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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

Active Publication Date: 2014-04-23
吉林省盛世华鑫生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no preparation of sika deer antler thymosin β 10 related reports

Method used

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  • Sika deer antler thymosin beta10 recombinant protein, preparation method and application of protein
  • Sika deer antler thymosin beta10 recombinant protein, preparation method and application of protein
  • Sika deer antler thymosin beta10 recombinant protein, preparation method and application of protein

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention relates to sika deer antler thymosin beta10 recombinant protein, a preparation method and application of protein, belonging to the technical field of gene engineering. The method comprises the following steps: cloning deer antler thymosin beta10 genes, constructing a prokaryotic expression vector pET-28a-Tbeta10, performing inducible expression in host bacteria E.coli BL21 and separating and purifying the recombinant protein. The prokaryotic expression of the sika deer antler thymosin beta10 is attempted for the first time by utilizing a gene engineering technology, the prokaryotic expression vector is successfully constructed, high-efficiency expression of the recombinant protein is induced, and the purification step is simple and rapid. The in-vitro activity research proves that the sika deer antler thymosin beta10 recombinant protein has an effect of obviously promoting proliferation of osteoblasts M3T3.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to a sika deer antler thymosin beta 10 Recombinant protein and its preparation method and use. Background technique [0002] Sika deer antler is the unossified and densely grown young horns of the sika deer stag, an animal of the deer family. It is a traditional Chinese medicine in my country. The chemical components in velvet antler mainly include amino acids, inorganic elements, proteins, lipids, sugar compounds, etc., of which the protein content accounts for more than 50%. Modern research shows that deer antler has various pharmacological effects such as anti-aging, anti-oxidation, enhancing body immunity, anti-tumor, anti-chemical drug damage, and anti-ulcer. In recent years, with the rapid development of biotechnology, more and more researchers have begun to pay attention to the biologically active proteins and polypeptides in velvet antler, which play an extreme...

Claims

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Application Information

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IPC IPC(8): C07K14/575C12N15/16C12N15/70A61K38/22A61P19/00
CPCC07K14/57581C12N15/70
Inventor 赵雨冷向阳张惠王伟楠李红艳姜先刚徐占炜
Owner 吉林省盛世华鑫生物科技有限公司
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