RESUMO
Siganus oraminl-amino acid oxidase (SR-LAAO), isolated from the serum of Siganus oramin, is an innate immune protein with significant antibacterial activity. The aim of this study was to express SR-LAAO in insect cells using a baculovirus expression system and evaluate the function of the recombinant SR-LAAO. To this end, an optimized sequence of the SR-LAAO gene was designed and synthesized, based on the codon bias of insect cells. Bacmid shuttle vectors and recombinant baculovirus were successfully constructed, and the recombinant baculovirus was transfected into Sf9 insect cells. The antibacterial activity and enzymatic characteristics of the recombinant SR-LAAO were investigated. The results showed that the pFastBac-optiSR-LAAO shuttle vectors and Bacmid-optiSR-LAAO were correctly constructed. The Sf9 insect cells exhibited significant cytopathic effects following infection with Bacmid-optiSR-LAAO and Bacmid; the specific PCR analysis proved that the recombinant baculovirus was successfully constructed. The immunofluorescence assay revealed that the recombinant baculovirus rSR-LAAO was abundantly expressed in infected Sf9 insect cells; the results of SDS/PAGE and Western blot analyses showed that a specific band appeared at about 60 kDa. Moreover, the crude rSR-LAAO enzyme displayed strong antibacterial activity against aquatic pathogens, particularly Streptococcus agalactiae and Streptococcus iniae. In addition, the results of catalase interference test implied that the antibacterial activity of rSR-LAAO was directly associated with (H2O2 production). The results of the rSR-LAAO enzymatic characteristics test indicated that the Km value with l-Lysine as a substrate was 16.61 mM when the temperature was under 37 °C, and the optimum pH was 7. The antibacterial activity of rSR-LAAO could be completely inhibited by 10 mg/mL of pepsin, trypsin, and proteinase K compared with both methanol and acetone. Adding an equal volume of ethanol had a minimal impact on the antibacterial activity of rSR-LAAO. The crude enzyme could maintain a high level of antibacterial activity against both Gram-positive and -negative bacteria from 4 °C to 30 °C. In the present study, SR-LAAO was successfully expressed in Sf9 cells using the baculovirus expression system, and provides basic references for further research into the role of LAAO in marine animals and the development of new antimicrobial drugs.
Assuntos
Proteínas de Peixes/genética , L-Aminoácido Oxidase/genética , Perciformes/genética , Animais , Anti-Infecciosos/imunologia , Baculoviridae/genética , Proteínas de Peixes/metabolismo , L-Aminoácido Oxidase/metabolismo , Perciformes/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9/imunologia , TransfecçãoRESUMO
Enterovirus A71 (EV-A71) is a positive-strand RNA virus that causes hand-foot-mouth disease and neurological complications in children and infants. Although the underlying mechanisms remain to be further defined, impaired immunity is thought to play an important role. The host zinc-finger antiviral protein (ZAP), an IFN-stimulated gene product, has been reported to specifically inhibit the replication of certain viruses. However, whether ZAP restricts the infection of enteroviruses remains unknown. Here, we report that EV-A71 infection upregulates ZAP mRNA in RD and HeLa cells. Moreover, ZAP overexpression rendered 293 T cells resistant to EV-A71 infection, whereas siRNA-mediated depletion of endogenous ZAP enhanced EV-A71 infection. The EV-A71 infection stimulated site-specific proteolysis of two ZAP isoforms, leading to the accumulation of a 40 kDa N-terminal ZAP fragment in virus-infected cells. We further revealed that the 3C protease (3Cpro) of EV-A71 mediates ZAP cleavage, which requires protease activity. Furthermore, ZAP variants with single amino acid substitutions at Gln-369 were resistant to 3Cpro cleavage, implying that Gln-369 is the sole cleavage site in ZAP. Moreover, although ZAP overexpression inhibited EV-A71 replication, the cleaved fragments did not show this effect. Our results indicate that an equilibrium between ZAP and enterovirus 3Cpro controls viral infection. The findings in this study suggest that viral 3Cpro mediated ZAP cleavage may represent a mechanism to escape host antiviral responses.
Assuntos
Cisteína Endopeptidases/metabolismo , Enterovirus Humano A/enzimologia , Interações Hospedeiro-Patógeno , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/metabolismo , Replicação Viral , Proteases Virais 3C , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Enterovirus Humano A/genética , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Células Musculares/metabolismo , Células Musculares/virologia , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/antagonistas & inibidores , Proteínas de Ligação a RNA/genética , Células Sf9/imunologia , Células Sf9/virologia , Transdução de Sinais , Spodoptera , Proteínas Virais/genéticaRESUMO
OBJECTIVES: To analyze the transcriptome of Spodoptera frugiperda 9 (Sf9) cells infected with AcMNPV or AcMNPV-BmK IT. RESULTS: A comprehensive transcriptome profile for Sf9 cells infected with AcMNPV or AcMNPV-BmK IT is shown. 43127572, 46529744 and 47235310 RNA-Seq profiles permitted the quantification of expression levels for control (C), AcMNPV-BmK IT treatment (ABT) and AcMNPV treatment (AT) groups. There were 997 up-regulated or down-regulated candidate genes with significant different expression level in these treatment groups. CONCLUSION: These results provide a broad spectrum of candidate genes that are critically involved in the molecular regulation mechanism of Sf9 cells infected with AcMNPV-BmK IT.