Sika deer antler thymosin β10 recombinant protein and its preparation method and use
A technology of recombinant protein and thymosin, which is applied in the field of genetic engineering and achieves the effects of broad application prospects and simple purification steps
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Embodiment 1
[0021] Example 1: Sika deer antler thymosin β 10 gene cloning
[0022] 1. Extract velvet total RNA: Weigh 1.2g of velvet sample, quickly grind it into a uniform fine powder in liquid nitrogen, add Trizol reagent at a ratio of 1:10, let it stand for 30 minutes, continue grinding for 2 minutes, and then transfer it to an EP tube , 1 mL per tube, add 200 μL of 5M NaCl each, shake fully, then add 200 μL of CHCl3, shake vigorously, let stand for 2 min, centrifuge, and take the upper layer. Repeated addition of CHCl 3 Extract and centrifuge until the middle layer is free of impurities. Transfer the supernatant to an RNase-free EP tube, add pre-cooled isopropanol at a ratio of 1:1, precipitate at -20°C for 30 min, centrifuge, discard the supernatant, wash the precipitate with 1 mL of 75% ethanol, and then wash with 100% Wash with absolute ethanol, and evaporate the ethanol to dryness. Detected by agarose gel electrophoresis, the bands were clear and bright without degradation.
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Embodiment 2
[0026] Example 2: Sika deer antler thymosin β 10 Construction of prokaryotic expression vector
[0027] Use EcoRI and SalI to sequence the correct recombinant vector pMD18-T-Tβ 10 and the expression vector pET-28a were subjected to double enzyme digestion, separated by agarose gel electrophoresis, the target gene and the vector pET-28a were recovered according to the gel recovery method, and transformed into E.coliBL21 competent cells after connection, plated and incubated at 37 Cultivate at ℃, screen positive single colonies, and identify with EcoRI and SalI double enzyme digestion, and detect by agarose gel electrophoresis, the two electrophoresis bands of the expression vector fragment and the target gene are clearly visible, and the molecular weights of the expression vector fragment and the target gene are both Consistent with the theoretical value, preliminary identification of the prokaryotic expression vector pET-28a-Tβ 10 build succeeds ( figure 2 ). After furthe...
Embodiment 3
[0028] Example 3: Sika deer antler thymosin β 10 expression and purification of
[0029] 1. Sika deer antler thymosin beta 10 Expression: the successful prokaryotic expression vector pET-28a-Tβ will be constructed 10 Transform into the host strain E.coliBL21, incubate at 37°C with shaking at 200r / min for 12 hours, then inoculate the bacterial liquid into new LB (containing 30μg / mLKan+) medium at a ratio of 1:100, and shake at 37°C , measure OD every 1h 600, reached its logarithmic growth phase after 3.5h of culture. At this time, IPTG induced expression was performed. By comparing the SDS-PAGE electrophoresis results of different IPTG induction concentrations and different induction times, the optimal IPTG induction concentration was 0.5mmol / L, and the optimal induction time was 4h ( image 3 ). After the expression bacteria were subjected to wall-breaking treatment, the supernatant and the precipitate were respectively detected by SDS-PAGE electrophoresis, showing Tβ 10...
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