| Selenium is an essential trace element in biological life activities, playing a biological function in the form of selenoproteins. Selenoprotein W (SelW) is a member of the selenoprotein families, expressed in various tissues of many animals and acted as an oxidoreductase in mammals. At present, the studies about SelW are mainly carried out in mammalian, e.g. primates and rodents. However, little is known about of the SelW, especially in birds and the research in vitro have not been reported, as well as the study of chicken SelW biological functions also have been less, except for the distribution of the organization.. According to the molecular biology technology, chicken SelW eukaryotic cell expression vector containing SECIS of3’UTR area and green fluorescent protein expression vector were structured in this study. Throughing the green fluorescent protein expression vector, the cell translation conditions were optimized, and chicken SelW eukaryotic cell expression vector was translated into CHO-K1. The expression of SelW mRNA in translation cells were detected by RT-PCR and qPCR, simultaneously, SecS mRNA levels and SPS-1mRNA levels were also detected, and then, chicken SelW over-expression CHO-K1cell lines and nomal CHO-K1cell lines were acted as models, and the different concentrations of H2O2-induced apoptosis were monitored by AO/EB double staining and TUNEL assays, and the abundance of the Caspase-3, Caspase-8and Fas mRNA were detected by real-time quantitative reverse transcription PCR (qPCR). The results were displayed as follows:1. According to the known chicken SelW cDNA sequence, primers that can clone the chicken SelW SECIS were designed, and chicken SelW eukaryotic cell express vector pcDNA3.1/SelW was successfully constructed.2Throughing the extinction enzymes and DNA connection enzymes, green fluorescent protein gene of pEGFP-NI vector was connected to eukaryotic cell expression vector pcDNA3.1(+), and the restructuring green fluorescent protein expression vector pcDNA3.1/GFP was constructed. In optimization of translation trials, when the ratio of translation reagent and pcDNA3.1/GFP was8:2(μL:μg), the efficiency of translation was the highest.3Chicken SelW was successfully translated into CHO-K1cells in this study. The optimum condition in6-well culture plate was that8μL translation reagent and2μg expression vector were added into each well. SelW, SecS and SPS-1mRNA levels of pcDNA3.1/SelW translated cells and nomal cells were detected, after pcDNA3.1/SelW translated24h and48h, a SelW specificity strip was appeared in cells translated pcDNA3.1/SelW, hower untranslated cells were not shown. and at the same time, qPCR results were shown that SelW mRNA levels of translated cells were obviously higher than untranslated cells, which showed that chicken SelW over-expression cell lines were successfully constructed. We also found that SecS mRNA levels were significantly higher in chicken SelW translated cells, however SPS-1mRNA levels had no significant changes, so we assumed that the SecS was involved in the chicken SelW synthesis, however SPS-1was not.4Chicken SelW over-expression cell lines and nomal cell lines can be induced apoptosis by H2O2. Using the AO/EB double dye and TUNEL analysis, we found that the apoptosis rate of the chicken SelW translated cells was significantly lower than the untranslated cells induced by the same concentration of H2O2, and then, in the detection of Caspase-3, Caspase-8and Fas mRNA levels of the gene related to apoptosis, we found that the levels of Caspase-3, Caspase-8and Fas mRNA in chicken SelW translation cells were significantly lower than untranslated cells induced by the same concentration of H2O2, which assumed that SelW could reduce the oxidative damage induced by H2O2, had an important protective function in against oxidative damage and had an antioxidative function. |
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