RESUMO
Selenoprotein W (SelW) has been found to be ubiquitously expressed in tissues in vivo and was purified more than 18 years ago. However, little in vitro research has been performed on SelW from birds. To detect the mRNA levels of chicken SelW in cultured cell lines, chicken SelW cDNA was cloned into an expression vector. The chicken SelW expression construct was then transfected into CHO-K1 cells. Using RT-PCR and real-time quantitative reverse transcription PCR, we detected the expression of the chicken SelW mRNA. Moreover, the selenocysteine-synthase (SecS) and selenophosphate synthetase-1 (SPS-1) mRNA levels were analyzed. The expression of SelW was detected in SelW-transfected cells; no expression was observed in control cells. Significant increases in the SelW mRNA levels were obtained in chicken SelW-transfected cells relative to control cells. SecS mRNA levels were significantly increased in chicken SelW transfected cells. No significant difference in the SPS-1 level was observed. Our findings show that chicken SelW could be studied in vitro and that SecS and SPS-1 may have potential roles in SelW biosynthesis.
Assuntos
Expressão Gênica , Fosfotransferases/biossíntese , Selenoproteína W/biossíntese , Transferases/biossíntese , Animais , Células CHO , Galinhas , Cricetinae , Cricetulus , Fosfotransferases/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Selenoproteína W/genética , Transferases/genéticaRESUMO
Selenoprotein W (SelW) is expressed in various tissues of many animals and acts as an oxidoreductase in mammals. However, little is known about the role of the SelW in birds. To investigate the role of the chicken SelW on H(2)O(2)-induced apoptosis in CHO-K1 cells, overexpression of a chicken SelW cell lines (CHO-K1/SelW) were constructed. Using acridine orange/ethidium bromide (AO/EB) double staining and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assays, as well as WST-1 cell viability assay, we monitored the extent of the H(2)O(2)-induced apoptosis and detected the abundance of the caspase-3, caspase-8, and fas mRNA by real-time quantitative reverse transcription PCR (qPCR). We here found that overexpression of SelW cells, compared with the wild-type cells, resulted in a markedly decrease in sensitivity to H(2)O(2)-induced oxidative stress and had a lower apoptotic cell death in AO/EB and TUNEL assays. Cell viability revealed that overexpression of SelW cells had higher cell viability than wild-type cells. qPCR results found that overexpression of SelW cells had a lower levels of caspase-3, caspase-8, and fas mRNA than wild-type cells. Taken together, our findings suggested that SelW could reduce the oxidative damage induced by H(2)O(2) and had an important protective function in against oxidative damage.
Assuntos
Apoptose/efeitos dos fármacos , Galinhas/genética , Peróxido de Hidrogênio/farmacologia , Selenoproteína W/genética , Animais , Apoptose/genética , Células CHO , Caspase 3/genética , Caspase 8/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Expressão Gênica/efeitos dos fármacos , Marcação In Situ das Extremidades Cortadas , Oxidantes/farmacologia , Estresse Oxidativo/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Selenoproteína W/metabolismo , Receptor fas/genéticaRESUMO
We used site-directed mutagenesis to mutate two key amino acid residues, Glu164 and Arg167, of abrin A chain (ABRA), creating a mutant ABRA(E164AR167L). The mutant ABRA(mABRA) encoded by mABRA(E164AR167L) was expressed in the cytoplasm of Escherichia coli, and used to develop an effective vaccine to protect mice against native abrin intoxication. The cytotoxicity of mABRA was dramatically reduced as compared to that of recombinant ABRA(rABRA) and native abrin, but the antigenicity and immunogenicity remained the same. Balb/c mice were vaccinated with purified mABRA, and survival was evaluated after challenge with native abrin. Mice that were given three vaccinations developed a protective immune response that was 100% protective against an intraperitoneal (i.p.) administration of 10×LD50 of native abrin. Furthermore, the sera from immunized mice provided complete passive protection for naive mice. This study describes the generation of a substantial amount of mABRA from E. coli and the potential application of mABRA as an effective vaccine candidate for humans, to protect against a high-dose of native abrin.
