RESUMO
Glutathione S-transferases (GSTs) are essential components of the cellular detoxification system because of their capability to protect organisms against the toxicity of reactive oxygen species (ROSs). Four different GSTs (MrMuGST1-MrMuGST4) showing similarities with Mu-type GSTs were cloned from the hepatopancreas of Macrobrachium rosenbergii. These four GSTs have 219, 216, 218 and 219 amino acids in length, respectively. MrMuGST1-MrMuGST4 proteins all have a G-site in the N-terminus and an H-site in the C-terminus. Phylogenetic analysis reveals that four Mu-type GSTs are classified into two different clades (MrMuGST2 one clade; MrMuGST1, MrMuGST3 and MrMuGST4 other clades). Nonetheless, no site under positive selection was detected but rapid evolution was found in the few of MuGST genes. Reverse transcription-polymerase chain reaction (RT-PCR) results showed that MrMuGST1 and MrMuGST2 transcripts were expressed in all detected tissues, however, MrMuGST3 and MrMuGST4 were just mainly expressed in hepatopancreas and intestines. Quantitative RT-PCR analysis showed that MrMuGST1 and MrMuGST2 were down-regulated upon Vibrio anguillarum challenge, whereas MrMuGST3 and MrMuGST4 were quickly up-regulated 2 h after the Vibrio challenge. Our results imply that different Mu-type GSTs may respond to Vibrio challenge with different manners.
Assuntos
Glutationa Transferase/genética , Palaemonidae/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Glutationa Transferase/química , Glutationa Transferase/metabolismo , Hepatopâncreas/metabolismo , Imunidade Inata , Dados de Sequência Molecular , Palaemonidae/metabolismo , Filogenia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de Sequência/veterinária , Vibrio/fisiologiaRESUMO
Toll receptor was first discovered in Drosophila and has an important function in the innate immunity of invertebrates. In this study, the Toll receptor HcToll1 from Hyriopsis cumingii with a full length of 3810 bp consisting of a 3687 bp ORF that encodes a total of 1228 amino acids protein was selected for further study. The HcToll1 protein consisted of a signal peptide, 17 LRR domains, 2 LRRCT domains, 1 LRRNT domain, 1 TM domain, and 1 TIR domain. Phylogenetic analysis results showed that HcToll1 was clustered in one group together with other mollusca tolls. RT-PCR analysis results showed that HcToll1 was expressed in all tested tissues such as hemocytes, hepatopancreas, gills, and mantle. qRT-PCR analysis results showed that HcToll1 expression was increased by the presence of Escherichia coli, Vibrio anguillarum, Staphyloccocus aureus, and White Spot Syndrome Virus (WSSV). Over-expression of HcTIR could up-regulate expression of drosomycin gene in Drosophila S2 cells. The results of our study indicated that HcToll1 is a functional Toll and it has an important function in the generation of innate immune responses of H. cumingii against microbial challenge.
Assuntos
Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/imunologia , Unionidae/genética , Unionidae/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/fisiologia , Regulação da Expressão Gênica , Dados de Sequência Molecular , Especificidade de Órgãos , Filogenia , RNA/genética , RNA/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Receptor 1 Toll-Like/química , Unionidae/química , Vibrio/fisiologia , Vírus da Síndrome da Mancha Branca 1/fisiologiaRESUMO
Oncolytic viruses armed with therapeutic transgenes of interest show great potential in cancer immunotherapy. Here, a novel oncolytic adenovirus carrying a signal regulatory protein-α (SIRPα)-IgG1 Fc fusion gene (termed SG635-SF) was constructed, which could block the CD47 'don't eat me' signal of cancer cells. A strong promoter sequence (CCAU) was chosen to control the expression of the SF fusion protein, and a 5/35 chimeric fiber was utilized to enhance the efficiency of infection. As a result, SG635-SF was found to specifically proliferate in hTERT-positive cancer cells and largely increased the abundance of the SF gene. The SF fusion protein was effectively detected, and CD47 was successfully blocked in SK-OV3 and HO8910 ovarian cancer cells expressing high levels of CD47. Although the ability to induce cell cycle arrest and cell death was comparable to that of the control empty SG635 oncolytic adenovirus in vitro, the antitumor effect of SG635-SF was significantly superior to that of SG635 in vivo. Furthermore, CD47 was largely blocked and macrophage infiltration distinctly increased in xenograft tissues of SK-OV3 cells but not in those of CD47-negative HepG2 cells, indicating that the enhanced antitumor effect of SG635-SF was CD47-dependent. Collectively, these findings highlight a potent antitumor effect of SG635-SF in the treatment of CD47-positive cancers.