Sika deer antler thymosin beta10 recombinant protein, preparation method and application of protein
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 , 1mL per tube, add 200μL of 5M NaCl, shake fully, then add 200μL of CHCl3, shake vigorously, let stand for 2min, 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.
[0023] 2...
Embodiment 2
[0026] Example 2: Sika deer antler thymosin β 10 Construction of prokaryotic expression vector
[0027] Use EcoR I and Sal I 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, connected and transformed into E.coli BL21 competent cells, plated and placed on Cultivate at 37°C, screen positive single colonies, identify by EcoR I and Sal I 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 expression vector fragment and the target gene The molecular weights were consistent with the theoretical value, and the prokaryotic expression vector pET-28a-Tβ was initially identified 10 build succeeds ( figure 2 ). After further sequencing, compa...
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 10Transform into the host strain E.coli BL21, incubate at 37°C with shaking at 200r / min for 12 hours, inoculate the bacterial liquid into new LB (containing 30 μg / mL Kan+) medium at a ratio of 1:100, and inoculate at 37°C Shake culture, measure OD every 1h 600 , reached its logarithmic growth phase at 3.5 hours of culture. At this time, IPTG induced expression was carried out, and by comparing the SDS-PAGE electrophoresis results of different IPTG induction concentrations and different induction times, the optimal IPTG induction concentration was optimized to be 0.5mmol / L, and the optimal induction time was 4h ( image 3 ). After the expression bacteria were broken, the supernatant and the precipitate were subjected to SDS-PAGE electrophoresis detection, showing...
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