Preparation method and application of selenium-enriched protein of bacteria
A technology of selenoproteins and selenoproteins, which is applied in the field of preparation and application of bacterial selenoproteins, can solve problems not involved in the synthesis and application of selenoproteins, achieve enhanced ability to resist pathogenic microorganisms, improve yield and quality, The effect of high organic selenium content
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Embodiment 1
[0026] Since the conclusion that selenium can enhance immunity is obtained from mammalian experiments, whether selenium can really enhance fish immunity has not been confirmed. In order to confirm that selenium can indeed enhance fish immunity, we added 199 medium (ThermoFisher Scientific) containing 10% fetal bovine serum to a 25ml culture flask, and inoculated carp epithelial cells (EPC cells), placed in CO 2 Incubate at 28°C for 24 hours in an incubator. After replacing the fresh medium, the red nano-element selenium (Se 0 ) (0, 10, 20, 50 μg / ml), continue to culture for 48 hours. Cell RNA was extracted using Trizol kit (Ambion) after the cells were collected. Then use RNA as a template and use PrimeScript TM II 1 st cDNA was synthesized with StrandcDNA Synthesis Kit (Takara Company). After the cDNA was diluted 10 times, the expression levels of internal reference β-actin (β-actin) and interferon IFN gene were detected by SYBR Green fluorescent quantitative PCR metho...
Embodiment 2
[0028] In order to verify whether endogenous spirulina of tea trees can efficiently synthesize selenoproteins, endogenous spirulina of tea trees was inoculated into LB medium containing different concentrations of sodium selenate (0, 20, 50, 100 mM), and cultured at 37°C for 12 hours . Bacterial cells were collected by centrifugation at 4°C and 8000 rpm, and washed twice with PBS (phosphate buffer, pH 7.2). Use RNAiso TM Kit (Takara company) to extract total RNA, and then use Prime Script TM RT reagent kit (Takara Company) was used to synthesize cDNA, and then the relative expression levels of 15 genes in the selenoprotein synthesis pathway of Endospira spirulina were detected by conventional semi-quantitative PCR method. The 16S rRNA gene of endophytic Spirulina spirulina in tea trees was used as an internal reference, and the results are shown in figure 2 . Under the stimulation of selenate, Endospira spirulina of tea tree can highly express glucose-6-phosphate 1-dehyd...
Embodiment 3
[0030] Inoculate 5 mL of nutrient broth medium (10 g of tryptone, 3 g of beef powder, 5 g of NaCl, and add water to 1000 mL) of the endophytic Helix spirochete strains stored at -80 °C, shake at 150 rpm, and incubate overnight at 28 °C. The activated bacterial solution was inserted into LB liquid medium containing 0, 5, 10, 20, and 50 mM sodium selenate at a ratio of 1:100 (10 g tryptone, 5 g yeast extract, 10 g NaCl, added water to 1000 mL, pH7. In 2), the rotating speed of the shaker is 250 rpm, and the temperature is 37° C. for 24 hours. The bacterial cells were collected by centrifugation at 6000 rpm and 4° C. for 15 minutes, and suspended with 0.1 M PBS (phosphate-balanced saline, pH 7.4). Repeat centrifugation and washing twice under the same conditions to remove residual components in the culture. The bacteria collected by centrifugation were suspended in solution I (0.1mM EDTA (ethylenediaminetetraacetic acid), 50mM TrisHCl, pH8.0), and the bacteria were disrupted by ...
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