RESUMO
In this study, we describe an optimized method of obtaining virus-like particles (VLPs) of the recombinant hepatitis C virus (HCV) core protein (HCcAg) expressed in yeast cells (Pichia pastoris), which can be used for the construction of diagnostic test systems and vaccine engineering. The described simplified procedure was developed to enable in vitro self-assembly of HCcAg molecules into VLPs during protein purification. In brief, the HCcAg protein was precipitated from yeast cell lysates with ammonium sulfate and renatured by gel filtration on Sephadex G-25 under reducing conditions. VLPs were self-assembled after the removal of the reducing agent by gel filtration on Sephadex G-25. Protein purity and specificity were evaluated by SDS-PAGE and immunoblotting analysis. The molecular mass of VLPs and their relative quantity were measured by HPLC, followed by confirmation of VLPs production and estimation of their shape and size by transmission electron microscopy. As a result, we obtained recombinant HCcAg preparation (with ~90% purity) in the form of VLPs and monomers, which has been used to produce hybridomas secreting monoclonal antibodies (mAbs) against HCcAg.
Assuntos
Anticorpos Monoclonais Murinos/imunologia , Hepacivirus , Anticorpos Anti-Hepatite C/imunologia , Saccharomycetales , Vacinas de Partículas Semelhantes a Vírus , Proteínas do Core Viral , Vacinas contra Hepatite Viral , Animais , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Saccharomycetales/genética , Saccharomycetales/metabolismo , Vacinas de Partículas Semelhantes a Vírus/biossíntese , Vacinas de Partículas Semelhantes a Vírus/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/isolamento & purificação , Proteínas do Core Viral/biossíntese , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Proteínas do Core Viral/isolamento & purificação , Vacinas contra Hepatite Viral/biossíntese , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/isolamento & purificaçãoRESUMO
Elevated concentrations of circulating casomorphins (CM), the exogenous opioid peptides from milk casein, may contribute to the pathogenesis of autism in children. Because several mass spectrometry studies failed to detect casomorphins in autistic children, it was questioned whether these peptides can be detected in body fluids by mass spec. Here we demonstrated, using a novel high sensitivity ELISA method, that autistic children have significantly higher levels of urine CM-7 than control children. The severity of autistic symptoms correlated with concentrations of CM-7 in the urine. Because CMs interact with opioid and serotonin receptors, the known modulators of synaptogenesis, we suggest that chronic exposure to elevated levels of bovine CMs may impair early child development, setting the stage for autistic disorders.
