RESUMO
Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.
Assuntos
Anticorpos Antivirais , Proteínas do Capsídeo , Circovirus , Escherichia coli , Proteínas Recombinantes , Vacinas de Partículas Semelhantes a Vírus , Animais , Circovirus/imunologia , Circovirus/genética , Suínos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Vacinas Virais/genética , Desenvolvimento de Vacinas , Antígenos Virais/imunologia , Antígenos Virais/genética , Imunoglobulina G/sangue , Análise Custo-Benefício , Feminino , Interferon gama/metabolismo , Imunogenicidade da VacinaRESUMO
OBJECTIVE: To compare the efficacy of ceftriaxone and that of cefoperazone plus sulbactam (sulperazon) in controlling infection, in scavenging bacteria from bile, and in their costs when treating acute suppurative cholangitis with choledochostomy. METHODS: Patients were randomly assigned to two groups: the ceftriaxone group (R-group, n=95) and sulperazon group (S-group, n=95). Before choledochostomy, both groups received one intravenous dose of the corresponding antibiotics: and 2 g ceftriaxnoe for the R-group, 2 g sulperazon, containing 1 g cefoperazone and 1 g sulbactam, for the S-group. After the operation, the patients in the R-group received ceftriaxone 2 g i.v. q.d.; the patients in the S-group received sulperazon 2 g i.v. b.i.d.. In addition, all patients in both groups received metronidazole 0.5 g daily before and after the operation. The efficacy was evaluated by efficiency in controlling infection and the persisting days of symptoms due to infection, fever and leukocytosis; the persisting days was compared using the life table method to calculate the "cumulative probability of persistence of symptoms (CPPS)". The two groups were also compared in regards to their biliary bacterial clearance rates and the costs directly attributable to the antibiotics. RESULTS: The efficiency in controlling infection was 98.9% (94/95) in both groups. However, the CPPS of the R-group decreased more rapidly than that of the S-group, Log-Rankchi2=6.7901, P=0.0092. Biliary bacterial clearance rate on post-operative day 3 was 72.0% (36/50) for the R-group, 41.3% (19/46) for the S-group, P=0.0037. Cost directly attributable to the antibiotics were (1788.29 +/- 518.46) yuan (RMB) for the R-group, and (3768.74 +/- 820.55) yuan for the S-group, F=395.51, P=0.0000. CONCLUSION: Both ceftriaxone and sulperazon are effective in treating acute suppurative cholangitis when used before and after choledochostomy. Ceftriaxone is superior in expediting symptom relief and bacterial clearance from bile, and is more cost-effective.