Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombin...Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.展开更多
Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It has been previously described as a carrier of peptides or proteins to increase their immunogenicity in different animal species,...Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It has been previously described as a carrier of peptides or proteins to increase their immunogenicity in different animal species, but its activity has never been evaluated in chickens. In this work, the use of BLS to improve the antibody response against bovine rotavirus (BRV) VP8d protein in laying hens was assessed. VP8d is the inner domain of the VP8 spike protein which preserves the sialic acid binding activity and the neutralizing epitopes present in the viral protein. Hens were immunized three times with 2 μg of VP8d alone or fused to BLS. Hens inoculated with BLSVP8d developed higher antibody titers (evaluated by ELISA and viral neutralization test) than hens immunized either with VP8d alone or the mixture of VP8d and BLS. Furthermore, IgY antibodies against BLSVP8d were able to fully protect mice against challenge with virulent BRV in a dose-depent-manner. Overall, these results demonstrate that BLS is a potent immonumodulator that enhances the antibody response in hens, thus increasing the concentration of specific IgY in the egg yolk, one of the main issues to be adressed in order to improve the use of the IgY technology.展开更多
Chlamydia trachomatis (C.tr) infections are the most prevalent bacterial sexually transmitted infections worldwide. They are often asymptomatic and therefore underdiagnosed as there is no routine screening surveillanc...Chlamydia trachomatis (C.tr) infections are the most prevalent bacterial sexually transmitted infections worldwide. They are often asymptomatic and therefore underdiagnosed as there is no routine screening surveillance. This case supports the possibility of sexual abuse as a route of transmission of C.tr. It is well known that nearly one third of sexually assaulted children are at risk for infection by a sexually transmitted agent. This is why in cases of sexual abuse, it is standardized that C.tr positive results by Nucleic Acid Amplification Techniques (NAATs) should be confirmed looking for another C.tr target;for this reason, we used a Polimerase Chain Reaction (PCR) directed to cryptic plasmid of C.tr. Confirmation was specified by the use of another PCR with a different genetic target (ompA) and sequencing. We concluded that our patient’s oral lesions were probably originated by her father’s sexual abuse.展开更多
文摘Foot-and-mouth disease is a highly contagious disease that produces severe economic losses in the livestock industry. This disease is being controlled by the use of an inactivated vaccine. However, the use of recombinant empty capsids as a subunit vaccine has been reported to be a promising candidate because it avoids the use of virus in the vaccine production. A plasmid containing the capsid precursor P12A and protease 3C sequences of foot-and-mouth disease virus (FMDV) was constructed and used to compare transient and stable expression in mammalian cells. When BHK-21 cells were transfected with the recombinant vector, protease 3C cleaved the capsid precursor P12A into the structural proteins VP0, VP1 and VP3. A sucrose gradient demonstrated that the structural proteins assembled into different subviral particles. Attempts to generate a stable cell line only allowed isolating low-level-expressing clones, probably due to the effect of protease 3C on the cells. Moreover, the recombinant protein yield achieved in transient expression assays was much higher than the one achieved in stable expression assays. Results indicate that mammalian cells are a good strategy to produce recombinant FMDV subviral particles. However, the alternative approach of transient gene expression in scalable systems should be used instead of the standard method that involves the generation of a stable cell line.
文摘Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It has been previously described as a carrier of peptides or proteins to increase their immunogenicity in different animal species, but its activity has never been evaluated in chickens. In this work, the use of BLS to improve the antibody response against bovine rotavirus (BRV) VP8d protein in laying hens was assessed. VP8d is the inner domain of the VP8 spike protein which preserves the sialic acid binding activity and the neutralizing epitopes present in the viral protein. Hens were immunized three times with 2 μg of VP8d alone or fused to BLS. Hens inoculated with BLSVP8d developed higher antibody titers (evaluated by ELISA and viral neutralization test) than hens immunized either with VP8d alone or the mixture of VP8d and BLS. Furthermore, IgY antibodies against BLSVP8d were able to fully protect mice against challenge with virulent BRV in a dose-depent-manner. Overall, these results demonstrate that BLS is a potent immonumodulator that enhances the antibody response in hens, thus increasing the concentration of specific IgY in the egg yolk, one of the main issues to be adressed in order to improve the use of the IgY technology.
文摘Chlamydia trachomatis (C.tr) infections are the most prevalent bacterial sexually transmitted infections worldwide. They are often asymptomatic and therefore underdiagnosed as there is no routine screening surveillance. This case supports the possibility of sexual abuse as a route of transmission of C.tr. It is well known that nearly one third of sexually assaulted children are at risk for infection by a sexually transmitted agent. This is why in cases of sexual abuse, it is standardized that C.tr positive results by Nucleic Acid Amplification Techniques (NAATs) should be confirmed looking for another C.tr target;for this reason, we used a Polimerase Chain Reaction (PCR) directed to cryptic plasmid of C.tr. Confirmation was specified by the use of another PCR with a different genetic target (ompA) and sequencing. We concluded that our patient’s oral lesions were probably originated by her father’s sexual abuse.
