SS2. SS2 strain SC-19 used in this study was isolated from a sick pig during the epidemic outbreak in Sichuan province of China in 2005. SC-19 was grown in tryptic soy broth (TSB) or on tryptic soy agar (TSA) plates (Difco, Detroit, MI) with 5% newborn bovine serum (Sijiqing Biological Engineering Materials Co. Ltd, Hangzhou, China) at 37 °C. Escherichia coli DH5α (TaKaRa, Dalian, China) and E. coli BL21 (DE3) (Novagen, Shanghai, China) were used for cloning and expression of the recombinant protein HP0245EC, respectively. Escherichia Vitamin B12 coli was grown routinely in Luria–Bertani (LB) broth or on LB agar (Oxoid, Basingstoke, UK) supplemented with kanamycin (50 μg mL−1) at 37 °C. Chromosomal DNA was isolated from broth-grown SS2 strain SC-19 as described previously (Smith et al., 2001).
The DNA responsible for the extracellular region of HP0245 (hp0245EC) was amplified with the forward primers 5′-CGTACAGAATTCGGTGCTAGTCGAACGTTG-3′ and reverse primer 5′-CGTATCGTCGACGGTCATAAGAATTTCAAGTTG-3′ (the underlined letters indicate enzyme cut sites EcoR I and Sal I, respectively) according to the published sequence (GenBank accession no. NC009442). PCR was performed at 94 °C for 5 min; 94 °C for 1 min, 56 °C for 1 min, 72 °C for 1 min, for 30 cycles; and at 72 °C for 10 min with PrimeSTAR HS DNA polymerase (TaKaRa). The product cut with EcoR I/Sal I (TaKaRa) was cloned into pET-28a (+) (Novagen) and transferred to E. coli DH5α. The positive clone was confirmed by DNA sequencing. pET-28a-hp0245EC was transferred to E. coli BL21 (DE3) for expression. The recombinant protein was induced at 37 °C in cultures grown at log phase by adding 0.1 mM isopropyl-β-d-thiogalactopyranoside (Sigma, St. Louis, MO) for 3 h. The recombinant protein HP0245EC formed as inclusion body was purified according to the method of Sambrook & Russell (2006).