Histopathological Analysis of Lung Tissues Lung tissues extracted from immunized mice or normal mice were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) before being paraffin-sectioned and stained with a hematoxylin-eosin (HE) staining kit (Solarbio) used in accordance with the manufacturers instructions. fusion antigens were significantly higher than those induced by antigens alone. Subsequent challenges with either a nonlethal or lethal dose of revealed that this vaccine candidates have strong prophylactic effects against infection. Therefore, these candidate pertussis vaccines should be examined in further trials against Furthermore, the AB5 subunit-based vaccine strategy may provide a new option for vaccine design. 2. Materials and Methods 2.1. Strains, Culture Conditions, and Plasmids Bp148 stain was provided by the Beijing Childrens Hospital. Bp148 was cultured at 37 C in pertussis SS liquid culture or charcoal medium (10 g of peptone, 4 g of charcoal, 5 g of sodium chloride, 0.001 g of niacin, and 12 g of agar dissolved to a final volume of 1 L, to which 20% defibrinated sheep blood was added). The expression strain, BL21 (DE3) qualified cells, was purchased from Transgen Biotech Co. Ltd. (Beijing, China) and cultured in LuriaCBertani (LB) broth or LB solid medium (made up of 1.5% agar) at 37 C. To induce protein expression, cells were first cultured in LB broth at 37 C until the absorbance at 600 nm (OD600) reached 0.6, after which isopropyl -d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 1 mM, and the culture was grown for another 10 h at 30 C. Kanamycin (50 g/mL) was used for plasmid selection when required. 2.2. Construction of Expression Vectors The sequences of Fim2, PtxS1, and FHA1877C2250, searched from the genome of for 10 min, dissolved in 1 SDS (50 mM Tris-HCl (pH 6.8), 1.6% (for 10 min, resuspended in A1 solution (0.5 M NaCl, 13.3 mL of Tris-HCl, 8 M urea, and 10 mM imidazole, pH 7.5), and subjected to high-pressure homogenization followed by centrifugation. We then collected the resulting sediment and Slit2 dissolved it in A1 solution; this suspension was centrifuged Qstatin at 8000 for 10 min at 4 C. Finally, the resulting supernatant was collected Qstatin for purification. Ten column volumes of A1 solution were used to equilibrate the nickel column, after which the protein sample was loaded at a flow rate of 4 mL/min before A1 solution was used to wash away unbound protein until the ultraviolet absorbance (280 nm) was close to 0 mAU. Finally, five column volumes of buffer B1 (0.5 M Qstatin NaCl, 13.3 mL of Tris-HCl, 8 M urea, and 0.5 M imidazole, pH 7.5) were used for elution and collection of the target protein. The eluted protein was then placed in a dialysis bag and incubated at 4 C for 3 days for renaturation. During this period, the urea concentration was constantly decreased, and the protein was finally dialyzed in 1phostphate-buffered saline (PBS) (pH 8.0) solution. After renaturation, samples were filtered using a 0.22-M filter and aliquoted before freezing and storage at ?80 C. After purification, a SUMO-specific protease was used to cleave the 6His usually tag and SUMO tag (if present) of the proteins. 2.5. Animal Immunization Experiments Five-week-old, specific pathogen-free female BALB/c mice were purchased from SPF Biotechnology (Beijing, China) and reared in the Laboratory Animal Centre of the Academy of Military Medical Sciences. Mice were housed with a constant ambient temperature (23 3 C) and humidity (55 5%). Food, bedding, and water were changed every 4 days. All animal experiments were approval by the Ethics Committee of the Academy of Military Medical Sciences. Subcutaneous immunization was conducted on Days 0, Qstatin 14, and 28 in mice. The immunization dose was 100 L (2 g of pertussis antigen and 100 g.